CA2432441C - Method for protecting a substance liable to oxidative deterioration - Google Patents
Method for protecting a substance liable to oxidative deterioration Download PDFInfo
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- CA2432441C CA2432441C CA2432441A CA2432441A CA2432441C CA 2432441 C CA2432441 C CA 2432441C CA 2432441 A CA2432441 A CA 2432441A CA 2432441 A CA2432441 A CA 2432441A CA 2432441 C CA2432441 C CA 2432441C
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- 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
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Abstract
Antioxidative protection is conferred on a substance liable to oxidative deterioration, by admixing it with at least one compound selected from sub-groups (.alpha.), (.beta.) and (.gamma.), namely: (.alpha.) rosmarinic acid;
(.beta.) salts of the carboxylic acid function in rosmarinic acid; (.gamma.) esters and amides of the carboxylic acid function in rosmarinic acid; subject to certain provisos.
(.beta.) salts of the carboxylic acid function in rosmarinic acid; (.gamma.) 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 dichloride, 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 71,11,12-trihydroxy-6,10-(epoxymethano)abieta- 8,11,13-trien-20-one.
US 4,638,095 (Chang et al.) describes the isolation from rosemary of the antioxidant "rosmaridiphenol", 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 8.4-11.8, 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 CO2 at 350-bar and subsequent fractionation, proposes to prepare antioxidants from a similar source by a multistep process utilizing extraction with CO2 at 80-300 bar, treating the extraction residue with a CI-4 alcohol or C5_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-insolubles 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
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 dichloride, 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 71,11,12-trihydroxy-6,10-(epoxymethano)abieta- 8,11,13-trien-20-one.
US 4,638,095 (Chang et al.) describes the isolation from rosemary of the antioxidant "rosmaridiphenol", 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 8.4-11.8, 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 CO2 at 350-bar and subsequent fractionation, proposes to prepare antioxidants from a similar source by a multistep process utilizing extraction with CO2 at 80-300 bar, treating the extraction residue with a CI-4 alcohol or C5_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-insolubles 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
2 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) HO OtOH OH
HO OH
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 antiinflammatory 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-lipoxygenase inhibitor is also featured in JP 1121217, where, extracted from perilla 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.
In US 4,354,035 (Christ et al.), there is described a process for isolating rosmarinic acid (of formula depicted below) HO OtOH OH
HO OH
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 antiinflammatory 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-lipoxygenase inhibitor is also featured in JP 1121217, where, extracted from perilla 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.
3 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 carnosic 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
Although the Viani patent, for example, mentions rosmarinic acid, together with carnosic 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
4 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 (a), ((3) and (y), namely:
(a) rosmarinic acid;
(R) salts of the carboxylic acid function in rosmarinic acid;
(y) 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 ((X), (13 and (y), together with any other antioxidant(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 antiinflammatory 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-lipoxygenase 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 antioxidant(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 ((x), (0) and (y), namely: (a) rosmarinic acid; (a) salts of the carboxylic acid function in rosmarinic acid; (y) 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 (a), ((3 and (y), together with any other antioxidant(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 (a), ((3) and (y), namely: (a) rosmarinic acid; (13) salts of the carboxylic acid function in rosmarinic acid; (y) 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 (a), ((3 and (y), together with any other antioxidant(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 rosmarinate, preferably isolated from tissue of plants of the Labiatae family, most preferably by use of an aqueous extractant; and (ii) a rosmarinate 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), (il) 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, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic 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 antioxidant(s) according to the invention, an amount of antioxidant(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 0-carotene.
More especially, food compositions which contain fats or oils comprising fatty acids or their esters, either saturated or unsaturated, may be preserved using antioxidant(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-(1958). 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 the like.
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, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic 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 a-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.
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 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 Na2CO31NaHCO3 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 %) run 4 residue extracted with 125 ml water for minutes at 100 C +0.4 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'-azobis (2-amidinopropane).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 l 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 1 cm is packed with MacroPrep-methyl (hydrophobic interaction 40 pm, Bio-Rad) was washed exhaustively with water and ethanol, and then used for purification of the water extract Iyophilizate (see Example 1(f), above). 200 mg of the Iyophilizate 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% 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 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 Rosmarinate 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) 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:
(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 rosmarinate, and the formula weight (282) for C18H15NaO8 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 g (hrs) Solubility Toxicity ----------------------------------------------------------------------------(prior art:) Vitamin E 444 lipid 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-solubilized 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 (thiobarbituric 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,
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 (a), ((3) and (y), namely:
(a) rosmarinic acid;
(R) salts of the carboxylic acid function in rosmarinic acid;
(y) 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 ((X), (13 and (y), together with any other antioxidant(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 antiinflammatory 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-lipoxygenase 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 antioxidant(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 ((x), (0) and (y), namely: (a) rosmarinic acid; (a) salts of the carboxylic acid function in rosmarinic acid; (y) 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 (a), ((3 and (y), together with any other antioxidant(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 (a), ((3) and (y), namely: (a) rosmarinic acid; (13) salts of the carboxylic acid function in rosmarinic acid; (y) 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 (a), ((3 and (y), together with any other antioxidant(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 rosmarinate, preferably isolated from tissue of plants of the Labiatae family, most preferably by use of an aqueous extractant; and (ii) a rosmarinate 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), (il) 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, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic 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 antioxidant(s) according to the invention, an amount of antioxidant(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 0-carotene.
More especially, food compositions which contain fats or oils comprising fatty acids or their esters, either saturated or unsaturated, may be preserved using antioxidant(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-(1958). 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 the like.
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, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic 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 a-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.
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 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 Na2CO31NaHCO3 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 %) run 4 residue extracted with 125 ml water for minutes at 100 C +0.4 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'-azobis (2-amidinopropane).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 l 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 1 cm is packed with MacroPrep-methyl (hydrophobic interaction 40 pm, Bio-Rad) was washed exhaustively with water and ethanol, and then used for purification of the water extract Iyophilizate (see Example 1(f), above). 200 mg of the Iyophilizate 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% 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 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 Rosmarinate 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) 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:
(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 rosmarinate, and the formula weight (282) for C18H15NaO8 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 g (hrs) Solubility Toxicity ----------------------------------------------------------------------------(prior art:) Vitamin E 444 lipid 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-solubilized 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 (thiobarbituric 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 rosmarinate; 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 rosmarinate. 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 rosmarinate 0.005%^ 7 sodium rosmarinate 0.013%= 8 ----------------------------------------------------------------------as 0.2% of the above concentrate = as 0.5% of the above concentrate EXAMPLE 7: Antioxidant Effect in Emulsions containing (3-Carotene (3-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 aliquots 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 f3-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 rosmarinate. Each sample was diluted 1:10 and the ascorbic acid content was analyzed by HPLC on a 250 cm x 4 mm RP-1 8 column, and eluting with 30:70 acetonitrile/water at a flow rate of 1 ml/min. 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 acid) -------- - ---- - ------------------ - - - ---------- ------- - -- - - - ------------ - ---- - ------- - --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 rosmarinate solution ("Organox os") contained 17% propylene glycol, 65% lecithin and 17% sodium rosmarinate (50%
pure). Additionally, oxidation at 4 C without antioxidant was noted for comparison.
The results were as follows:
Essential oil % Organox os % rosmarinate oxidation T( BA) 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 0.31 Onm 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 % Organox os % rosmarinate 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 0.080nm 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 % Organox os % rosmarinate oxidation (TBA) 5 ml, 30 C 0 0 1.8nm 5 ml, 30 C 0.1 0.008 1.3nm 5 ml, 30 C 0.155 0.013 1.0nm 5 ml, 30 C 0.2 0.017 0.9nm 5 ml, 30 C 0.25 0.021 0.65nm 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.
----------------------------------------------------------------------------------------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 rosmarinate; 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 rosmarinate. 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 rosmarinate 0.005%^ 7 sodium rosmarinate 0.013%= 8 ----------------------------------------------------------------------as 0.2% of the above concentrate = as 0.5% of the above concentrate EXAMPLE 7: Antioxidant Effect in Emulsions containing (3-Carotene (3-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 aliquots 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 f3-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 rosmarinate. Each sample was diluted 1:10 and the ascorbic acid content was analyzed by HPLC on a 250 cm x 4 mm RP-1 8 column, and eluting with 30:70 acetonitrile/water at a flow rate of 1 ml/min. 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 acid) -------- - ---- - ------------------ - - - ---------- ------- - -- - - - ------------ - ---- - ------- - --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 rosmarinate solution ("Organox os") contained 17% propylene glycol, 65% lecithin and 17% sodium rosmarinate (50%
pure). Additionally, oxidation at 4 C without antioxidant was noted for comparison.
The results were as follows:
Essential oil % Organox os % rosmarinate oxidation T( BA) 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 0.31 Onm 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 % Organox os % rosmarinate 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 0.080nm 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 % Organox os % rosmarinate oxidation (TBA) 5 ml, 30 C 0 0 1.8nm 5 ml, 30 C 0.1 0.008 1.3nm 5 ml, 30 C 0.155 0.013 1.0nm 5 ml, 30 C 0.2 0.017 0.9nm 5 ml, 30 C 0.25 0.021 0.65nm 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 (14)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A substance liable to oxidative deterioration, which is characterized by the fact that it incorporates as antioxidant for that substance:
an extract from tissue of plants of the Labiatae family obtained by aqueous extraction at a temperature of less than 90 C and comprising sodium rosmarinate or a derivative of said sodium rosmarinate, said derivative being selected from the group consisting of rosmarinate salts other than the sodium salt, rosmarinic acid and its esters and amides, wherein said extract is characterized in that it is completely water soluble under weakly acid, neutral or alkaline conditions; and wherein said substance is selected from the group consisting of:
pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions, food compositions adapted for human consumption, and ingredients, liable to oxidative deterioration of any of said compositions.
an extract from tissue of plants of the Labiatae family obtained by aqueous extraction at a temperature of less than 90 C and comprising sodium rosmarinate or a derivative of said sodium rosmarinate, said derivative being selected from the group consisting of rosmarinate salts other than the sodium salt, rosmarinic acid and its esters and amides, wherein said extract is characterized in that it is completely water soluble under weakly acid, neutral or alkaline conditions; and wherein said substance is selected from the group consisting of:
pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions, food compositions adapted for human consumption, and ingredients, liable to oxidative deterioration of any of said compositions.
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 sodium rosmarinate and rosmarinate salts other than the sodium salt; or (f) said antioxidant constitutes about 0.0001 to about 1.0% by weight of said substance.
(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 sodium rosmarinate and rosmarinate salts other than the sodium salt; or (f) said antioxidant constitutes about 0.0001 to about 1.0% by weight of said substance.
3. A food composition according to claim 1 or claim 2, which is selected from sugar-based confectionery, manufactured cereals, fruit or vegetable products, beverages or beverage concentrates, ground meat products and vegetable analogues thereof.
4. A food composition according to claim 3, which comprises at least one additional ingredient selected from the water-soluble vitamins thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic 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.
5. Use of an extract of tissue of plants of the Labiatae family obtained by aqueous extraction at a temperature of less than 90 C and comprising sodium rosmarinate or a derivative of said sodium rosmarinate, said derivative being selected from the group consisting of rosmarinate salts other than the sodium salt, rosmarinic acid and its esters and amides, wherein said aqueous extract is characterized as completely water-soluble under weakly acid, neutral or alkaline conditions; as an antioxidant in admixture with a substance liable to oxidative deterioration, in at least a minimum amount necessary to confer antioxidative protection on said substance, wherein said substance is selected from the group consisting of pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions, food compositions adapted for human consumption an ingredient, liable to oxidative deterioration, adapted to be incorporated into any one of compositions.
6. Use according to claim 5, 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 sodium rosmarinate and rosmarinate salts other than the sodium salt; or (f) said antioxidant constitutes about 0.0001 to about 1.0% by weight of said substance.
(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 sodium rosmarinate and rosmarinate salts other than the sodium salt; or (f) said antioxidant constitutes about 0.0001 to about 1.0% by weight of said substance.
7. Use according to claim 6, 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.
8. Use according to claim 7, wherein said food composition comprises at least one additional ingredient selected from the water-soluble vitamins thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic 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 prostagiandins, and physiologically compatible derivatives of said fatty acids, food colorants and pigments, acceptable dispersing and suspending agents, and water.
9. Method for treating a substance liable to oxidative deterioration in order to confer antioxidative protection on said substance, which comprises admixing said substance with an extract of tissue of plants of the Labiatae family obtained by aqueous extraction at a temperature of less than 90 C and comprising sodium rosmarinate or a derivative of said sodium rosmarinate, said derivative being selected from the group consisting of rosmarinate salts other than the sodium salt, rosmarinic acid and its esters and amides, wherein said aqueous extract is characterized as completely water-soluble under weakly acid, neutral or alkaline conditions;
wherein the extract comprising sodium rosmarinate or the derivative of said sodium rosmarinate is present in at least a minimum amount necessary to confer said protection on said substance, and wherein said substance is selected from the group consisting of pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions, food compositions adapted for human consumption and ingredients, liable to oxidative deterioration, adapted to be incorporated into any one of said compositions.
wherein the extract comprising sodium rosmarinate or the derivative of said sodium rosmarinate is present in at least a minimum amount necessary to confer said protection on said substance, and wherein said substance is selected from the group consisting of pharmaceutical compositions, cosmetic compositions, animal feedstuff compositions, food compositions adapted for human consumption and ingredients, liable to oxidative deterioration, adapted to be incorporated into any one of said compositions.
10. Method according to claim 9, 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 sodium rosmarinate and rosmarinate salts other than the sodium salt, preferably obtained by cation-exchange with said sodium salt; or (f) said antioxidant constitutes about 0.0001 to about 1.0% by weight of said substance.
(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 sodium rosmarinate and rosmarinate salts other than the sodium salt, preferably obtained by cation-exchange with said sodium salt; or (f) said antioxidant constitutes about 0.0001 to about 1.0% by weight of said substance.
11. Method according to claim 9 or claim 10, 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.
12. Method according to claim 11, wherein said food composition comprises at least one additional ingredient selected from the water-soluble vitamins thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic 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.
13. A substance according to claim 2, wherein said sodium rosmarinate and rosmarinate salts other than the sodium salt are obtained by cation exchange with said sodium salt.
14. Use according to claim 6, wherein said sodium rosmarinate and rosmarinate salts other than the sodium salt are obtained by cation exchange with said sodium salt.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IL1999/000693 WO2000039248A1 (en) | 1998-12-24 | 1999-12-22 | Method for protecting a substance liable to oxidative deterioration |
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| Publication Number | Publication Date |
|---|---|
| CA2432441A1 CA2432441A1 (en) | 2000-07-06 |
| CA2432441C true CA2432441C (en) | 2011-03-01 |
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ID=27772945
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2432441A Expired - Fee Related CA2432441C (en) | 1999-12-22 | 1999-12-22 | Method for protecting a substance liable to oxidative deterioration |
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| Country | Link |
|---|---|
| CA (1) | CA2432441C (en) |
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1999
- 1999-12-22 CA CA2432441A patent/CA2432441C/en not_active Expired - Fee Related
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| CA2432441A1 (en) | 2000-07-06 |
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