AGAROPHYTON CHILENSIS EXTRACT, RICH IN FREE FATTY ACIDS, AS A NUTRACEUTICAL OR NUTRITIONAL SUPPLEMENT, SUITABLE FOR MODULATING PPAR# ACTIVITY

Abstract

Agarophyton chilensis extract, enriched with free fatty acids and PPARγ modulators, comprising palmitic acid, stearic acid, myristic acid, oleic acid, and 8-hydroxyeicosatetraenoic acid (8-HETE). Method for obtaining the extract. Nutraceutical composition, comprising the extract enriched with free fatty acids and PPARγ modulators, wherein said extract is useful for treating or preventing health problems, which require neuroprotection, wherein said neuroprotection involves activation of PPARγ receptors.

Claims

1. Fraction of an Agarophyton chilensis oleoresin extract-enriched with free fatty acids and PPARγ modulators, and free of neutral lipids, wherein it comprises palmitic acid, stearic acid, myristic acid, oleic acid, and 8-hydroxyeicosatetraenoic acid (8-HETE).

2. The Agarophyton chilensis extract enriched with free fatty acids and PPARγ modulators according to claim 1, wherein it comprises: TABLE-US-00008 12 to 26 wt. % of palmitic acid; 3.7 to 4.9 wt. % of stearic acid; 2.2 to 5.8 wt. % of myristic acid; 0.8 to 6.8 wt. % of oleic acid; 0.2 to 0.5 wt. % of 8-HETE.

3. The Agarophyton chilensis extract enriched with free fatty acids and PPARγ modulators according to claim 2, wherein it further comprises: 0.017 to 0.009% of 9-HETE and 0.016 to 0.034% of the group of further eicosanoids formed by Leukotriene B4, 12-hydroxy-eicosatetraenoic acid, 5,12 di-hydroxy-eicosatetraenoic acid, 8-hydroxy-eicosapentaenoic acid, 14,15-epoxy-eicosatrienoic acid, 8-hydroxy-eicosatrienoic acid, and 11-hydroxy-eicosatetraenoic acid.

4. The Agarophyton chilensis extract enriched with free fatty acids and PPARγ modulators according to any one of claims 1 to 3, wherein it comprises per milligram of extract B 191.7 micrograms of palmitic acid, 42.5 micrograms of stearic acid, 39.2 micrograms of myristic acid, 37.6 micrograms of oleic acid, 3.5 micrograms of 8-HETE, 1.3 micrograms of 9-HETE, and 0.25 micrograms of further eicosanoids.

5. Method of obtaining the extract enriched with free fatty acids and PPARγ modulators according to claim 1, wherein an oleoresin obtained by dichloromethane extraction from the lyophilized and ground biomass of the Agarophyton chilensis algae, wherein that the method comprises the steps of: a) providing an oleoresin extracted with dichloromethane from the lyophilized and ground biomass of the Agarophyton chilensis algae; b) extracting by aminopropyl column chromatography said oleoresin dissolved in a load solvent (hexane, diethyl ether, acetic acid), and eluting with a solvent mixture A (chloroform: 2-isopropanol, 2:1), to remove the neutral components; c) eluting from the column of step b) with a solvent mixture B (chloroform, methanol, acetic acid) to obtain an eluate or Extract B; d) drying Extract B protected from light, evaporating with a N.sub.2 stream and storing at −80° C.; e) optionally resuspending Extract B in DMSO 99.9% and storing at inert atmosphere at −80° C.

6. The method according to claim 5, wherein the load solvent mixture contains hexane: diethyl ether: acetic acid in a ratio of 100:3:0.3.

7. The method according to claim 5, wherein the solvent mixture A contains chloroform: 2-isopropanol in a ratio of 2:1.

8. The method according to claim 5, wherein the solvent mixture B contains chloroform, methanol and acetic acid in a ratio of 100:2:2.

9. Nutraceutical composition, wherein it comprises the Agarophyton chilensis extract enriched with free fatty acids and PPARγ modulators according to claim 1.

10. The nutraceutical composition according to claim 9, wherein it further comprises further active components.

11. The nutraceutical composition according to claim 10, wherein the active components are selected from oils, antioxidants, vitamins and drugs.

12. The nutraceutical composition according to claim 10, wherein it further comprises Omega 3 oil.

13. Use of the Agarophyton chilensis extract enriched with free fatty acids and PPARγ modulators according to claim 1, wherein it is for preparing a nutraceutical composition useful for the treatment or prevention of health problems requiring neuroprotection, wherein said neuroprotection involves activation of PPARγ receptors.

14. The use according to claim 13, wherein the nutraceutical composition is useful for the treatment or prevention of chronic or acute neurological diseases; chronic or acute inflammatory disorders; stroke.

15. The use according to claim 13, wherein the nutraceutical composition is useful for preventing, mitigating and/or treating ischemia or stroke.

16. The use according to claim 13, wherein the nutraceutical composition does not cause an increase in adipogenesis.

17. The use of the enriched extract according to claim 1, wherein it is for preparing a nutritional supplement, useful as neuroprotector.

18. The use according to claim 18, wherein the nutraceutical supplement is useful for preventing, mitigating and/or treating ischemia or stroke.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1: It shows a flowchart for the processes for obtaining Extract B from Step 2 to Step 4 from Extract A.

[0052] FIG. 2: It shows the Extract B recovery percentage (grams of Extract B per 100 g of Extract A) obtained from 8 independent chromatographic separations, carried out in different months of the year (from April 2017 to February 2018). The dotted line represents the mean recovery value of Extract B.

[0053] FIG. 3: It shows a comparison of the PPARγ activation induced by Extract A and by Extract B at the same concentrations (30 and 60 μg/mL). The values correspond to the mean of six determinations of independently produced extracts. *p<0.5 between treatment with Extract A 60 μg/mL and control; **p<0.01 between treatment with Extract B 30 μg/mL and control; ***p<0.001, between treatment with Extract B 60 μg/mL and control (Kruskal-Wallis).

[0054] FIG. 4: It shows a comparison of the PPARγ activation induced by 25 μM of FMOC-Leu (FMOC), 25 μM of INT-131 (INT), and 60 μg/mL of Extract B. The values correspond to the mean of six determinations of independently produced extracts. **p<0.01, ***p<0.001 ANOVA Tukey Test.

[0055] FIG. 5: It shows the PPARγ transcriptional activation induced by 1 μM of rosiglitazone in PC12 cells. The values correspond to the mean of three determinations of independently produced extracts. ***p<0.001 ANOVA Tukey Test between control and RGZ treatment.

[0056] FIG. 6: It shows that the PPARγ transcriptional activation induced by 60 μg/mL of Extract B is inhibited by co-treatment with 10 μM of the specific PPARγ inhibitor, T0070907 (T007), in PC12 cells. The values correspond to the mean of three determinations of independently produced extracts. ***p<0.001 ANOVA Tukey Test between Extract B and Extract B with PPARγ inhibitor.

[0057] FIG. 7: It shows the quantification of Oil Red O accumulation after a differentiation assay of 3T3-L1 cells into adipocyte cells, treated with 60 μg/mL of Extract A, with 60 μg/mL of Extract B, 25 μmL of FMOC-Leu (FMOC) or 1 μM of rosiglitazone (RGZ). The values correspond to the mean of three determinations of independently produced extracts. **p<0.01 in RGZ treatments relative to the control (Kruskal-Wallis).

[0058] FIG. 8: It shows the expression of mRNA expression of the differentiation gene, fabp4, after the differentiation assay of 3T3-L1 cells into adipocyte cells, treated with 60 μg/mL of Extract A, 60 μg/mL of Extract B, 25 μmL of FMOC-Leu (FMOC) or 1 μM of rosiglitazone (RGZ). The values correspond to the mean of four determinations of independently produced extracts. ***p<0.001 in RGZ treatments relative to the control (Kruskal-Wallis).

[0059] FIG. 9: It shows the survival of cortical neurons in culture after damage induced with oligomycin A; wherein 100% corresponds to survival found under basal conditions without treatment with oligomycin. Neurons were treated with Extract B in different concentrations (10-50 μg/mL) or with DMSO (white bar, 0) as carrier, and then they were damaged with oligomycin A. The values correspond to the mean of three independent determinations.

[0060] FIG. 10: It shows the survival of cortical neurons in culture after damage induced with oligomycin A; wherein 100% corresponds to survival found under basal conditions without treatment with oligomycin. It shows the comparison of the survival of neurons treated with Extract A or Extract B (10 and 50 μg/mL) after damage with oligomycin. ***p<0.001 in treatments with Extract B in all concentrations relative to the control (0) (ANOVA, Tukey Test).

DETAILED DESCRIPTION OF THE DISCLOSURE

[0061] The disclosure refers to a fatty acid enriched extract with the capability of transcriptionally activating and/or modulating the PPARγ receptor (Extract B) and having neuroprotective and antioxidant activity and further having the advantage of having no adipogenic activity. As described above, the PPARγ pharmacological agonists used are drugs called TZD, which have the disadvantage of increasing adipogenesis or having a capability of favoring cell differentiation into adipocytes.

[0062] Said enriched extract is obtained by chromatographic fractioning of an oleoresin from Agarophyton chilensis (Extract A), wherein degradation of oxylipins and antioxidants is minimized. Oxylipins are oxidized lipids derived from free fatty acids produced by phospholipases and lipoxygenases, acting as mediators of physiological responses to tissue stress in plants. The claimed extract comprises as main components: palmitic acid and 8-HETE, and it may also comprise stearic acid, myristic acid, oleic acid, and 9-HETE, together with further eicosanoids at lower ratios.

[0063] The claimed extract (Extract B) can be used in the prevention, mitigation or treatment of metabolic diseases, including diabetes, dyslipidemias, and chronic and acute neurological diseases, even ischemia, chronic and acute inflammatory disorders, and as insulin sensitizing antidiabetic agent, in pulmonary hypertension, stroke, or any disease that might be treated with PPARγ agonists, given that the activation of this receptor provides cellular and metabolic benefits which translate into a health benefit.

[0064] In a preferred embodiment, this extract is useful for preparing a nutraceutical composition of different types, which can be formulated as a nutraceutical supplement, in the form of tablets, syrups, pills, etc., wherein the supplement comprising said nutraceutical composition and has a neuroprotective, anti-inflammatory and antioxidant activity, especially having therapeutic, preventive or mitigating effects on ischemia or stroke. In particular, the nutraceutical supplement has therapeutic, preventive or mitigating effects on cerebrovascular disorders mediated by the activation of the PPARγ transcriptional factor.

[0065] In a second preferred embodiment, the nutraceutical extract is useful for preparing a nutritional supplement, wherein said nutritional supplement has neuroprotective activity, especially therapeutic, preventive or mitigating effects on ischemia or stroke. In particular, the nutritional supplement has therapeutic or preventive effects on cerebrovascular disorders mediated by the activation of the PPARγ transcriptional factor.

[0066] Methods

[0067] Preparation of the Claimed Extract A

[0068] The process for generating the enriched extract from Agarophyton chilensis is described in WO2014186913, which comprises the steps described as follows:

[0069] Natural Fresh Algae Collection and Transportation

[0070] The algae are collected from the coastal edge in Chile, preferably live and whole algae are collected. Preferably, the red algae (Agarophyton chilensis) are collected and selected manually, with a size being ≥60 cm long, thus preserving the growing algae. The collected and selected material is transported to the site of acclimatization and subsequent cultivation in thermo-insulated containers, with special care to maintain the moisture of the algae, adding enough seawater.

[0071] Acclimation and Cultivation of Algae Collected in Pools Under Standardized Conditions

[0072] Once the collected and selected algae arrive at destination, biomass is first washed with drinking water, weighted and provided in open culture tanks with a capacity of 4,600 liters, the tanks having aeration and continuous water replacement for their growth in the free-floating mode covering a cultivation area of 6 m.sup.2. The tanks and biomass are weekly washed with filtered seawater to avoid epiphytic loading and accumulation of organic matter. Said biomass can be maintained under culture conditions from two to twenty weeks before being processed for extraction of lipidic components and obtaining the extract enriched in fatty acids and oxidized fatty acids. Algae collection and culture is not season-dependent, as the enriched extracts produced in different seasons have the same PPARγ activating capability (data not shown).

[0073] Biomass Washing and Freezing

[0074] The process of extracting components comprises removing biomass from the culture tanks, conveying it to the processing site, maintaining temperature (>20° C.) and moisture. Excess seawater is removed from the algae by centrifugation and is put in vacuum sealed bags for freezing (−20° C.).

[0075] Algae Tissue Disruption and Dehydration Through Lyophilization

[0076] The algae are thawed in vacuum in cold water, then they are removed from the bag, and washed with a saline buffer solution (NaCl (137 mmol/L), KCl (2.7 mmol/L), Na.sub.2HPO.sub.4.2 H.sub.2O (10 mmol/L), KH.sub.2PO.sub.4 (2.0 mmol/L), pH 7.4), and excess water is removed by centrifugation.

[0077] Then, the algae from the former step is (manually or automatically) chopped at ambient temperature (20-24° C.), obtaining 1-5 mm pieces. The chopped algae are put in petri dishes, trays or bags, and maintained in the freezer at −80° C. for at least 24 hours. The previously frozen algae is lyophilized for 12 to 48 hours, preferably 24 hours; at a temperature of −20° C. to −90° C., preferably at −50° C.; at a pressure of 0.1 Pa (0.001 mbar) to 3 Pa (0.03 mbar), preferably at a pressure of 1.4 Pa (0.014 mbar). Once the lyophilized product is obtained, it is stored in vacuum bags at −20° C.

[0078] Algae Grinding and Solid-Liquid Extraction of Lipidic Components: (Extract A, Based on the Technique Described in WO2014186913)

[0079] The vacuum lyophilized biomass from the former step is allowed to reach ambient temperature (20-24° C.), then it is weighted and ground to a fine powder with a particle size of ≤0,5 mm. For the process of obtaining Extract A, an Erlenmeyer flask (500 ml) is used, which is added with an amount of the finely ground algae, and dichloromethane (CH.sub.2Cl.sub.2) is incorporated, at a ratio of 1/2 to 1/10 by weight of finally ground algae/volume of CH.sub.2Cl.sub.2, preferably 1:3.6 w/v. Each container is exposed to an atmosphere saturated with N.sub.2, and sealed. Immediately, it is incubated with horizontal stirring at a temperature of 45° C. to 25° C. (34° C.); for a term of 2 hours to 10 minutes (30 minutes).

[0080] Thereafter, the mixture is decanted and filtered under vacuum in a sintered glass filter and Whatman No. 1 paper. The liquid phase is received in another container. The solid phase is resuspended in the solvent, and the previous extraction procedure is repeated, at least once. The filtered liquid is concentrated in a rotary evaporator at a temperature between 37-39° C., resulting in a semi-finished oleoresin with traces of the solvent, dichloromethane. This product is resuspended in a minimum volume of cyclohexane, quickly frozen at −80° C. and lyophilized for 24 hours, finally obtaining Extract A.

[0081] Extract A is stored under saturated atmosphere of inert gas (Ar) in order to avoid oxidation processes, at −20° C.

[0082] Obtaining the Oily Extract Enriched With Saturated, Unsaturated and Oxidized Free Fatty Acids, and with Antioxidant Capability (Extract B).

[0083] The produced Extract A is subjected to a solid phase chromatography extraction to concentrate saturated, unsaturated free fatty acids, and oxidized fatty acids, to obtain Extract B.

[0084] Extract B is obtained by solid phase chromatography separation, using aminopropyl columns, with organic solvent mixtures as mobile phases, through which sequential elutions are performed, wherein in a first step the neutral lipid components are extracted with solvents being less polar, such as eluents, and then the polar lipid components are extracted (rich in free fatty acids) with solvents having a higher polarity.

[0085] The separation of the sample by the aminopropyl column is carried out with a sample load of 5 wt. % relative to the stationary phase. FIG. 1 shows a scheme for obtaining Extract B from steps 2 to 4. Example 1 describes details about the process for obtaining Extract A and Extract B.

[0086] Preparation of the Claimed Extract B

[0087] The Steps for Obtaining Extract B are Detailed Next:

[0088] The oleoresin obtained by dichloromethane extraction from the lyophilized and ground biomass ofAgarophyton chilensis is provided, which is subjected to the following steps: [0089] a) extracting by aminopropyl column chromatography said oleoresin dissolved in the load solvent, consisting of hexane, diethyl ether, and acetic acid, and eluting with a solvent mixture A, consisting of chloroform and 2-isopropanol, to remove the neutral components; [0090] b) eluting the column from step a) with a solvent mixture B, consisting of chloroform, methanol and acetic acid, to obtain an eluate corresponding to Extract B; [0091] c) drying Extract B protected from light, by evaporation with N.sub.2 stream, and storing at −80° C.; [0092] d) optionally resuspending Extract B in DMSO 99.9% and storing in an inert atmosphere at −80° C.

[0093] Wherein the solvent mixtures used in the claimed method are as follows:

TABLE-US-00003 Mixture Components Ratio Load solvent mixture hexane:diethyl ether: 100:3:0.3 acetic acid Solvent mixture A chloroform:2-isopropanol 2:1 Solvent mixture B chloroform:methanol: 100:2:2 acetic acid

[0094] Through the process for obtaining Extract B according to the present disclosure, a product recovery with a yield of 12% to 25% is achieved (FIG. 2).

[0095] Optionally, Extract B obtained by chromatographic separation is resuspended in DMSO 99.9% in a concentration of 60 mg/mL and stored in an inert atmosphere at −80° C.

[0096] Analysis of Extract A and B components

[0097] Characterization of Extract A

[0098] During the development of the claimed disclosure, Extract A was assessed to determine the content of antioxidants, such as tocopherols and carotenoids. Additionally, the use of in vitro tests allowed establishing the antioxidant capability of Extract A and Extract B.

[0099] Measurements of six different Extracts A show that this Extract has plenty of antioxidant molecules: tocopherols (total: 6673 μg/g of Extract A), the most abundant molecule being γ-tocopherol (5232 μg/g of Extract A) and β-carotene (1538 μg/g of Extract A). The presence of lycopene was not detected in Extract A.

[0100] Concerning the content of the different fatty acids present in Extract A, the representative composition shows 51.7% of saturated fatty acids, 18.2% of monounsaturated fatty acids, 24.7% of polyunsaturated fatty acids, and 2.2% of trans fatty acids, palmitic acid being the most abundant fatty acid, with a content of 39%. The full composition is exhibited in Table 1.

TABLE-US-00004 TABLE 1 Composition of fatty acids in Extract A of Agarophyton chilensis Type Fatty acid (%) Saturated Tridecanoic 1.15 Myristic 3.90 Palmitic 39.20 Margaric 1.30 Stearic 4.0 Polyunsaturated Linoleic 3.39 Arachidonic (AA) 19.40 Alpha-linolenic (ALA) 0.61 Eicosapentaenoic (EPA) 0.66 Docosahexaenoic (DHA) 0.10 Monounsaturated Oleic 14.2 Trans — 2.25

[0101] Characterization of Extract B

[0102] On the other hand, a lipidomic assay was carried out (Lipidomics Center, Wayne University) to establish the presence of oxidized fatty acid and fatty acid derivatives. Surprisingly, it was found that Extract B is enriched with 8-hydroxy-eicosatetraenoic acid (8-HETE), which is an arachidonic acid (AA) oxidized derivative produced by lipoxygenase, representing 90% of fatty acid oxidized derivatives (Table 2). Moreover, it was determined that per milligram of Extract B, there is a significant presence of 3.5 μg of 8-HETE, 0.1 μg of 9-HETE and 0.25 μg of a mixture of equivalent amount of other arachidonic acid oxidized derivatives, including Leukotriene B4 [LTB4], 12-Hydroxy-Eicosatetraenoic Acid [12-HETE], 5,12 di-Hydroxy-Eicosatetraenoic Acid [5(s), 12(s)-DiHETE], 8-Hydroxy-Eicosapentaenoic Acid [8-HEPE], 14,15-Epoxy-Eicosatrienoic Acid [14(15)-EpETrE], 8-Hydroxy-Eicosatrienoic Acid [8-HETrE], and 11-Hydroxy-Eicosatetraenoic Acid [11-HETE] (Table 2).

TABLE-US-00005 TABLE 2 Identification of oxidized fatty acid derivatives present in the free fatty acid moiety. The content of oxidized fatty acids (eicosanoids) present in Extract B (n = 3) was assessed. μg/mg Eicosanoid Extract B 8-HETE 3.55 9-HETE 0.13 LTB4 0.06 12-HETE 0.05 5(s), 12(s)-DiHETE 0.04 8-HEPE 0.03 14(15)-EpETrE 0.04 8-HETrE 0.016 11-HETE 0.014

[0103] Among the fatty acids contained in Extract B, the more abundant ones were found to be palmitic acid, followed by stearic acid, oleic acid and myristic acid (Table 3).

TABLE-US-00006 TABLE 3 Profile of the most abundant fatty acids present in Extract B (n = 3). μg/mg Fatty acid Extract B Palmitic (C16:0) 191.7 Stearic (Cl8:0) 42.5 Myristic (Cl4:0) 39.2 Oleic (C18:l) 37.6 Dodecanoic (Cl2:0) 3.8 Lignoceric (C24:0) 3.7 Palmitoleic (Cl6:1) 2.0 Linoleic (C16:2w6) 0.5 Arachidonic (C20:4w6) 2.5 Eicosapentaenoic (C20:5w3) 0.09

[0104] In view of the values described in Tables 1, 2 and 3, it should be noted that in Extract A, the most abundant polyunsaturated fatty acid is AA. In turn, Extract B has a low ratio of this polyunsaturated fatty acid, wherein the content of AA is 0.2% w/w, while Extract A contains 1.9% w/w of AA (Table 1), and it is enriched with its oxidized derivatives 8-HETE and 9-HETE. AA is part of cell membranes, being part of glycerolipids in algae. It is known that AA is a prostaglandin precursor; hence, it has been involved in proinflammatory pathways. Therefore, the biological activities of Extract B could be different and superior to those of Extract A, since the former is not enriched with AA. In addition, as mentioned above, 8-HETE acid acts as natural ligand of PPARs, capable of activating both PPARα and PPARγ.

[0105] The extract enriched with free fatty acids and PPARγ modulators ofAgarophyton chilensis of the disclosure contains 19.2% of palmitic acid; 4.2% of stearic acid; 3.9% of myristic acid; 3.8% of oleic acid; 0.35% of 8-HETE; 0.013% of 9-HETE; and 0.025% of further arachidonic acid derivative oxidized fatty acids: LTB4, 12-HETE, 5(s),12(s)-DiHETE, 8-HEPE, 14(15)-EpETrE, 8-HEPE, 8-HETrE, 11-HETE.

[0106] It is important to indicate that 20-HETE acid, as well as other hydroxy acids, prostaglandins and leukotrienes were detected in poor abundancy or not detected at all in the analyses (data not shown). In particular, 20-HETE acid has been described as a potent vasoconstrictor involved in the development of hypertension and other pathologies related to inflammation; hence, its absence prevents the use of Extract B from showing undesirable effects associated with said compound. Furthermore, leukotriene B4 was found in low concentrations of 0.006% w/w with a variation between 0.01-0.006%.

[0107] Finally, based on the results obtained for the components of the claimed extract, it is possible to state that Extract B, Agarophyton chilensis, contains low amounts of prostaglandins as compared to other related red algae, such as Agarophyton vermiculophylum, which is probably due to a low activity of cyclooxygenase (Honda et al., 2019).

[0108] In view of the foregoing, it can be concluded that Extract B is advantageous for use as nutraceutical product, since the low content of AA and derivatives of the same would barely cause undesirable side effects associated with said components.

[0109] Furthermore, Extract B derived from Agarophyton chilensis does not have detectable prostaglandins, unlike those that can be detected in a methanolic extract of Agarophyton vermiculophylum (Rempt et al., 2012).

[0110] Extract B, compared to Extract A, is enriched with natural ligands of PPARγ, since in PPARγ transcriptional activation assays in PC12 cells, a capability of activating PPARγ is observed in more than twice the activation as compared to the results achieved when using Extract A, as shown in FIG. 3, which exhibits a comparison of the PPARγ transcriptional activation with Extract A and with Extract B in the same concentrations.

[0111] The following examples provide the preferred exemplified embodiments of the disclosure, which do not delimit the scope of the disclosure, which can be carried out considering variations related to the details described herein.

[0112] In Vitro Assessments and Bioassays

[0113] PPARγ Activation Assay in PC12 Cells

[0114] The dry Extracts (A and B) were resuspended in DMSO (99.9%) at a maximum dilution of 100 mg/mL, in a preferred dilution range of 10, 20, 25, 30, 35, 40, 45, 50, 55, 60 μg/mL. Dilutions are stored in an inert atmosphere between −20 and 80° C. until the moment of using them in the cellular tests.

[0115] In the PPARγ activation assay, an assay with the reporter gene gal4 PPARγ is performed, which allow assessing the activation of PPARγ induced by ligand, to which end concentrations of 30 and 60 μg/mL were used. PC12 cells were seeded in a 48-well plate at a density of 100,000 cells/well/250 μL of maintenance medium containing the medium RPMI1640® (Roswell Park Memorial Institute-1640 Medium) supplemented with 10% of horse serum (HS), 5% of fetal bovine serum (FBS) and 1× antibiotic/antimicotic (Ab-Am). Then, the DNA (0.8 μg luciferase plasmid (MH100tkLUC), 0.8 μg PPARγGAL4 plasmid and 0.053 μg β-galactosidase plasmid, (β-Gal)) were mixed in OptiMEM® (Opti-Minimun Essential Medium). This constitutes the DNA mixture. In parallel, lipofectamine 2000 was diluted in OptiMEM®. This constitutes the Lipofectamine® mixture. Both mixtures were kept at room temperature for 5 minutes, then were mixed at equal ratios and incubated for 20 minutes at room temperature, while the medium of PC12 cells was replaced with 200 μL of transfection medium (RMPI 1640 supplemented with 2% FBS and 2% HS, without antibiotics) per well. After the 20 min incubation, each well was added with 50 μL of the DNA-lipofectamine mixture. Then, the DNA-lipofectamine mixture was incubated for 6 hours at ambient temperature. Subsequently, the medium of the cells was replaced with a treatment medium (RPMI 1640 supplemented with 2% FBS, 2% HS and 1× Ab-Am). Afterwards, the treatment was carried out with the extracts and its corresponding DMSO control in the treatment medium for 16 hours. In the case of the assays with PPARγ inhibitors, a preincubation for 30 minutes was performed with the PPARγ inhibitor, T0070907 (10 μM), and the inhibitor was maintained for the corresponding 16 hours of treatment (n=3, for each condition). After the preceding incubation, the treatment medium was removed and the cells were lysed with 100 μL of lysis buffer 1× CCLB® (Cell Culture Lysis Buffer, luciferase kit, dilution of stock 5× in water 3×). The lysate was collected, centrifuged for 1 minute at 14000 rpm, the β-galactosidase and luciferase activity in the supernatant was measured, and the times of PPARγ activation was calculated relative to the DMSO control (final concentration in the medium of 0.1%). In parallel, the activity of β-galactosidase was assessed (colorimetry). The luciferase activity values were relativized to the beta-galactosidase activity. The activity results were expressed as times of increase in relation to the activity obtained with control cells (treated only with DMSO). With this assay, both Extract A and Extract B were observed as having PPARγ activators. As shown in FIG. 3, Extract A triggers 3.7 and 7.8 times the receptor activity at 30 μg/mL and 60 μg/mL, respectively (n=6), while Extract B triggers 5.9 and 20.2 times the activity at 30 μg/mL y 60 μg/mL, respectively (n=6), wherein the activity of the last concentration was significantly higher (p<0.01, ANOVA, Tukey post-test) than the one shown by the negative control (cells treated with 0.1% DMSO carrier). This activation level is similar to the level observed with SPPARM-type PPARγ partial activators, such as FMOC-Leu and INT-131 (FIG. 4). Treatment with 25 μM of FMOC-Leu or INT-131 triggered an increase in the PPARγ transcriptional activity in a significantly greater manner as compared to the negative control, being 14.7 and 8.1 times higher (n=10, p<0.01 ANOVA, Tukey post-test), respectively. On the contrary, TZD, rosiglitazone, called a full agonist, at the concentration of 1 μM increased 109 times the PPARγ transcriptional activity (n=9) (FIG. 5). The PPARγ transcriptional activity assay by the PPARγGAL4 system is a highly specific cell assay system. It shows that PPARγGAL4 activation is only mediated by the binding of PPARγ agonists to the nuclear receptor ligand binding domain. This specificity is evidenced when incubating with the PPARγ pharmacological inhibitor, T0070907 (T007). As shown in FIG. 6, simultaneous treatment of PC12 cells transfected with plasmids that allow assessing PPARγ transcriptional activity with Extract B and the inhibitor T0070907 significantly prevented activation induced by Extract B, thereby reducing its activation 3.4 times relative to the control (n=2 in quadruplicate). P<0,01. ANOVA, Tukey post-test.

[0116] Induction Assays of cell Differentiation into Adipocytes

[0117] In order to assess the effect of the claimed extracts on differentiation of fibroblasts into adipocytes, 3T3-L1 cells are cultured at low passage, avoiding cells from reaching confluence. In order to differentiate 3T3-L1 cells, 600,000 cells were seeded in a 35 mm plate in a 2 mL volume of maintenance medium for staining with Oil-Red-O. In parallel, 90,000 cells per well were seeded in a 250 μL volume of maintenance medium, in a 48-well plate, for a viability assay using tetrazolium salt (MTT), and in a 12-well plate 210,000 cells per well were seeded in a 1 mL volume for extraction of mRNA. Thereafter, the maintenance medium was replaced. It was kept for 48 hours, and then it was replaced to a differentiation medium I (base differentiation medium supplemented with 1 μg/mL insulin, 0.5 mM isobutylmethylxanthine (IBMX), and 0.1 μg/mL dexamethasone) in the absence or presence of the following PPARγ activators: rosiglitazone, RGZ (1 μM), FMOC-Leu (25 μM) or with Extracts A (60 μg/mL) or B (60 μg/mL).

[0118] The treatments were incubated for 48 hours, and then were changed to a differentiation medium II (base differentiation medium supplemented with 1 μg/mL insulin) for another 48 hours. Subsequently, they were kept in the base differentiation medium (DMEM supplemented with 10% FBS and 1× penicillin/streptomycin).

[0119] On the 7th day of in vitro culture, mRNA was extracted to assess gene expression of differentiated adipocytes, to which end the cultured and differentiated cells were lysed on the 7th day of culture, and mRNA was extracted in HiBind® RNA mini column, according to the manufacturer's instructions. The extraction quality was checked through integrity in gel and any genomic contaminant was removed with the DNAase enzyme. Complementary DNA (cDNA) was generated by reverse transcription of mRNA (RT-MLV and random primer). Then, the resulting cDNA was amplified by qPCR with SRYBRGreen. Three reference genes (GAPDH, ribosomal subunit 18S and b-actin) were simultaneously amplified to quantify expression of the gene of interest encoding FAPB-4 (Fatty-Acid-Binding-Protein 4), which is a protein that increases transcription during adipogenesis.

[0120] On the 10th day, staining with Oil Red O (staining for neutral lipids) was performed, to assess lipid (triglyceride) accumulation inside the differentiated cells, which is a feature of the differentiation of fibroblast into adipocytes. To this end, 3T3-L1 cells were washed two times with lx sterile phosphate buffered saline (PBS) at 37° C. Cells were fixed with 4% paraformaldehyde (PFA) 4% in 1× PBS for 1 hour at ambient temperature, and then were washed with PBS and deionized water. Isopropanol 60% was added for 6 minutes, and then were allowed to dry completely. Thereafter, the Oil Red O solution (6:4) was added, 1 ml per 35 mm well, for 2 hours at ambient temperature.

[0121] The next day images were captured by light microscopy, using 4× and 10× lenses of cell staining. Then, the isopropanol solubilized staining was quantified by spectrophotometry at 490 nm.

[0122] Furthermore, a viability assay with MTT was carried out, wherein cells were washed with PBS and incubated with 0.5 mg/mL of MTT in PBS at 37° C. for 2 hours. After removing MTT, DMSO (99.9%) was added to lyse cells and dissolve MTT/MTT formazan. In order to assess absorbance at 570 nm and 620 nm in a spectrophotometer, 100 μl of this solution were moved to a 96-well plate. The difference between absorbances was calculated, and the survival relative to the control (DMSO) was determined.

[0123] The differentiating phenotype is observed in the accumulation of staining for neutral lipids, Red-Oil-O. FIG. 7 shows that both Extract A and Extract B exhibited an accumulation close to that of the control (Extract A: 1.1 times; Extract B: 1.2 times; n=4). It should be noted that these values were close to the values obtained with the treatment with the SPPARM-type agonist FMOC-Leu 25 μM (Fmoc: 1.1; n=4) and that these values are compared to those obtained with the treatment with the full agonist (rosiglitazone), which shows a significant accumulation of neutral lipids compared to the control, more than double in comparison to the control (RGZ: 2.5 times; n=4; p<0.5, Kruskal-Wallis test). The lack of staining with the agonists present in Extracts A and B is not due to the effect on cell viability, since the MTT assay shows that all treatments exhibit a cell survival over 90% (RGZ: 96.5%; FMOC: 100%; Extract A: 96%; Extract B: 92.4%; n=3).

[0124] The observation made in view of the staining is correlated to the expression of the adipocyte differentiation marker, the fabp4 gene. As shown in FIG. 8, only those cells treated with the agonist rosiglitazone display a significant induction of this marker (n=3; p<0.5, Kruskal-Wallis test), while the treatment both with the SPPARM-type agonist FMOC-Leu and with Extracts A and B show a low expression of this marker, close to the control DMSO (RGZ: 14.7 times; FMOC: 1.7 times; Extract A: 1.1 times; Extract B: 2.2 times; n=3).

[0125] Determining the Antioxidant Capacity of the Extract

[0126] The antioxidant capacity of Extracts A and B (resuspended in DMSO) was established with the Oxiselect™ Total Antioxidant Capacity (TAC) kit (Cell Biolabs), in order to assess the antioxidant capacity of lipid samples. In this method, copper is reduced by the samples or standard, where the reduced copper interacts as a chromogen to form a compound that absorbs at 490 nm. As standard, a uric acid solution (serial dilutions) freshly prepared in DMSO 50% in water from a 60 mM stock solution in NaOH 1N, with distilled water, was used.

[0127] The Extracts were mixed with the reaction buffer in methanol, according to the manufacturer's instructions. The mixture was incubated for 5 minutes with orbital shaking, then the stopper solution was added, and the reaction was quantified by measuring absorbance in spectrophotometer at 490 nm.

[0128] All reactions were produced in duplicate. In order to carry out the comparison to other natural products, the same process described in the preceding sections was performed, to generate an oily extract from commercial maqui and spirulina powder samples. The results were expressed as uric acid equivalents/100 mg of sample.

[0129] The antioxidant capacity of the Extracts, which is directly proportional to the increase in absorbance, was expressed as copper reducing equivalents and it was found that 5 mg of Extract A has an antioxidant capacity similar to that obtained with 20 μg/mL (micrograms per milliliters) of pure α-Tocopherol. In turn, the total antioxidant capacity of Extract B is 1.8 times the total antioxidant capacity of oleoresin (Table 4).

TABLE-US-00007 TABLE 4 Total antioxidant capacity of Extracts A and B of Agarophyton chilensis mg Uric Acid Eq/100 mg Extract Sample Mean Extract A 430 Extract B 760 Spirulina oily extract 344 Maqui oily extract 305

[0130] Neuroprotection

[0131] Neuroprotection Assay

[0132] To assess the neuroprotective capacity of Extract B, a model of neuronal damage caused by chemical ischemia was used, preferably the model of damage caused by oligomycin A. Oligomycin A is a macrolide that blocks oxidative phosphorylation, inhibiting mitochondrial ATP synthase.

[0133] In order to assess the neuroprotective capacity, a primary culture of cortical neurons of stage E18 rat embryos was used. To this end, adult female rats were acquired, pregnant at stage 18, from the CINBIOT bioterium of the Faculty of Biological Science, Pontificia Universidad Católica de Chile. The rats are maintained at a temperature of 20° C. with water and feed ad libitum under the conditions approved by the ethical committee for the management of laboratory animals, in accordance with the guidelines established by the Chilean Commission for Scientific and Technological Research, CONICYT. Euthanasia was performed by CO.sub.2 inhalation, pursuant to the work protocol, to obtain the cortical neurons. Embryonic brain cortexes were placed in Hank's Balanced Salt Solution (HBSS®) in ice and were incubated with trypsin for 15 minutes at 37° C. for disaggregation, then were washed 3 times with HBSS and Minimum Essential Medium (MEM) was added as adhesion medium with 10% horse serum (HS). The tissue was mechanically disaggregated. Then, 110,000 cells/well were seeded in 48-well plates. The following day (DIV01), the medium was replaced with a maintenance medium (Neurobasal 2% B27®, 1× penicillin/streptomycin). Subsequently, the culture was treated with 1 μM Ara-C (Cytokine (β/δ Arabinofuranoside) to remove the contaminant glia, and each 2 days ⅓ of the medium was replaced with neurobasal medium 2% B27, the cortical neurons were maintained in culture for 8 days, in controlled atmosphere at 37° C., then the neurons were treated with Extract B (10, 25 and 50 μg/mL) dissolved in the culture medium, using DMSO as carrier, and as preservative of the extract, vitamin E was used (10 μM in all concentrations tested).

[0134] On the 9th of culture, the challenge with oligomycin A was carried out. Firstly, an incubation at 37° C. with oligomycin A 2 μM was conducted for 30 minutes, then it was changed to a neuronal maintenance medium plus extract. Thereafter, neuronal viability was determined, by removing the entire culture medium, it was washed once with PBS and incubated with 0.5 mg/mL of MTT in neurobasal medium without phenol red at 37° C. for 2 hours to establish the mitochondrial activity, wherein MTT (yellow) is transformed into MTT formazan (dark blue), when reduced by live cells. After removing MTT, pure DMSO was added, to lyse neurons and dissolve MTT/MTT formazan. Subsequently, 100 μl of this solution were moved to a 96-wel plate to measure absorbance at 570 nm and 620 nm in spectrophotometer. The difference between absorbances (570-620) was calculated, and the survival was estimated relative to the control treated with DMSO and without damage caused by oligomycin A. The presence of MTT formazan is quantified at 570 nm. The neuronal survival is expressed as percentage of the control (DMSO).

[0135] The results obtained from the above-mentioned assay indicated that the treatment with oligomycin A reduced neuronal viability at 22.5% (FIG. 9). It should be noted that the treatment of neurons with Extract B (10, 20 and 50 μg/mL) increases neuronal survival, obtaining a viability between 42-53% relative to the control treated with DMSO 0.1%. Therefore, the treatment with Extract B in all concentrations tested has a statistically significant neuroprotective effect relative to the control (p<0.001 ANOVA, Tukey Post-test). On the other hand, it was verified that the treatment with only vitamin E (10 μM) does not provide protection against oligomycin A (data not shown). Upon comparing the neuronal protection of Extract B to Extract A, it is observed that Extract A has a lower protective effect as the one obtained in the same concentration with the treatment with Extract B (one-way ANOVA, Tukey post hoc; p<0.05; FIG. 10).

[0136] This example shows that Extract B has a protective capacity against neuronal damage induced with oligomycin A, indeed the treatment of cortical neurons with Extract B significantly reduced the neuronal death induced by oligomycin A allowing substantial protection of neuronal viability. All together these results indicate that this extract has a surprising greater neuroprotective capacity than Extract A (FIGS. 9 and 10).

[0137] This viability assay shows a high reproducibility among assays, even though it is carried out in a primary culture, and due to its characteristics, it shows per se a natural variability.