Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/3463—Organic compounds; Microorganisms; Enzymes
  • A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/3463—Organic compounds; Microorganisms; Enzymes
  • A23L3/3481—Organic compounds containing oxygen
  • A23L3/3508—Organic compounds containing oxygen containing carboxyl groups
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/3463—Organic compounds; Microorganisms; Enzymes
  • A23L3/3544—Organic compounds containing hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/105—Plant extracts, their artificial duplicates or their derivatives

Definitions

• Antioxidant composition comprising quercetagetin and gallic acid
• the present invention belongs to the field of antioxidants.
• antioxidants for preserving foods, feeds, pharmaceutical or cosmetic compositions are particularly useful for preserving lipid-containing compositions.
• oxidation is highly relevant. It particularly affects fats, leading to lipid oxidation.
• lipid oxidation is a major cause of stored products spoilage, causing flavor deterioration, color alteration and bad odors.
• Polyunsaturated fatty acids (PUFA) with labile double bonds are oxidized and the decomposition of PUFA produces secondary oxidative compounds, such as hexanal, pentanal, heptanal, and octanal. These volatile aldehydes mostly contribute to the development of oxidative rancidity known as warmed-over flavor (WOF).
• Secondary oxidative compounds are not only responsible for quality deterioration of food, but ingested oxidative products can also be a risk for human and animal health.
• lipid- containing products such as food for human consumption, animal feed, pharmaceuticals or even cosmetic products.
• antioxidant compounds have been preferably employed because of their potential health benefits and safety. Some examples are tocopherols and phospholipids such as lecithin. Likewise, many natural plant extracts are under strong investigation because of their high antioxidant content, including flavonoids and other polyphenols.
• antioxidants used so far present various disadvantages, for instance, tocopherol based antioxidants are expensive and their efficacy vary as a preservative.
• the inventors have developed a new combination of compounds for improving the oxidative stability of various types of compositions.
• the combination of the invention comprises a mixture of quercetagetin and gallic acid at specific ratios.
• the invention provides a combination comprising quercetagetin and gallic acid at a weight ratio from 65: 1 to 1.2: 1 , wherein the gallic acid is in acid form, or in the form of a salt or an ester thereof.
• the invention provides a food, animal feed, pharmaceutical, veterinarian, or cosmetic composition comprising the combination as defined above.
• the invention provides the use of the combination as defined above as an antioxidant in a food, animal feed, pharmaceutical, veterinarian, or cosmetic composition.
• the invention provides a method for preparing the combination as defined above, comprising the steps of providing quercetagetin; adding gallic acid;
• Fig. 1 shows the antioxidant efficacy of a Tagetes erecta extract (quercetagetin), gallic acid and combinations thereof at the concentrations indicated.
• the y-axis represents the protection factor (PF) (calculated as explained in the examples below), and the x-axis represents the concentration of active substances (antioxidants) tested.
• the values obtained with gallic acid are represented by empty squares, TE extract by empty circles, a mixture of TE extract and gallic acid at a weight ratio 15:1 by black triangles, at a weight ratio 5:1 by black circles, at a weight ratio 1.67:1 by empty triangles, and at a weight ratio 1 :1.8 by black triangles.
• N 4-8 replicates per concentration of antioxidants tested.
• Two- way Anova (Test Tukey), * P ⁇ 0.05 vs. TE, # P ⁇ 0.01 vs. gallic acid.
• Fig. 2 shows the antioxidant efficacy of quercetagetin, Tagetes erecta extract, gallic acid and combinations thereof at the concentrations indicated.
• the y-axis represents the protection factor (PF) (calculated as explained in the examples below), and the x-axis represents the concentration of active substances (antioxidants) tested.
• PF protection factor
• the values obtained with gallic acid are represented by empty circles, quercetagetin by empty squares, TE extract by empty triangles, a mixture of TE extract and gallic acid at a weight ratio 15:1 by black triangles pointing down, a mixture of TE extract and gallic acid at a weight ratio 5:1 by black triangles pointing up, and a mixture of quercetagetin and gallic acid at a weight ratio 9:1 by black circles.
• N 4-14 replicates per concentration of antioxidants tested. Two-way Anova (Test Tukey), different letters in each assessed concentration represents significant differences (P ⁇ 0.05).
• the present invention provides a combination of quercetagetin, and gallic acid at a weight ratio from 65: 1 to 1.2: 1 , wherein when the gallic acid is in acid form or in the form of a salt or an ester thereof. Where the gallic acid is in the form of a salt or ester thereof, the weight ratio relates to the corresponding amount of gallic acid.
• weight ratio refers to the relation of weights of the molecules or compounds indicated. For instance, a weight ratio (weight/weight) of 65:1 between quercetagetin and gallic acid refers to 65 units of quercetagetin and 1 unit of gallic acid.
• % by weight or“% w/w” of a component refers to the amount of the single component relative to the total weight of the composition or, if specifically mentioned, of other component.
• the weight of an extract refers to the dry extract weight or mass.
• Quercetagetin is also known as 2-(3,4-dihydroxyphenyl)-3,5,6,7-tetrahydroxychromen-4- one, and has a CAS number 90-18-6. Quercetagetin has the formula (I):
• Gallic acid is also known as 3,4,5-trihydroxybenzoic acid, and has a CAS number 149-91- 7.
• Gallic acid has the formula (II):
• gallates Several salts and esters of gallic acid, which are called gallates, are known to also have antioxidant activity and, thus, can be used in the combinations of the invention.
• alkyl esters of gallic acid such as methyl gallate, propyl gallate, octyl gallate, and dodecyl (lauryl) gallate, or gallic acid salts such as sodium gallate, can be combined with quercetagetin to obtain a synergic antioxidant combination.
• Quercetagetin and gallic acid are commercially available and can be obtained from several chemical suppliers.
• the gallic acid ester is an alkyl ester, such as a C1-C12 alkyl ester.
• the gallic acid ester is selected from the group consisting of methyl gallate, ethyl gallate, propyl gallate, octyl gallate, dodecyl gallate, and mixtures thereof.
• the preparation of the gallic acid salts can be carried out by methods known in the art.
• gallic acid esters can be prepared by methods known in the art. In general, gallic acid esters are prepared, for example, by reacting the free acid form of the gallic acid with a stoichiometric amount of the appropriate alcohol at high temperatures. In another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the gallic acid is in acid form.
• the weight ratio between quercetagetin and gallic acid or the salt or ester thereof is from 65:1 to 1.2:1, from 64:1 to 1.2:1, from 63:1 to 1.2:1, from 62:1 to 1.2:1, from 61.2:1 to 1.2:1, from 60:1 to 1.2:1, from 59:1 to 1.2:1, from 58:1 to 1.2:1, from 57:1 to 1.2:1, from 56:1 to 1.2:1, from 55:1 to 1.2:1, from 54:1 to 1.2:1, from 53:1 to 1.2:1, from 52:1 to 1.2:1, from 51.2:1 to 1.2:1, from 50:1 to 1.2:1, from 49:1 to 1.2:1, from 48:1 to 1.2:1, from 47:1 to 1.2:1, from 46:1 to 1.2:1, from 45:1 to 1.2:1, from 44:1 to 1.2:1, from 43:
• the weight ratio between quercetagetin and gallic acid or the salt or ester thereof is from 10:1 to 1.2:1, from 10:1 to 1.3:1, from 10:1 to 1.4:1, from 10:1 to 1.5:1, from 10:1 to 1.6:1, from 10:1 to 1.7:1, from 10:1 to 1.8:1, from 10:1 to 1.9:1, from 10:1 to 2:1, from 10:1 to 2.1:1, from 10:1 to 2.2:1, from 10:1 to 2.3:1, from 10:1 to 2.4:1, from 10:1 to 2.5:1, from 10:1 to 2.6:1, from 10:1 to 2.7:1, from 10:1 to 2.8:1, or from 10:1 to 2.9:1.
• the combination consists of
• quercetagetin and gallic acid at a weight ratio from 65:1 to 1.2:1.
• the combination comprises
• the combination comprises quercetagetin and gallic acid at a weight ratio from 65:1 to 1.2:1, more particularly from 20:1 to 1.5:1, more particularly, from 10:1 to 1.5:1, more particularly, from 10:1 to 2:1, more particularly, from 4:1 to 2:1, or even more particularly, from 8.6:1 to 2.85:1. More particularly, the combination comprises quercetagetin and gallic acid at a weight ratio from 49:1 to 1.5:1. More particularly, the combination comprises quercetagetin and gallic acid at a weight ratio from 9: 1 to 1.5: 1.
• the quercetagetin is provided in the form of a plant extract.
• plant extract or“extract” refers to concentrated preparations of various parts of plants obtained by isolating the active constituents, such as antioxidants, from the plant by suitable means.
• suitable means for their isolation can be used, for example, aqueous solutions, alcoholic solutions, organic solvents, microwave or supercritical fluids extraction.
• Said extracts contain one or more active ingredients and may be incorporated in compositions in a variety of forms, including a pure or semi-pure component, a solid or liquid extract, or a solid plant matter.
• the plant extract comprises quercetagetin in an amount equal to or higher than 30 %w/w of dry extract mass, more in particular, equal to or higher than 40 %w/w of dry extract mass, even more in particular, equal to or higher than 50 %w/w of dry extract mass, and even more in particular, from 35 to 60 %w/w of dry extract mass.
• the plant extract is a Tagetes erecta extract.
• the Tagetes erecta extract is a flower bagasse extract.
• the Tagetes erecta flower bagasse extract comprises quercetagetin in an amount from 35 to 60 %w/w of dry extract mass.
• Tagetes erecta extract it is understood a concentrated preparation of compounds obtained from the Tagetes erecta plant.
• the flower bagasse is the remaining residue after extracting the oleoresin from the flowers.
• the main antioxidant compound of the Tagetes erecta flower bagasse extract is quercetagetin, which accounts for more than 94% of its antioxidant (see the examples below).
• Tagetes erecta also known as Mexican marigold, Aztec marigold, African marigold, or simply, marigold
• Tagetes erecta is an herbaceous plant belonging to the family Asteraceae. This plant has been usually used in traditional medicine and is a common ornamental plant around the world.
• Marigold oleoresins are used by the industry as a source of natural carotenoids to produce, for instance, pigments to be added in feed for broiler chicken or laying hens.
• Carotenoid-rich oleoresin is obtained from dried marigold flowers with hexane, generating high amounts of defatted flower residues or bagasse. These residues are currently used as a fiber source for animals, as fertilizer or simply discarded.
• an additional advantage of the combinations of the invention is that quercetagetin can be obtained from abundant and inexpensive compounds that are commonly discarded in industry, such as the Tagetes erecta bagasse extract, which is highly enriched in quercetagetin.
• the plant extract is selected from the group consisting of an aqueous extract, an alcoholic extract, and an organic extract.
• the plant extract is an alcoholic extract.
• aqueous extract refers to a plant extract where the extraction has been performed using water as the only solvent.
• organic extract refers to a plant extract where the extraction has been performed using an organic solvent that is not an alcohol.
• alcoholic extract refers to a plant extract where the extraction has been performed using a mixture of water and an alcohol, such as methanol or ethanol.
• the extract is a dry extract.
• the weight ratio of the extract and gallic acid should be adjusted depending on the amount of quercetagetin contained in the extract to obtain the ratios of the present invention. For example, as shown in the examples below, where a Tagetes erecta extract containing quercetagetin at 57 % w/w is used, a combination of the extract and gallic acid at a weight ratio of 5:1 has to be made in order to obtain a 2.85:1 weight ratio of quercetagetin and gallic acid.
• the combination comprises a Tagetes erecta extract and gallic acid in a weight ratio from 65:1 to 1.8:1 , more in particular from 18:1 to 3:1 , even more in particular from 16:1 to 4:1 , and even more in particular, from 15:1 to 5:1.
• a Tagetes erecta extract and gallic acid in a weight ratio from 65:1 to 1.8:1 , more in particular from 18:1 to 3:1 , even more in particular from 16:1 to 4:1 , and even more in particular, from 15:1 to 5:1.
• the combination comprises a Tagetes erecta extract and added gallic acid in a weight ratio from 90:1 to 1.8:1 , more in particular from 40: 1 to 3: 1 , even more in particular from 20: 1 to 4: 1 , and even more in particular, from 15:1 to 5:1.
• the combination comprises a Tagetes erecta alcoholic extract and added gallic acid in a weight ratio from 90:1 to 1.8:1 , more in particular from 40: 1 to 3: 1 , even more in particular from 20: 1 to 4: 1 , and even more in particular, from 15:1 to 5:1.
• the combination comprises a Tagetes erecta flower bagasse alcoholic extract and added gallic acid in a weight ratio from 90:1 to 1.8:1 , more in particular from 40:1 to 3:1 , even more in particular from 20:1 to 4:1 , and even more in particular, from 15:1 to 5:1.
• the invention provides in a second embodiment a food, animal feed, pharmaceutical, veterinarian, or cosmetic composition comprising the combination as defined above.
• the compositions of the invention can additionally incorporate other active ingredients which reinforce the beneficial effects of the combination of the invention.
• A“food composition”, as used herein, refers to any solid, semi-solid, or liquid composition, also a drinkable liquid, which is suitable for human consumption.
• the term“animal feed composition” refers to any solid, semi-solid, or liquid composition, also a drinkable liquid, which is suitable for non-human animal consumption; therefore, it also encompasses pet food. Both food and feed compositions are meant to also encompass the raw materials and feedstock used for their elaboration, as well as dietary supplements.
• the term“dietary supplement” refers to a preparation intended to supplement the diet and provide nutrients, such as vitamins, minerals, fiber, fatty acids, or amino acids, that may be missing or may not be consumed in sufficient quantity in a subject’s diet.
• Cosmetic composition refers to compositions for improving the appearance or hygiene of subjects, comprising cosmetically acceptable excipients or carriers.
• cosmetically acceptable refers to that excipients or carriers suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic response, among others.
• pharmaceutical composition or“veterinarian composition” refer to compositions used for improving or maintaining the health of subjects or animals, respectively, and comprise pharmaceutically acceptable excipients or carriers.
• compositions comprising the combination of the invention can be presented in any form, for example, solid or liquid.
• the combination of the invention is particularly useful to prevent the oxidation of composition comprising lipids.
• the food, animal feed optionally in combination with any of the embodiments provided above or below, the food, animal feed,
• composition comprises lipids. More particularly, the composition comprises an amount of lipids equal to or higher than 1 % w/w, even more particular equal to or higher than 10 % w/w.
• lipids refers to organic molecules that are insoluble in water and soluble in nonpolar solvents, for example, fatty acids, steroids, or triacylglycerides (fats).
• the combination of the invention is especially useful for preventing the oxidation of fats.
• the food, animal feed, pharmaceutical, veterinarian, or cosmetic composition comprises vitamins or carotenoids.
• the food, animal feed, pharmaceutical, veterinarian, or cosmetic composition comprises the combination of the invention in an amount from 0.001 % w/w to 1 % w/w, more in particular from 0.005% w/w to 0.5% w/w, and even more in particular, from 0.01 % w/w to 0.1 % w/w.
• the food, animal feed, pharmaceutical, veterinarian, or cosmetic composition comprises quercetagetin in an amount from 20 ppm to 5000 ppm. More particularly, from 25 ppm to 500 ppm, and even more particularly, from 50 ppm to 200 ppm.
• the food, animal feed, pharmaceutical, veterinarian, or cosmetic composition comprises gallic acid in an amount from 20 ppm to 5000 ppm. More particularly, from 25 ppm to 500 ppm, and even more particularly, from 50 ppm to 200 ppm.
• the invention provides in a third aspect the use of the combination as defined above as an antioxidant in a food, animal feed, pharmaceutical, veterinarian, or cosmetic composition.
• the combination of the invention is useful for increasing the oxidative stability of any composition that comprises oxidation-sensitive molecules, for instance lipids.
• the combination as defined above is used as an antioxidant in a food, animal feed, pharmaceutical, veterinarian, or cosmetic composition, wherein the compositions comprises lipids, more particularly, it comprises an amount of lipids equal to or higher than 1 % w/w, even more particular equal to or higher than 10 % w/w.
• the invention provides in a fourth aspect a method for preparing the combination as defined above, comprising the steps of:
• the suitable liquid carrier is selected from the group consisting of an edible carrier and a pharmaceutically acceptable carrier.
• the suitable liquid carrier is selected from group consisting of alcohol (such as ethanol or isopropanol), organic acid, ester (such as citrates,
• A“pharmaceutical acceptable carrier” as used herein, refers to a carrier suitable for use in contact with the tissue or organ of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
• An“edible carrier” refers to a carrier that can be ingested by animals, including humans, without significant deleterious health consequences.
• the bagasse of Tagetes erecta was obtained by subjecting the flowers to oleoresin extraction (H. B. Sowbhagya et at.,“Natural Colorant from Marigold-Chemistry and Technology”, Food Reviews International, 2004, vol. 20, pp. 33-50).
• the bagasse was subjected to alcoholic solvent extraction according to standard procedures.
• the extraction may be carried out using as solvent methanol and/or ethanol in water at volume ratios ranging from 1 :3 to 3:1 , a range of temperatures from 30 to 50 °C during periods of time from 0.5 to 2 hours, and with a solid-to-liquid ratio from 1 :30 to 1 :50 (w/v), depending on the characteristics of the raw materials.
• the solid-liquid intermediate product was later filtered and the extract was spray dried to obtain a concentrated yellow to brown powder.
• the set of operating conditions for concentration-filtration and spray drying of the extract are dependent on the specific characteristics of each batch of extract, which is influenced by quality of the original crop where obtained. Ingredients added to the preparation to be spray dried also determine operating conditions as well.
• the solid residue and viscosity is standardized among extract batches. A two-fluid nozzle with a cap-orifice diameter of 0.5 mm was used.
• the aqueous dispersion is mixed for approximately 30 min before spray-drying within this reference range of operating conditions: drying air inlet temperature, 100-170°C;
• atomization air volumetric flowrate 300-800 l/h; feed volumetric flowrate: 3-15 ml/min; feed concentration, 3-10% (w/w); drying air volumetric flowrate: 25-50 m 3 /h.
• quercetagetin represents practically all the antioxidant substances of the extract (94.24 % of the assessed active substances in these samples). Quercetin represents a 4.39% of the assessed phenolics and flavonoids assessed and gallates including methyl-gallate and gallic acid represent a 1.37% (1.17% and 0.20%,
• Rancimat assays were performed with 892 Professional Rancimat according to manufacturer’s instructions (Metrohm, Switzerland), in order to assess the antioxidant efficacy of quercetagetin (provided in the form of a Tagetes erecta bagasse extract above described) in combination with various antioxidants.
• the temperature of the heating block was fixed at 120 °C.
• 60 ml_ of deionized water was added to each cell and its conductivity was determined until it was stable (1.5 - 2 h).
• the antioxidant protection factors were determined by the ratio of the induction period with antioxidant to the induction period of the respective negative control (without antioxidant). The higher the antioxidant protection factor, the better the efficacy of the antioxidant assessed. SAS Software version 9.1.3 (SAS institute) was used to perform all statistical analysis. Results with TE extract combined with gallic acid
• n 2 (replicates per each concentration)

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