WO2000039248A1 - Method for protecting a substance liable to oxidative deterioration - Google Patents

Method for protecting a substance liable to oxidative deterioration Download PDF

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Publication number
WO2000039248A1
WO2000039248A1 PCT/IL1999/000693 IL9900693W WO0039248A1 WO 2000039248 A1 WO2000039248 A1 WO 2000039248A1 IL 9900693 W IL9900693 W IL 9900693W WO 0039248 A1 WO0039248 A1 WO 0039248A1
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Prior art keywords
substance
antioxidant
compositions
food
acid
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PCT/IL1999/000693
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French (fr)
Inventor
Rena Reznik
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Rad Natural Technologies Ltd.
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Priority claimed from IL12772498A external-priority patent/IL127724A0/en
Priority claimed from IL12859399A external-priority patent/IL128593A0/en
Application filed by Rad Natural Technologies Ltd. filed Critical Rad Natural Technologies Ltd.
Priority to CA2432441A priority Critical patent/CA2432441C/en
Priority to AU16787/00A priority patent/AU1678700A/en
Priority to EP99959658A priority patent/EP1246884A1/en
Publication of WO2000039248A1 publication Critical patent/WO2000039248A1/en

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    • 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
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
    • 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
    • A23L3/3481—Organic compounds containing oxygen
    • A23L3/3508—Organic compounds containing oxygen containing carboxyl groups
    • A23L3/3517—Carboxylic acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/37—Esters of carboxylic acids
    • A61K8/375—Esters of carboxylic acids the alcohol moiety containing more than one hydroxy group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K15/00—Anti-oxidant compositions; Compositions inhibiting chemical change
    • C09K15/04—Anti-oxidant compositions; Compositions inhibiting chemical change containing organic compounds
    • C09K15/06—Anti-oxidant compositions; Compositions inhibiting chemical change containing organic compounds containing oxygen
    • C09K15/08—Anti-oxidant compositions; Compositions inhibiting chemical change containing organic compounds containing oxygen containing a phenol or quinone moiety
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B5/00—Preserving by using additives, e.g. anti-oxidants
    • C11B5/0021—Preserving by using additives, e.g. anti-oxidants containing oxygen
    • C11B5/0035—Phenols; Their halogenated and aminated derivates, their salts, their esters with carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation

Definitions

  • the present invention relates to protecting a substance liable to oxidative deterioration and to materials which are so protected
  • antioxidant material is isolated from rosemary by extraction with a non-polar solvent, removal of the solvent and steam distillation, giving an aqueous dispersion, which was filtered, antioxidant being obtained from the solid material by further processing including extraction with aqueous alkali at pH at least 10 5
  • An isolated antioxidant is 7 ⁇ , 11 , 12-t ⁇ hydroxy-6, 10-(epoxymethano)ab ⁇ eta- 8,11 ,13-t ⁇ en-20-one
  • US 4,877,635 (Todd) describes a process for producing an oil-soluble extract of a Labiatae herb claimed to contain essentially all of the antioxidant substances in the herb, in the course of which acetone- or methyl ethyl ketone-insolubles, defined as pro-oxidant substances, are, so far as practicable, removed by precipitation US 5,023,017 (Todd) relates to a stable Labiatae antioxidant solution, having a pH 84-11 8, prepared from an initial solvent extract of the herb, and claimed to contain (besides ⁇ 75% water, and an edible alcohol and/or polyol) essentially all of the antioxidant substances in the herb, which is preferably rosemary, sage or thyme
  • rosmarinic acid is valuable in view of its antnnflammatory properties, see e g US 4,329,361 (Zenk et al )
  • rosmarinic acid is also known for its use in skin-treatment or cosmetic compositions, see e g JP 63162611 , JP 9067251 and US 5,393,526 (Castro)
  • rosmarinic acid is claimed to be useful in a composition for protection against erythema and inflammatory reactions caused by exposure to UV rays, and to have antioxidant, antibacterial and antifungal activity, see FR 2652001 (cf JP 06145034)
  • the action of rosmarinic acid as a 5-l ⁇ poxygenase inhibitor is also featured in JP 1121217, where, extracted from pe ⁇ lla species, it is used as a constituent of an antiallergic food
  • a still further object of the invention is to provide a process protection for a substance liable to oxidative deterioration by inclusion of water-soluble antioxidant material prepared by permitting maximum recovery of the industrially useful components from plants of the Labiatae family, such components including essential oils, completely water-soluble antioxidant material and known components which are both water-insoluble and organic solvent soluble, such as vitamin E and carnosic acid
  • the present invention accordingly provides in one aspect a substance liable to oxidative deterioration, which is characterized by the fact that it incorporates as antioxidant for that substance, at least one compound selected from sub-groups ( ⁇ ), ( ⁇ ) and ( ⁇ ), namely ( ⁇ ) rosmarinic acid,
  • the invention provides use of at least one compound selected from sub-groups ( ⁇ ), ( ⁇ ) and ( ⁇ ), namely ( ⁇ ) rosmarinic acid, ( ⁇ ) salts of the carboxylic acid function in rosmarinic acid, ( ⁇ ) esters and amides of the carboxylic acid function in rosmarinic acid, in admixture with a substance liable to oxidative deterioration, in at least a minimum amount necessary to confer antioxidative protection on said substance, provided that when said substance is a dermato-cosmetic composition, said at least one compound is used in an amount less than 0 1 % of the admixture, and that said at least one compound excludes mixtures in a form as extracted from natural sources and comprising any compound in sub-groups ( ⁇ ), ( ⁇ and ( ⁇ ), together with any other ant ⁇ ox ⁇ dant(s)
  • the invention provides a method for treating a substance liable to oxidative deterioration, in order to confer antioxidative protection on said substance, which comprises admixing said substance with at least one compound selected from sub-groups ( ⁇ ), ( ⁇ ) and ( ⁇ ), namely ( ⁇ ) rosmarinic acid, ( ⁇ ) salts of the carboxylic acid function in rosmarinic acid, ( ⁇ ) esters and amides of the carboxylic acid function in rosmarinic acid, in at least a minimum amount necessary to confer said protection on said substance, provided that when said substance is a dermato-cosmetic composition, said at least one compound is used in an amount less than 0 1 % of the admixture, and that said at least one compound excludes mixtures in a form as extracted from natural sources and comprising any compound in sub-groups ( ⁇ ), ( ⁇ and ( ⁇ ), together with any other ant ⁇ ox ⁇ dant(s)
  • the proviso as to the dermato-cosmetic composition excludes the disclosure of FR 2,652,
  • FIG. 1 illustrates chromatographic fractionation of antioxidant material from rosemary, having utility in accordance with an embodiment of the invention
  • FIG. 2 illustrates chromatographic fractionation of antioxidant material from oregano, having utility in accordance with an embodiment of the invention
  • antioxidant in a form selected from completely water-soluble materials containing a major proportion of non-antioxidant diluent, and which are solid at ambient temperatures, and aqueous solutions thereof,
  • said antioxidant comprises at least one of
  • a sodium rosma ⁇ nate preferably isolated from tissue of plants of the Labiatae family, most preferably by use of an aqueous extractant
  • the substance incorporating antioxidant according to the invention is selected from sub-groups (I), (II) and (III), namely
  • compositions (I) pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption,
  • compositions selected from pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption,
  • compositions selected from pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption, which composition comprises an ingredient liable to oxidative deterioration
  • Such food composition may be e g , selected from sugar-based confectionery, manufactured cereals, fruit or vegetable products, beverages or beverage concentrates, ground meat products and vegetable analogues thereof
  • Such food composition may comprise at least one additional ingredient selected from the water-soluble vitamins thiamine, ⁇ boflavin, niacin, py ⁇ doxine, pantothenic acid, biotin, fo c acid, cobalamin and ascorbic acid, the oil-soluble vitamins retinol, calciferol, tocopherol and menadione, in combined form the elements sodium, potassium, calcium, phosphorus, magnesium, chlorine, sulfur, iron, copper, iodine, manganese, cobalt, zinc molybdenum, fluorine, selenium and chromium, unsaturated fatty acids which are known to metabolized in the body to prostaglandins, and physiologically compatible derivatives of said fatty acids, food colorants and pigments, acceptable dispersing and suspending agents, and
  • an amount of ant ⁇ ox ⁇ dant(s) effective to prevent oxidation of the ingredient liable to oxidative deterioration should be used Generally, from about 0 0001 to about 1 %, preferably from about 0 0005 to about 0 1% by weight of the food composition may be used, depending on the nature of the composition and the type of oxidative activity which is to be inhibited
  • Colorants or pigments, used in food compositions, which are liable to oxidative deterioration are for example, polyene pigments and colorants such as ⁇ -carotene
  • compositions which contain fats or oils comprising fatty acids or their esters, either saturated or unsaturated may be preserved using ant ⁇ ox ⁇ dant(s) according to the invention
  • fatty acids are well known and are listed in Noller, Textbook of Organic Chemistry, 2nd Ed pp 108-113 and 138-146 (1958), which is incorporated by reference herein
  • Typical but non-limiting examples of food compositions, including fats and oils adapted for use therein, are soybean oil, corn oil, cottonseed oil, olive oil, butter, margarine, salad creams and mayonnaises, dairy products, ice cream, frozen vegetables, soups, fried foods and
  • a particular aspect of the present invention resides in the use of rosmarinic acid and its derivatives as described herein, for the purpose of antioxidative protection of vitamins and their analogs and functional derivatives of the vitamins and the analogs
  • the water-soluble vitamins thiamine, ⁇ boflavin, niacin, py ⁇ doxine, pantothenic acid, biotin, fo c acid, cobalamin and ascorbic acid the oil-soluble vitamins retinol, calciferol, tocopherol and menadione, and their analogs and functional derivatives thereof, especially such analogs and derivatives having analogous biological activity
  • vitamins which are themselves known to be antioxidants in particular ascorbic acid (including its salts and esters) and ⁇ -tocopherol (including its esters) can themselves be protected from deterioration by use of rosmarinic acid and its derivatives
  • the substance liable to oxidative deterioration, the oxidation which is inhibited in accordance with the present invention includes the essential oils, such as those derived, for example, from citrus fruit and flowers
  • the antioxidants utilized in accordance with the invention are selected from rosmarinic acid, salts of the carboxylic acid function in rosmarinic acid, and esters and amides of the carboxylic acid function in rosmarinic acid Rosmarinic acid may be isolated from natural sources e g as described in certain of the above-mentioned published documents, or it (and indeed its salts, esters and amides) may be chemically synthesized It may also be made by enzymatic or cell culture procedures, see e g US 5,011 ,775 (Rao) and published documents mentioned therein, the contents of this patent and of such documents, being incorporated herein by reference
  • Example 1 shows inter alia that the prior art practice of obtaining antioxidant material from Labiatae family plants, from organic solvent soluble extracts, while rejecting aq fractions, and/or rejecting acetone-insolubles, results in substantial loss of potentially valuable antioxidant Moreover, Example 1 (c) shows surprisingly, that contrary to Viani and Todd, useful completely water-soluble antioxidant material can be obtained by alkaline extraction
  • the method is based on the rate of oxidation of linoleic acid (LA) to its conjugated diene hydroperoxide (Pryor et al , in J Org Chem , 1993, 58 (13) 3521 -3532), and 2,2'-azob ⁇ s (2-am ⁇ d ⁇ nopropane) 2HCI (ABAP) is used to provide a constant rate of radical production
  • LA linoleic acid
  • ABAP 2,2'-azob ⁇ s (2-am ⁇ d ⁇ nopropane) 2HCI
  • a 1 % LA aq emulsion was prepared with 1 % Tween 20 and 0 05M sodium phosphate buffer (pH 7 4)
  • a control sample contained 0 025 ml of the LA emulsion + 2 87 ml of the buffer + 0 1 ml 0 05M ABAP
  • a test sample contained 0 05 ml of the LA emulsion + 2 7-2 8
  • a column of 8cm x 1cm is packed with MacroPrep-methyl (hydrophobic interaction 40 ⁇ m, Bio-Rad) was washed exhaustively with water and ethanol, and then used for purification of the water extract lyophilizate (see Example 1(f), above) 200 mg of the lyophilizate was dissolved in 1 ml water, and the solution was loaded on the MacroPrep column that had been pre-equilibrated with water Elution was carried out using a 0 1 % acetic acid/ 70 30 ethanol in water gradient starting with a 100 0 ratio at 0 minutes and ending with a 0 100 ratio at 220 minutes, and at a flow rate of 2 0 ml/minute Peaks are monitored by UV absorption at 280nm The column effluent was collected after 85 minutes (193 ml) and the active material was found to be eluted between 85 to 100 minutes (36 ml, 30-40%o ethanol (balance water))
  • a concentrate was first prepared containing 40% lecithin, 40% oil (e g soybean oil), 20% propylene glycol and 4% based on the foregoing mixture of 65% ( ⁇ 2 6% pure) sodium rosmannate Rosmarinic acid can alternatively be substituted for the sodium salt
  • the thus-prepared concentrate can then be used in a bulk oil which it is desired to protect against oxidative deterioration
  • oil is soybean oil
  • test results at 60°C were as follows Antioxidant Concentration Days until oxidation
  • Vitamin E 0 06% 4

Abstract

Antioxidative protection is conferred on a substance liable to oxidative deterioration, by admixing it with at least one compound selected from sub-groups (α), (β) and (η), namely: (α) rosmarinic acid; (β) salts of the carboxylic acid function in rosmarinic acid; (η) esters and amides of the carboxylic acid function in rosmarinic acid; subject to certain provisos.

Description

METHOD FOR PROTECTING A SUBSTANCE LIABLE TO OXIDATIVE DETERIORATION
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to protecting a substance liable to oxidative deterioration and to materials which are so protected
Artificial antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary-butylhydroxyquinone (TBHQ) and propyl gallate (PG) are known However, in recent years, there has developed a movement towards avoiding using artificial additives, particularly where foodstuffs and medicaments are concerned Much effort has therefore been invested in obtaining antioxidants from plants and especially from the Labiatae plant family
Thus, in US 3,950,266 (Chang et al ), there is described a process for extracting antioxidant material from rosemary or sage, using organic solvents (examples of which are hexane, benzene, ethyl ether, chloroform, ethylene dichloπde, dioxane and methanol) boiling at up to 100°C, and subsequently carrying out various steps of purification It appears that in all practical examples, before further purification, the crude antioxidant was washed with water, and then bleached with active carbon It will be appreciated that antioxidant material produced in this manner will be solvent-soluble and water-insoluble
By contrast, according to US 4,012,531 (Viani), extraction of plant material is carried out in absence of organic solvents, under mildly alkaline conditions, using a basic aqueous buffer at pH from 7 to about 11 5, preferably in an inert atmosphere Examples describe the extraction of rosemary, sage and parsley at pH 8 6-9 3, at 55-90°C
In US 4,450,097 (Nakatani et al ), antioxidant material is isolated from rosemary by extraction with a non-polar solvent, removal of the solvent and steam distillation, giving an aqueous dispersion, which was filtered, antioxidant being obtained from the solid material by further processing including extraction with aqueous alkali at pH at least 10 5 An isolated antioxidant is 7β, 11 , 12-tπhydroxy-6, 10-(epoxymethano)abιeta- 8,11 ,13-tπen-20-one
US 4,638,095 (Chang et al ) describes the isolation from rosemary of the antioxidant "rosmaπdiphenol", which is structurally a dibenzocycloheptene derivative This compound was obtained by chromatographic separation (and appeared in the 75 25 diethyl ether/hexane fraction) of a product made by a procedure including solvent extraction and steam distillation
US 4,877,635 (Todd) describes a process for producing an oil-soluble extract of a Labiatae herb claimed to contain essentially all of the antioxidant substances in the herb, in the course of which acetone- or methyl ethyl ketone-insolubles, defined as pro-oxidant substances, are, so far as practicable, removed by precipitation US 5,023,017 (Todd) relates to a stable Labiatae antioxidant solution, having a pH 84-11 8, prepared from an initial solvent extract of the herb, and claimed to contain (besides <75% water, and an edible alcohol and/or polyol) essentially all of the antioxidant substances in the herb, which is preferably rosemary, sage or thyme
US 5,433,949 (Kahleyss et al ), after referring to US 5,017,397 which describes preparation of antioxidants by extracting Labiatae species with C02 at 350-1000 bar and subsequent fractionation, proposes to prepare antioxidants from a similar source by a multistep process utilizing extraction with C02 at 80-300 bar, treating the extraction residue with a Ci alcohol or Cs 7 hydrocarbon followed by active carbon, and washing out the resultant solvent extract with water to remove color, aromatics and remaining solvent
It is evident that the various methods known to the prior art for obtaining antioxidants from plants of the Labiatae family have for the most part been based on the assumption that the desired materials are to be found in water-insoluble extracts obtained by extracting with non-aqueous solvents, particularly water-immiscible solvents, and in many of the relevant patents, aqueous fractions are rejected Moreover, in Todd '635, acetone-msolubles are removed, being considered to contain undesired pro-oxidants, and in Todd '017 it is again emphasized that the acetone insoluble materials do not contain antioxidants Although, as has been noted above, the Viani patent describes direct extraction of the plants with buffered aqueous alkali, the product on evaporation gives a mixture of the desired product with inorganic materials, separation from which entails at least a further step involving crystallization, ion-exchange treatment or acidification Moreover, the present inventor has found that the product precipitated on acidification on Viani's alkaline solution is essentially water-insoluble
In US 4,354,035 (Christ et al ), there is described a process for isolating rosmarinic acid (of formula depicted below)
Figure imgf000005_0001
from essentially water-immiscible organic solvent extracts of an aqueous extract made by extracting Melissa officinalis with water at 80-100°C, and acidifying to pH 2-2 5 This patent does not suggest that an industrially useful water-soluble product could be made directly from aqueous extracts of Melissa officinalis, without requiring use of water-immiscible organic solvent extracts
According to the Christ et al patent, rosmarinic acid is valuable in view of its antnnflammatory properties, see e g US 4,329,361 (Zenk et al ) However, rosmarinic acid is also known for its use in skin-treatment or cosmetic compositions, see e g JP 63162611 , JP 9067251 and US 5,393,526 (Castro) Moreover, rosmarinic acid is claimed to be useful in a composition for protection against erythema and inflammatory reactions caused by exposure to UV rays, and to have antioxidant, antibacterial and antifungal activity, see FR 2652001 (cf JP 06145034) The action of rosmarinic acid as a 5-lιpoxygenase inhibitor is also featured in JP 1121217, where, extracted from peπlla species, it is used as a constituent of an antiallergic food
Mention should also be made of the Albeck et al and Grossman et al patents, in particular U S Patents Nos 4,857,325, 4,923,697, 4,986,985, 4,997,666 and 5,124,167, which relate to the preparation of antioxidants by aqueous extraction of plant material specifying certain plant families and species, and utilization of such antioxidants These patents neither disclose nor suggest that antioxidants might be obtained by similarly extracting plants of the Labiatae family, nor do they identify the chemical structure of the antioxidants
The entire contents of the above-mentioned U S and other Patents, or published patent applications, are incorporated herein by reference Contrary to what is to be expected from the prior art, it has surprisingly been found by the present inventor that useful water-soluble and acetone-insoluble antioxidant material may be obtained from plants of the Labiatae family by extraction, which may be carried out at ambient temperatures, with aqueous extractant, by a process in which use of buffered alkali and a step of separation from admixed salts, need not arise The water-soluble antioxidant material extracted and the extraction process are the subject of our copending patent application 97074
Although the Viani patent, for example, mentions rosmarinic acid, together with camosic acid and pro-oxidant flavones, as extracted from rosemary at pH 8 5, there was no inference drawn either in this patent or in FR 2652001 or JP 1121217, or in other prior art, that rosmarinic acid (or its derivatives) could per se be viable for antioxidant applications, i e to inhibit oxidation of other substances
Considering also, in general, the vast effort that has in the past been devoted to extraction of plants of the Labiatae family, the awareness that useful oil-soluble antioxidants are present in the extracts and the knowledge that rosmarinic acid may also be extracted from these plants, it is surprising that there has been, to the best of Applicants' knowledge, no recognition that this acid or its salts have worthwhile antioxidant activity per se, enabling it to be used as an industrial antioxidant and consequently there appear to be no existing patents on this subject
It is therefore an object of the present invention to provide protection for a substance liable to oxidative deterioration by inclusion in the substance of rosmarinic acid or a derivative thereof, and to provide substances so protected
It is moreover an object of the present invention to provide protection for a substance liable to oxidative deterioration by inclusion of water-soluble antioxidant material extractable from the plants in question, by using weakly acidic, neutral or alkaline aqueous extractant
It is a further object of the present invention to provide such protection by inclusion of a material which is highly efficient in terms of antioxidant activity, as compared with many known antioxidants used in industry
Also, in view of much of the prior art on this subject, the inventor has unexpectedly found that conventionally rejected aqueous residues from the plant material in question, which has already been solvent extracted to remove water-insoluble solutes (including antioxidants such as carnosic acid), may nevertheless afford useful antioxidant material Thus, it is yet a further object of the present invention to provide protection for a substance liable to oxidative deterioration by inclusion of water-soluble antioxidant material prepared by a process for extraction of water-soluble antioxidant material from aqueous residues of known extraction processes wherein plants of the Labiatae family have already been subjected to extraction to remove water-insoluble solutes
A still further object of the invention is to provide a process protection for a substance liable to oxidative deterioration by inclusion of water-soluble antioxidant material prepared by permitting maximum recovery of the industrially useful components from plants of the Labiatae family, such components including essential oils, completely water-soluble antioxidant material and known components which are both water-insoluble and organic solvent soluble, such as vitamin E and carnosic acid
Other objects of the invention will become apparent from the description which follows
SUMMARY OF THE INVENTION
The present invention accordingly provides in one aspect a substance liable to oxidative deterioration, which is characterized by the fact that it incorporates as antioxidant for that substance, at least one compound selected from sub-groups (α), (β) and (γ), namely (α) rosmarinic acid,
(β) salts of the carboxylic acid function in rosmarinic acid, (γ) esters and amides of the carboxylic acid function in rosmarinic acid, provided that said substance excludes a pharmaceutical formulation, cosmetic composition, animal feedstuff, and a foodstuff and a food composition adapted for human consumption, in any of which said at least one compound was incorporated as a pharmacologically active ingredient in presence or absence of any other pharmacologically active ingredient, and that said at least one compound excludes mixtures in a form as extracted from natural sources and comprising any compound in sub-groups (α), (β and (γ), together with any other antιoxιdant(s)
The above proviso, in which "pharmacologically active ingredient" includes such ingredient intended for skin improvement or protection, excludes from the scope of the invention, for example, the disclosures of US 4 329,361 , where rosmarinic acid was used in a daily dose of 50-1000 mg for its antnnflammatory properties, use of rosmarinic acid or its salts in skin cosmetics based on its wound treating effect (JP 63162611 ) or anti-aging effect (US 5,393,526), use of rosmarinic acid in a composition for protection against erythema and inflammatory reactions caused by exposure to UV rays (FR 2652001 ), and inclusion of rosmarinic acid in an antiallergic food connected with its 5-lιpoxygenase inhibiting activity (JP 1121217)
The above proviso further excludes from the scope of the invention use of mixtures of rosmarinic acid or any of its derivatives with any other antιoxιdant(s) (such as carnosic acid or carnosates) in a form as extracted from natural sources (see e g , Viani)
In another aspect, the invention provides use of at least one compound selected from sub-groups (α), (β) and (γ), namely (α) rosmarinic acid, (β) salts of the carboxylic acid function in rosmarinic acid, (γ) esters and amides of the carboxylic acid function in rosmarinic acid, in admixture with a substance liable to oxidative deterioration, in at least a minimum amount necessary to confer antioxidative protection on said substance, provided that when said substance is a dermato-cosmetic composition, said at least one compound is used in an amount less than 0 1 % of the admixture, and that said at least one compound excludes mixtures in a form as extracted from natural sources and comprising any compound in sub-groups (α), (β and (γ), together with any other antιoxιdant(s)
In yet another aspect, the invention provides a method for treating a substance liable to oxidative deterioration, in order to confer antioxidative protection on said substance, which comprises admixing said substance with at least one compound selected from sub-groups (α), (β) and (γ), namely (α) rosmarinic acid, (β) salts of the carboxylic acid function in rosmarinic acid, (γ) esters and amides of the carboxylic acid function in rosmarinic acid, in at least a minimum amount necessary to confer said protection on said substance, provided that when said substance is a dermato-cosmetic composition, said at least one compound is used in an amount less than 0 1 % of the admixture, and that said at least one compound excludes mixtures in a form as extracted from natural sources and comprising any compound in sub-groups (α), (β and (γ), together with any other antιoxιdant(s) The proviso as to the dermato-cosmetic composition excludes the disclosure of FR 2,652,001 , in which rosmarinic acid is present in an amount of 0 1-5 0%
The term "antioxidative protection" in the present specification and claims is intended to include partial as well as substantially total inhibition of oxidation
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates chromatographic fractionation of antioxidant material from rosemary, having utility in accordance with an embodiment of the invention
Figure 2 illustrates chromatographic fractionation of antioxidant material from oregano, having utility in accordance with an embodiment of the invention
DETAILED DESCRIPTION OF THE INVENTION The substance incorporating antioxidant according to the invention may be further characterized by at least one of the following features
(a) it incorporates additionally an emulsifying, dispersing or suspending agent,
(b) it incorporates said antioxidant in a form selected from completely water-soluble materials containing a major proportion of non-antioxidant diluent, and which are solid at ambient temperatures, and aqueous solutions thereof,
(c) it is in the form of a powder, tablet, capsule, solution, emulsion, concentrate, syrup, suspension, gel or dispersion,
(d) said antioxidant has been subjected to chromatographic enrichment or purification, prior to incorporation in said substance,
(e) said antioxidant comprises at least one of
(i) a sodium rosmaπnate, preferably isolated from tissue of plants of the Labiatae family, most preferably by use of an aqueous extractant, and
(n) a rosmaπnate salt other than the sodium salt, preferably obtained by cation-exchange with said sodium salt, (f) said antioxidant constitutes about 0 0001 to about 1 0% by weight of said substance
It is presently preferred that the substance incorporating antioxidant according to the invention is selected from sub-groups (I), (II) and (III), namely
(I) pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption,
(II) an ingredient, liable to oxidative deterioration, adapted to be incorporated into a composition selected from pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption,
(III) a composition selected from pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption, which composition comprises an ingredient liable to oxidative deterioration
Where the substance is a food composition, this may be e g , selected from sugar-based confectionery, manufactured cereals, fruit or vegetable products, beverages or beverage concentrates, ground meat products and vegetable analogues thereof Such food composition may comprise at least one additional ingredient selected from the water-soluble vitamins thiamine, πboflavin, niacin, pyπdoxine, pantothenic acid, biotin, fo c acid, cobalamin and ascorbic acid, the oil-soluble vitamins retinol, calciferol, tocopherol and menadione, in combined form the elements sodium, potassium, calcium, phosphorus, magnesium, chlorine, sulfur, iron, copper, iodine, manganese, cobalt, zinc molybdenum, fluorine, selenium and chromium, unsaturated fatty acids which are known to metabolized in the body to prostaglandins, and physiologically compatible derivatives of said fatty acids, food colorants and pigments, acceptable dispersing and suspending agents, and water
When food compositions are preserved with antιoxιdant(s) according to the invention, an amount of antιoxιdant(s) effective to prevent oxidation of the ingredient liable to oxidative deterioration, e g an oil or fat, or a colorant or pigment, should be used Generally, from about 0 0001 to about 1 %, preferably from about 0 0005 to about 0 1% by weight of the food composition may be used, depending on the nature of the composition and the type of oxidative activity which is to be inhibited
Colorants or pigments, used in food compositions, which are liable to oxidative deterioration, are for example, polyene pigments and colorants such as β-carotene
More especially, food compositions which contain fats or oils comprising fatty acids or their esters, either saturated or unsaturated, may be preserved using antιoxιdant(s) according to the invention The fatty acids are well known and are listed in Noller, Textbook of Organic Chemistry, 2nd Ed pp 108-113 and 138-146 (1958), which is incorporated by reference herein Typical but non-limiting examples of food compositions, including fats and oils adapted for use therein, are soybean oil, corn oil, cottonseed oil, olive oil, butter, margarine, salad creams and mayonnaises, dairy products, ice cream, frozen vegetables, soups, fried foods and
A particular aspect of the present invention resides in the use of rosmarinic acid and its derivatives as described herein, for the purpose of antioxidative protection of vitamins and their analogs and functional derivatives of the vitamins and the analogs In this context there may be mentioned the water-soluble vitamins thiamine, πboflavin, niacin, pyπdoxine, pantothenic acid, biotin, fo c acid, cobalamin and ascorbic acid, the oil-soluble vitamins retinol, calciferol, tocopherol and menadione, and their analogs and functional derivatives thereof, especially such analogs and derivatives having analogous biological activity It is of particular interest, in accordance with an embodiment of this aspect of the invention, that vitamins which are themselves known to be antioxidants, in particular ascorbic acid (including its salts and esters) and α-tocopherol (including its esters) can themselves be protected from deterioration by use of rosmarinic acid and its derivatives
The substance liable to oxidative deterioration, the oxidation which is inhibited in accordance with the present invention, includes the essential oils, such as those derived, for example, from citrus fruit and flowers
The antioxidants utilized in accordance with the invention are selected from rosmarinic acid, salts of the carboxylic acid function in rosmarinic acid, and esters and amides of the carboxylic acid function in rosmarinic acid Rosmarinic acid may be isolated from natural sources e g as described in certain of the above-mentioned published documents, or it (and indeed its salts, esters and amides) may be chemically synthesized It may also be made by enzymatic or cell culture procedures, see e g US 5,011 ,775 (Rao) and published documents mentioned therein, the contents of this patent and of such documents, being incorporated herein by reference
Without prejudice to the generality of the present invention, it is presently preferred to make rosmarinic acid salts for use in the present invention, as described and claimed in copending application 97074, and as exemplified below
The invention will be illustrated by the following non-limiting examples
EXAMPLE 1 Aqueous extraction of raw rosemary plant tissue
(a) A mixture of the dried superficial growing parts of rosemary plants (i e stems, leaves and flowers), 2 g, was homogenized with 10 ml water and maintained at ambient temperature (about 24°C) for one hour The pH of the mixture during this extraction was about 6 5 The supernatant was separated from residual plant tissue and lyophilized to give 0 2 g (10% yield) of a crude completely water-soluble product containing about 10% antioxidant-active substance, the activity of crude product being 430 units/mg
(b) When the extraction in (a) was repeated, except that citric acid was added initially to give pH 2, it was found that the supernatant had a pH of about 7 and that the crude completely water-soluble lyophilized product had approximately the same antioxidant activity as before
(c) When the extraction in (a) was repeated, except that NaOH was added initially to give pH 11 , it was found that the resultant supernatant had a pH of about 7 and that the crude completely water-soluble lyophilized product had approximately the same antioxidant activity as before
(d) When the extraction in (a) was repeated, except that the rosemary plant tissue had been treated with steam and extracted with ether, prior to aqueous extraction, it was found that the crude completely water-soluble lyophilized product had approximately the same antioxidant activity as before
(e) (comparative example) Rosemary plant tissue (5 g) was vigorously stirred with 50 ml Na2Cθ3 NaHC03 buffer at pH 8 6 and 90°C, essentially as described in Example 2 of US 4,012,531 (Viani) Extraction of a portion of the reaction mixture with aqueous acetone and lyophilization of the product (after evaporation of the acetone), at this stage, gave a product containing only 5% of the antioxidant material of the present invention (as identified by a sharp peak on HPLC separation and elution with aqueous methanol at between 14 and 15 minutes retention time), thus showing that the desired product remained dissolved in the alkaline medium A separate portion of the reaction mixture was then acidified as suggested by Viani and as carried out by Todd (see Example 2 of US 5,023,017), both Viani and Todd utilizing only the precipitate as "antioxidant" The precipitate was filtered off and was found to be almost completely water-insoluble The filtrate, after concentration, extraction with aqueous acetone and lyophilization of the product (after evaporation of the acetone) gave a completely water-soluble antioxidant material, 28% pure in the desired product, according to HPLC This experiment demonstrates inter alia that Viani's aqueous alkaline extract did not contain a completely water-soluble solute.
(f) Rosemary or oregano plant tissue was first subjected to the action of steam at 100°C for about one hour to recover essential oils by steam distillation Water was added to the residue and the mixture was allowed to stand at 60-100°C for about 2-45 minutes, filtered and the filtrate containing about 4% solute was concentrated to about 40% solute, again filtered and spray-dried The yellowish powder product contained about 9 5% antioxidant active substance and could be used as such or was subject to chromatographic purification It was found that while a relatively long work-up time resulted in a darker-colored product (light brown to brown), the high antioxidant activity of the product (about 430 units/mg) was independent of its color Yield of the powder was as follows
Run Extraction conditions Yield from 25 g plant tissue
1 20 minutes at 80°C 0 7 g (2 8%)
2 45 minutes at 80°C 1 2 g (4 8%)
3 20 minutes at 100°C 1 2 g (4 8%)
4 45 minutes at 100°C 1 4 g (5 6 %)
5 run 4 residue extracted with 125 ml water for
15 minutes at 100°C +04 g (+1 6%)
6 run 5 residue extracted with 125 ml water for
15 minutes at 100°C +0 28 g (+1 1 %)
4-6 (combined) 2 08 g (8 3%)
Example 1 shows inter alia that the prior art practice of obtaining antioxidant material from Labiatae family plants, from organic solvent soluble extracts, while rejecting aq fractions, and/or rejecting acetone-insolubles, results in substantial loss of potentially valuable antioxidant Moreover, Example 1 (c) shows surprisingly, that contrary to Viani and Todd, useful completely water-soluble antioxidant material can be obtained by alkaline extraction
EXAMPLE 2 Method for determination of antioxidant activity
The method is based on the rate of oxidation of linoleic acid (LA) to its conjugated diene hydroperoxide (Pryor et al , in J Org Chem , 1993, 58 (13) 3521 -3532), and 2,2'-azobιs (2-amιdιnopropane) 2HCI (ABAP) is used to provide a constant rate of radical production A 1 % LA aq emulsion was prepared with 1 % Tween 20 and 0 05M sodium phosphate buffer (pH 7 4) A control sample contained 0 025 ml of the LA emulsion + 2 87 ml of the buffer + 0 1 ml 0 05M ABAP, a test sample contained 0 05 ml of the LA emulsion + 2 7-2 8 ml of the buffer + 0 1 ml 0 05M ABAP + 0 05-0 1 ml of the sample under test Absorption at 234nm is followed for 5 minutes to establish the uninhibited rate of autoxidation The test antioxidant in a concentration of 1 mg/ml is then checked to get 50% inhibition, which defines one unit (e g , if 20 μl inhibits 50%, this is one unit and thus the antioxidant activity is 1000/20 = 50 units/mg) The test antioxidant was added and the inhibited reaction was followed until the antioxidant was consumed and the rate of the absorption change at 234 nm reverted to that observed at the outset
EXAMPLE 3 Chromatographic purification and identification of antioxidant
A column of 8cm x 1cm is packed with MacroPrep-methyl (hydrophobic interaction 40 μm, Bio-Rad) was washed exhaustively with water and ethanol, and then used for purification of the water extract lyophilizate (see Example 1(f), above) 200 mg of the lyophilizate was dissolved in 1 ml water, and the solution was loaded on the MacroPrep column that had been pre-equilibrated with water Elution was carried out using a 0 1 % acetic acid/ 70 30 ethanol in water gradient starting with a 100 0 ratio at 0 minutes and ending with a 0 100 ratio at 220 minutes, and at a flow rate of 2 0 ml/minute Peaks are monitored by UV absorption at 280nm The column effluent was collected after 85 minutes (193 ml) and the active material was found to be eluted between 85 to 100 minutes (36 ml, 30-40%o ethanol (balance water))
The progressive increase in purity of the above operations was determined by HPLC on RP-18, using a water/methanol gradient starting with a 100 0 ratio at 0 minutes and ending with a 30 70 ratio at 15 minutes, and at a flow rate of 1 ml/minute Results are shown, together with other relevant purification data, in Figs 1 and 2 (where "RA" denotes rosmarinic acid, sodium salt) and in the following table, where "% purity" means % in the product in question, of antioxidant material identified by a sharp peak on HPLC separation and elution with aqueous methanol at between 10 68-10 72 mins retention time
Fraction Amount crude % Purity Rosmannate Yield antioxidant (mg) (mg) (%) rosemary extract 200 10 20 100 (Fig 1 ) 21 -23 4 81 3 2 16
24-30 15 98 14 7 7 35
31 -32 2 86 1 6 8 4 total = 97 7 oregano extract 200 10 20 100 (Fig 2) 18-22 18 87 15 75
23-27 15 33 4 6 23 total = 98 0
Both 1H and 13C NMR spectra were measured for the chromatographically pure antioxidant of the invention It was found that there were only very small shift differences in the chemical shift values for a d6 -DMSO solution, compared with corresponding data reported for rosmarinic acid (d6 -acetone for 1H and D20 for 13C), see Kelley, C J et al , J Org Chem 40 1804 (1975) and ibid , 41 449 (1976) These differences, taken together with the fact rosmarinic acid is acetone-soluble and the present compound is virtually acetone-insoluble, are consistent with identification of the present product as a carboxylate salt of rosmarinic acid The compound was identified as sodium rosmannate, and the formula weight (282) for CiβHisNaOβ was confirmed by mass spectra
EXAMPLE 4 Stability and comparative efficiency of antioxidants (a) Stability The lyophilizate obtained from the aqueous extract has a shelf-life with substantially unchanged antioxidant activity of more than two years at 24°C This lyophilizate (6 mg) was dissolved in 0 5 g glycerol, and 0 25 g of the solution was placed in a tube which was then incubated at 180°C for 15 minutes A second similar tube was used as a control at room temperature Following incubation, 1 5 ml water was added to each tube and the activity was checked There was found to be no change in the activity in the control and in the tube incubated at 180°C Moreover, an aqueous extract of rosemary at pH 4 could be kept at ambient temperature for at least 12 months, with retention of antioxidant activity (b) Comparative efficiency Ex 2 is followed, but it is carried out until antioxidant is consumed and the rate of change of absorption at 234 nm had reverted to that observed at the control Efficiency is calculated as the time 1 mg of antioxidant continues to inhibit oxidation of linoleic acid Results are shown in the following table
Antioxidant Efficiency of 10 μg (hrs) Solubility TOXICI
(prior art )
Vitamin E 444 pid no
Vitamin C 93 water no
BHT 814 lipid yes
Trolox* 650 water no
PG 500 lipid yes
TBHQ 432 lipid yes
Rosemary oil 201 lipid no present invention
98% pure 1413 water no
*water-solubιlιzed vitamin E
It may be concluded from the foregoing results that the antioxidant of the invention is considerably more efficient than the known antioxidants against which it has been tested This is true even of the lyophilizate obtained from the aqueous extract as described above and the efficiency of the product is seen to rise dramatically on further purification
EXAMPLE 5 Antioxidant Effect in Emulsions containing Soybean Oil
(a) Oil in water emulsions were prepared from soybean oil (10%), Tween 80 (7%),
Span 80 (3%), water (80%) and antioxidant (0 1 or 0 02%) Antioxidant performance at 50°C as measured by the TBA (thiobarbituπc acid) test were as follows Antioxidant Concentration (%) Days until oxidation
BHA 0 02 2
Vitamin E 0 1 1
Vitamin C 0 1 1
Rosemary Extract* 0 02 4
Rosemary Extract* 0 1 4
*see below
(b) Water in oil emulsions from soybean oil (85%), PGPR emulsifier (Croda, 5%), water (10%) and antioxidant (0 1 or 0 02%) gave test results at 100°C, as measured by the Rancimat (AOM) test, as follows
Antioxidant Concentration (%) Hours until oxidation
BHA 0 02 9 54
Vitamin E 0 1 9 58
Vitamin C 0 1 25 20
Rosemary Extract* 0 02 19 05
Rosemary Extract* 0 1 29 00
*water-soluble, containing 10% sodium rosmannate, the concentration of the latter in the emulsions is thus, respectively, 0 002 and 0 01
EXAMPLE 6 Antioxidant Effect in Bulk Oil
A concentrate was first prepared containing 40% lecithin, 40% oil (e g soybean oil), 20% propylene glycol and 4% based on the foregoing mixture of 65% (≡2 6% pure) sodium rosmannate Rosmarinic acid can alternatively be substituted for the sodium salt
The thus-prepared concentrate can then be used in a bulk oil which it is desired to protect against oxidative deterioration Where the oil is soybean oil, test results at 60°C were as follows Antioxidant Concentration Days until oxidation
(prior art )
None 4
Vitamin E 0 06% 4
BHA 0 02% 4
Rosemary oil 0 4% 6 present invention sodium rosmannate 0 005%α 7 sodium rosmannate 0 013%* 8
D as 0.2% of the above concentrate ♦ as 0.5% of the above concentrate
EXAMPLE 7 Antioxidant Effect in Emulsions containing β-Carotene β-Carotene (6 mg), linoleic acid (1 ml) and Tween 40 (2 ml) were dissolved in chloroform, the mixture was concentrated in a rotary evaporator, and the last traces of chloroform were removed by nitrogen A model emulsion was then prepared by adding double-distilled water (25 ml) to the residue and diluting to 500 ml with phosphate buffer (pH 7 0) A solution (2 ml) of the inventive antioxidant was mixed with 50 ml a quots of the emulsion, such that antioxidant concentration was 0 005, 0 01 or 0 02% Comparison samples contained BHA in concentrations of 0 01 and 0 02%, and ascorbic acid (0 1 %) A control sample contained no antioxidant but instead, 95% ethanol (2 ml) It was found that over a 100 hour time period, the inventive antioxidant afforded a similar order of protection to β-carotene as did BHA, whereas little protection was afforded by ascorbic acid
EXAMPLE 8 Stabilization of Ascorbic Acid
It is known that ascorbic acid is subject to oxidative deterioration, and in particular that its aqueous solutions are rapidly oxidized by air For the purpose of this example, ascorbic acid was dissolved in 50% ethanol, and the solution, in a closed bottle, was placed in an oven at 60°C for 24 hours The experiment was repeated with the addition of 0 035 mg or 0 07 mg sodium rosmannate Each sample was diluted 1 10 and the ascorbic acid content was analyzed by HPLC on a 250 cm x 4 mm RP-18 column, and eluting with 30 70 acetonitπle/water at a flow rate of 1 ml/mm The peak area absorption at 245 nm, compared with the initial area, corresponded with the amount of ascorbic acid in each case, the results being as follows
Sample HPLC peak area (245 nm) % ascorbic acid
(10 mg/ml ascorbic aci 'd) control 12,334,252 100 after 60°C, 24 hours 5,572,750 45 (no additive) after 60°C, 24 hours 8,384,409 68 (+0 035 mg rosmarinic ; acid*) after 60°C, 24 hours 11 ,130,666 90
(+0 07 mg rosmarinic acid*)
*Na salt
These results show that rosmarinic acid (and its derivatives) effectively protects ascorbic acid from decomposition under the given conditions, particularly at the higher concentration of 0 7% It may be inferred that the extent of this protection would be considerably greater under other conditions, e g at ambient temperatures and/or when the ascorbic acid is not necessarily in the form of an aqueous solution
EXAMPLE 9 Inhibition of oxidation of essential oils (a) orange essential oil
Different concentrations of romarinate solution were added to 5 ml orange essential oil The samples were kept at 30°C for seven days, and were then checked for peroxide using the TBA test The rosmannate solution ("Organox os") contained 17% propylene glycol, 65% lecithin and 17% sodium rosmannate (50% pure) Additionally, oxidation at 4°C without antioxidant was noted for comparison The results were as follows Essential oil % Orqanox os % rosmannate oxidation
(TBA)
5 ml, 30°C 0 0 0 525nm
5 ml, 30°C 0 1 0 008 0 445nm
5 ml, 30°C 0 2 0 017 0 370nm
5 ml, 30°C 0 25 0 021 0 340nm
5 ml, 30°C 0 3 0 025 0 310nm
5 ml, 4°C 0 0 0 320nm
(b) grapefruit oil
This test was carried out similarly to part (a), above, except that the samples were kept for eleven days The results were as follows
Essential oil %> Orqanox os % rosmannate oxidation
(TBA)
5 ml, 30°C 0 0 0 150nm 5 ml, 30°C 0 1 0 008 0 105nm 5 ml, 30°C 0 2 0 017 0 OSOnm 5 ml, 4°C 0 0 0 080nm
(c) orange oil (colorless)
This test was carried out similarly to part (a), above, except that the samples were kept for eleven days The results were as follows
Essential oil % Orqanox os % rosmannate oxidation
(TBA)
5 ml, 30°C 0 0 1 8πm
5 ml, 30°C 0 1 0 008 1 3nm
5 ml, 30°C 0 155 0 013 1 Onm
5 ml, 30°C 02 0 017 0 9nm
5 ml, 30°C 025 0 021 065nm
5 ml, 4°C 0 0 0 70nm
Conclusions By using a relatively small amount of antioxidant at 30°C, it is possible to achieve approximately the same level of inhibition of oxidation, in essential oils, for the given period, compared with refrigeration at 4°C
While the present invention has been particularly described with reference to certain embodiments, it will be apparent to those skilled in the art that many modifications and variations may be made The invention is accordingly not to be construed as limited in any way by such embodiments, rather its concept is to be understood according to the spirit and scope of the claims which follow

Claims

1 A substance liable to oxidative deterioration, which is characterized by the fact that it incorporates as antioxidant for that substance, at least one compound selected from sub-groups (α), (β) and (γ), namely (α) rosmarinic acid,
(β) salts of the carboxylic acid function in rosmarinic acid, (γ) esters and amides of the carboxylic acid function in rosmarinic acid, provided that said substance excludes a pharmaceutical formulation, cosmetic composition, animal feedstuff, and a food composition adapted for human consumption, in any of which said at least one compound was incorporated as a pharmacologically active ingredient in presence or absence of any other pharmacologically active ingredient, and that said at least one compound excludes mixtures in a form as extracted from natural sources and comprising any compound in sub-groups (α), (β and (γ), together with any other antιoxιdant(s)
2 A substance according to claim 1 , further characterized by at least one of the following features
(a) it incorporates additionally an emulsifying, dispersing or suspending agent,
(b) it incorporates said antioxidant in a form selected from completely water-soluble materials containing a major proportion of non-antioxidant diluent, and which are solid at ambient temperatures, and aqueous solutions thereof,
(c) it is in the form of a powder, tablet, capsule, solution, emulsion, concentrate, syrup, suspension, gel or dispersion,
(d) said antioxidant has been subjected to chromatographic enrichment or purification, prior to incorporation in said substance,
(e) said antioxidant comprises at least one of
(i) a sodium rosmannate, preferably isolated from tissue of plants of the Labiatae family, most preferably by use of an aqueous extractant, and
(II) a romannate salt other than the sodium salt, preferably obtained by cation-exchange with said sodium salt,
(f) said antioxidant constitutes about 0 0001 to about 1 0% by weight of said substance 3 A substance according to either claim 1 or claim 2, which is selected from sub-groups (I), (II) and (III), namely
(I) pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption,
(II) an ingredient, liable to oxidative deterioration, adapted to be incorporated into a composition selected from pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption,
(III) a composition selected from pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption, which composition comprises an ingredient liable to oxidative deterioration
4 A food composition according to claim 3, which is selected from sugar-based confectionery, manufactured cereals, fruit or vegetable products, beverages or beverage concentrates, ground meat products and vegetable analogues thereof
5 A food composition according to claim 4, which comprises at least one additional ingredient selected from the water-soluble vitamins thiamine, πboflavin, niacin, pyndoxine, pantothenic acid, biotin, fo c acid, cobalamin and ascorbic acid, the oil-soluble vitamins retinol, calciferol, tocopherol and menadione, in combined form the elements sodium, potassium, calcium, phosphorus, magnesium, chlorine, sulfur, iron, copper, iodine, manganese, cobalt, zinc molybdenum, fluorine, selenium and chromium, unsaturated fatty acids which are known to metabolized in the body to prostaglandins, and physiologically compatible derivatives of said fatty acids, food colorants and pigments, acceptable dispersing and suspending agents, and water
6 A substance according to claim 1 which comprises at least one essential oil 7 Use of at least one compound selected from sub-groups (α), (β) and (γ), namely
(α) rosmarinic acid,
(β) salts of the carboxylic acid function in rosmarinic acid, (γ) esters and amides of the carboxylic acid function in rosmarinic acid, in admixture with a substance liable to oxidative deterioration, in at least a minimum amount necessary to confer antioxidative protection on said substance, provided that when said substance is a dermato-cosmetic composition, said at least one compound is used in an amount less than 0 1 % of the admixture, and that said at least one compound excludes mixtures in a form as extracted from natural sources and comprising any compound in sub-groups ( ), (β and (γ), together with any other antιoxιdant(s)
8 Use according to claim 7, further characterized by at least one of the following features
(a) said admixture incorporates additionally an emulsifying, dispersing or suspending agent,
(b) said admixture incorporates said antioxidant in a form selected from completely water-soluble materials containing a major proportion of non-antioxidant diluent, and which are solid at ambient temperatures, and aqueous solutions thereof,
(c) said admixture is in the form of a powder, tablet, capsule, solution, emulsion, concentrate, syrup, suspension, gel or dispersion,
(d) said antioxidant has been subjected to chromatographic enrichment or purification, prior to admixture with said substance,
(e) said antioxidant comprises at least one of
(i) a sodium rosmannate, preferably isolated from tissue of plants of the Labiatae family, most preferably by use of an aqueous extractant, and
(n) a rosmannate salt other than the sodium salt, preferably obtained by cation-exchange with said sodium salt,
(f) said antioxidant constitutes about 0 0001 to about 1 0%> by weight of said substance 9 Use according to either claim 7 or claim 8, wherein said substance on which antioxidative protection is conferred is selected from sub-groups (I), (II) and (III), namely
(I) pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption,
(II) an ingredient, liable to oxidative deterioration, adapted to be incorporated into a composition selected from pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption,
(III) a composition selected from pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption, which composition comprises an ingredient liable to oxidative deterioration
10 Use according to claim 9, wherein said food composition is selected from sugar-based confectionery, manufactured cereals, fruit or vegetable products, beverages or beverage concentrates, ground meat products and vegetable analogues thereof
11 Use according to claim 9, wherein said food composition comprises at least one additional ingredient selected from the water-soluble vitamins thiamine, nboflavin, niacin, pyndoxine, pantothenic acid, biotin, fo c acid, cobalamin and ascorbic acid, the oil-soluble vitamins retinol, calciferol, tocopherol and menadione, in combined form the elements sodium, potassium, calcium, phosphorus, magnesium, chlorine, sulfur, iron, copper, iodine, manganese, cobalt, zinc molybdenum, fluorine, selenium and chromium, unsaturated fatty acids which are known to metabolized in the body to prostaglandins, and physiologically compatible derivatives of said fatty acids, food colorants and pigments, acceptable dispersing and suspending agents, and water
12 Use according to claim 7, wherein said substance comprises at least one essential oil 13 Method for treating a substance liable to oxidative deterioration, in order to confer antioxidative protection on said substance, which comprises admixing said substance with at least one compound selected from sub-groups (α), (β) and (γ), namely
(α) rosmarinic acid,
(β) salts of the carboxylic acid function in rosmarinic acid,
(γ) esters and amides of the carboxylic acid function in rosmarinic acid, in at least a minimum amount necessary to confer said protection on said substance, provided that when said substance is a dermato-cosmetic composition, said at least one compound is used in an amount less than 0 1 % of the admixture, and that said at least one compound excludes mixtures in a form as extracted from natural sources and comprising any compound in sub-groups (α), (β) and (γ), together with any other antιoxιdant(s)
14 Method according to claim 13, further characterized by at least one of the following features
(a) said admixture incorporates additionally an emulsifying, dispersing or suspending agent,
(b) said admixture incorporates said antioxidant in a form selected from completely water-soluble materials containing a major proportion of non-antioxidant diluent, and which are solid at ambient temperatures, and aqueous solutions thereof,
(c) said admixture is in the form of a powder, tablet, capsule, solution, emulsion, concentrate, syrup, suspension, gel or dispersion,
(d) said antioxidant has been subjected to chromatographic enrichment or purification, prior to admixture with said substance,
(e) said antioxidant comprises at least one of
(i) a sodium rosmannate, preferably isolated from tissue of plants of the Labiatae family, most preferably by use of an aqueous extractant, and
(II) a rosmannate salt other than the sodium salt, preferably obtained by cation-exchange with said sodium salt,
(f) said antioxidant constitutes about 0 0001 to about 1 0% by weight of said substance 15 Method according to either claim 13 or claim 14, wherein said substance on which antioxidative protection is conferred is selected from sub-groups (I), (II) and (III), namely
(I) pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption,
(II) an ingredient, liable to oxidative deterioration, adapted to be incorporated into a composition selected from pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption,
(III) a composition selected from pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions and food compositions adapted for human consumption, which composition comprises an ingredient liable to oxidative deterioration
16 Method according to claim 14, wherein said food composition is selected from sugar-based confectionery, manufactured cereals, fruit or vegetable products, beverages or beverage concentrates, ground meat products and vegetable analogues thereof
17 Method according to claim 15, wherein said food composition comprises at least one additional ingredient selected from the water-soluble vitamins thiamine, nboflavin, niacin, pyndoxine, pantothenic acid, biotin, fohc acid, cobalamin and ascorbic acid, the oil-soluble vitamins retinol, calciferol, tocopherol and menadione, in combined form the elements sodium, potassium, calcium, phosphorus, magnesium, chlorine, sulfur, iron, copper, iodine, manganese, cobalt, zinc molybdenum, fluorine, selenium and chromium, unsaturated fatty acids which are known to metabolized in the body to prostaglandins, and physiologically compatible derivatives of said fatty acids, food colorants and pigments, acceptable dispersing and suspending agents, and water
18 Method according to claim 13 wherein said substance comprises at least one essential oil 19 A substance according to claim 3, wherein said ingredient comprises at least one member selected from the group consisting of vitamins, their functional derivatives, and analogs thereof
20 Use according to claim 9, wherein said ingredient comprises at least one member selected from the group consisting of vitamins, their functional derivatives, and analogs thereof
21 Method according to claim 15, wherein said ingredient comprises at least one member selected from the group consisting of vitamins, their functional derivatives, and analogs thereof
PCT/IL1999/000693 1998-12-24 1999-12-22 Method for protecting a substance liable to oxidative deterioration WO2000039248A1 (en)

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EP99959658A EP1246884A1 (en) 1998-12-24 1999-12-22 Method for protecting a substance liable to oxidative deterioration

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IL127724 1998-12-24
IL12772498A IL127724A0 (en) 1998-12-24 1998-12-24 Method for protecting a substance liable to oxidative deterioration
IL128593 1999-02-18
IL12859399A IL128593A0 (en) 1999-02-18 1999-02-18 Method for protecting a substance susceptible to oxidative deterioration

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EP3425033A4 (en) * 2016-03-02 2019-10-23 Fuji Oil Holdings Inc. Fat or oil composition exhibiting anti-oxidation properties
US11957135B2 (en) 2016-03-02 2024-04-16 Fuji Oil Holdings Inc. Chocolate-like food containing polyunsaturated fatty acid

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WO2009013757A1 (en) * 2007-07-26 2009-01-29 Frutarom Ltd. Composition for protecting oils and fat
WO2015095545A1 (en) * 2013-12-20 2015-06-25 Abbott Laboratories Nutritional composition containing oxidizable oil and rosmarinic acid
WO2016057818A1 (en) * 2014-10-08 2016-04-14 Abbott Laboratories Nutritional compositions comprising an oxidizable component and water-soluble plant extract
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JP2017531429A (en) * 2014-10-08 2017-10-26 アボット・ラボラトリーズＡｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ Nutritional composition comprising an oxidizable component and a water-soluble plant extract
EP3425033A4 (en) * 2016-03-02 2019-10-23 Fuji Oil Holdings Inc. Fat or oil composition exhibiting anti-oxidation properties
US11957135B2 (en) 2016-03-02 2024-04-16 Fuji Oil Holdings Inc. Chocolate-like food containing polyunsaturated fatty acid

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