DD299089A5 - METHOD FOR DETERMINING ANTI-OXYDATING PROPERTIES OF A LIVING ORGANISM OR A POTENTIALLY AGGRESIVE MEDIUM BY FREE RADICALS - Google Patents

Abstract

The invention relates to a method for the determination or evaluation of antioxidant activities of a living organism or of a potentially aggressive agent using free radicals as a means of inducing cell dissolution. According to the invention, a free-radical generator and a cell material are brought together in a suitable liquid biological medium. The cell material is selected from human, animal and plant cells, from fragments of these cells or from synthetic cell walls and fragments containing their liposomes, the cell material having previously been contaminated by a potentially aggressive agent. The release of free radicals is induced starting from the free radical generator. The dissolution of the cell material by the free radicals is evaluated in terms of a control sample containing the uncontaminated cell material. {Living organism; oxidation-inhibiting properties; potentially aggressive agent; free radicals; Induction; Zellaufloesung}

Description

For this 4 pages drawings

Field of application of the invention

This invention relates to a novel method for determining the antioxidant properties of a living organism or a potentially aggressive agent by means of free radicals.

In particular, it deals with the method of evaluating the oxidation-inhibiting state of cells of a living organism on the one hand and the oxidative or antioxidant activities of a chemical or physical agent on the other hand, which may be aggressive, i.e. the cell release initiated by the free radicals either increase or accelerate, or inhibit or delay.

Characteristic of the known state of the art

It is known that the free radicals in general exert an adverse effect on the organism and in particular on the cells of this organism. The free radicals attack the cell wall more or less quickly

Resistance of the cells, which gives them the enzymatic or molecular material (or equipment) of said cells. If the cell wall has been damaged, punctured or opened by free radicals, the cell contents spread outside the wall. See, in particular, the following documents: Chemical Abstracts 107,213235v, Chemical Abstracts 0107,232454g, Chemical Abstracts 100,99345J, Biological Abstracts 72 (# 9), page 5914, abstract no. 57169, (1981) and Biological Abstracts 73 (No. 12), page 8817, abstract No. 84420, (1982).

Abstract CA 107,21323Ev, referring to an article by M. MIKI et al., Arch. Biochem. Biophys., 258 (No.2), pp. 373-380 (1987), describes that alpha-tocopherol protects rat red blood cells from the dissolution initiated by dun-free radicals.

After the aforementioned summary CA 100,99345J, referring to an article by E. B. SPEKTOR and co-workers, Lab. DeIo (No. 1), page 26-28 (1984), the evaluation of the total antioxidant activity of a sample (blood plasma or cerebrospinal fluid) by absorption at 532 nm after induction of free radicals in a Zellmateriul (in the special case: membrane of erythrocytes ) determined by means of a UV lamp.

Object of the invention

The object of the invention is to provide an improved method for determining the antioxidant properties of a living organism or a potentially aggressive agent.

Explanation of the essence of the invention

The invention has for its object to add the cell material suitable free radicals in a novel way. According to the invention, a new technical solution is recommended, according to which (i) the free radicals are not produced in said cell material, thus originating from a free radical initiator added to the sclerosed cell material, and (ii) after Cellular material was previously contaminated with a potentially aggressive agent.

According to the invention, a novel method is proposed for the determination or evaluation of the antioxidant activities of a living organism or of a potentially aggressive agent, said method involving the use of free radicals as a means of initiating cell dissolution, characterized in that

1. In a suitable liquid biological medium, a free radical generator and a cell material (I) which has been selected from the complex are brought together

a) human, animal and plant cells,

b) fragments of said cells, and

c) synthetic walls and fragments containing their liposomes,

said cell material previously contaminated by a potentially aggressive agent (II);

2. initiating the release of free radicals from said free radical generator, and

3. evaluating the dissolution of the cell material by the free radicals relative to a control sample containing said uncontaminated cell material.

After this procedure, the oxidative state of Zellmateria !? I or the influence of the agent Il on the said material I determined.

In particular, the assessment of the dissolution of the cell material may be carried out in a "kinetic" manner (by periodic measurements on [constant volume] samples of liquid assay medium containing the free radical generator, the cell material and possibly the agent II to be tested) "Non-kinetic" manner (by measuring the dose-response type on samples from the liquid assay medium containing the cell material, optionally in conjunction with the agent to be tested II, as well as increasing aliquot quantities of the free radical generator). In the context of the kinetic evaluation, the resistance of the cell material I to free radicals is expressed by the time corresponding to a 50% dissolution of said cell material.

As part of the dose-response evaluation, the resistance of the cell material to free radicals is expressed by the concentration of the free-radical generator which initiates a 50% dissolution of said cell material. Among the free radical generators useful in the present invention, those which release the free radicals and which are often used in the field of polymerization to obtain macromolecules may be mentioned. Among these products, in particular, the generators of oxidative free radicals can be listed, such. As the benzoyl peroxide, the alkyl perbenzoates in C ₁ -C ₈ , preferably C ₃ -C ₄ (especially n-butyl perbenzoates, t-butyl perbenzoates, i-propyl perbonzoate and n-propyl perbenzoates), the dialkyl peroxydicarbonates or the alkyl groups containing 1 to 8 carbon atoms, preferably 3 to 4 carbon atoms (especially the diisopropyl peroxydicarbonate), the cumene-hydroxyperoxide, the azo-bis (isobutyronitrile), the 2,2'-azo-bis ( 2,4-dimethylvaleronitrile), the 2,2'-azobis (2-amidinopropane) and their possible addition salts, such as. As the chlorohydrates and their analogs. Among the producers of oxidation-promoting free radicals, those having a kinetics or reaction rate of order 0 (the release of the free radicals is constant over time) or better of order 1 are preferred (the release of the free radicals is linear in time). The inventively preferred producers of oxidation-promoting free radicals on the one hand, the 2,2'-azo-bis (2-amidinopropane) dichlorohydrate, which has a kinetics of order 1 in an aqueous medium and on the other hand, the 2,2'-azo-bis (2 , 4-dimethylvaleronitrile), which has a kinetics of order 1 in oily or organic medium. The release of the free radicals starting from a generator of free radicals is carried out according to a method known per se, for. By heat, light (especially the visible spectrum light or UV rays), protons, electrons, X-rays; Preferably, this release is conducted by photons or heat. For example, ranges the fact that 2,2'-azo-bis (2-amidinopropane) -dichlorhydrat to bring, in aqueous solution at the temperature of 37 ⁰ C to the release of free radicals after the reaction oxydationsfördernden

Cl ^- H ₂ N ⁺ CH ₃ CH ₃ ⁺ NH ₂ Cl "Cl ^- H ₃ N ⁺ CH ₃

CCN = N- CC 2 CC ♦ + N ₂

/ I 1 \ Δ ^/ \

H ₀ N CH ₀ CH, NH ₅ , H ₀ N CH,

... c. dot: c- ο

auszulosen.

The cell material I according to the invention comprises cells of human, animal, plant or synthetic origin or fragment of said cells, for example the cell walls.

Preferably, a material I is used which contains a colored or fluorescent marker which is released under the action of the free radicals.

In the case of the cell material I according to the invention, in particular pigmented living cells of human, animal or plant origin are used, d. h., That they contain a pigment or a dye, such as hemoglobin, chlorophyll, xanthophyll, carotene or anthocyanins whose release in the cell resolution is detectable, especially by measuring the fluctuation of the optical density using a spectrophotometer. The recommended live cells are derived from erythrocytes of animal origin, with the red blood cells preferably being taken from warm-blooded animals such as mammals and especially humans. The selection of red blood cells is based on the following criteria:

- the blood cells, such as. Erythrocytes have a relatively short half-life (80 days for red blood cells of human origin);

- the erythrocytes have all the molecular and enzymatic protection against radicals and are therefore considered as typical representatives of the other cells of the organism; and

- The accessibility and high availability of red blood cells, especially by simple blood sampling of a few milliliters.

When the red blood cells separated from their plasma are exposed to an oxidative free radical attack, the erythrocytes utilize all their enzymatic and molecular equipment to withstand this attack until the cell membrane or wall has changed sufficiently to release the cell contents. In the case of red blood cells, this release of the said content can be readily determined by spectrophotometry by measuring the amount of hemoglobin passing into the biological medium. The defense of the erythrocyte population tested is measured either by the time to release 50% p / p of the hemoglobin contained in the blood cells (kinetic evaluation) or by the concentration of the free radical generator (CHeos) causing the 50% hemolysis ( Evaluation of dose-effect type).

The cell material may also consist of cell walls or fragments of cell walls, preferably pigmented cells. The most suitable walls are those that contain a sufficient amount of pigments or dyes that can be released upon dissolution by the free radicals.

The cell material may also consist of a synthetic cell wall. In the synthetic cell walls, the substances containing liposomes in which a color-marking agent is enveloped, fixed or immobilized, which can be released upon dissolution by the free radicals, are preferred. The liposomes and membrane substances containing such colored markers are particularly advantageous since it is thus possible to avoid looking for healthy human, animal or plant test subjects for the control experiments and thus to ensure a better standardization of the determination method according to the invention.

According to the invention, the cell material preferably to be used should consist of red blood cells, better still of liposomes which conceal, fix or immobilize a color marker, which can be released in the course of dissolution by the free radicals. The tested free radical resistance is then determined either by the time to 50% dissolution, i. H. the time to release 50% p / p of the color marker (kinetic measurement), or expressed by the concentration of the producer of free radicals that induce a 50% dissolution (measurement dose-effect). A cell material contaminated by a potentially aggressive agent II is understood to be a cell material I as defined above which has been brought into contact with said agent II for at least 0.5 hours or at least 0.5 hours before the start of the method Invention has been exposed to the effect of said agent Il.

The bringing together of said cell material I with said agent II may consist in the introduction of the agent II into the living organism and then in the recovery of the pigmented cells contaminated by this agent II in order to use the method according to the invention. A contact may also be made which is incorporated into the cells of the material I in the agent II by a method known per se, either by injection (by a similar in vitro fertilization technology) or by osmosis.

The potentially aggressive agent II may be of a physical nature (irradiation with X-rays, beta rays, protons, etc.) or chemical nature (substances to be tested, reference substances, metabolites, etc.), but also of a physico-chemical nature (tobacco smoke, especially the emission free radicals implied by pyrolysis).

The biological experimental environment, which is an aqueous-liquid medium, contains the cell material I previously contaminated by the agent II and a free-radical generator and optionally one or more additives in the field of cell cultures and biological provisions are in use, especially a preservative. The agent II which contaminates said liquid experimental environment is introduced either alone or as such into said biological medium containing the cell material and the free radical generator at least 0.5 hours prior to the initiation of the free radical release reaction, or previously with the said cell material after contamination brought together. Contamination may result from the (deliberate or accidental) administration of said agent Ii (or one of its precursors) to the organism to which cells are taken or from the in vitro absorption of said agent II through the cell membrane.

In the case of living cells, as a result of contamination by administration to the organism, the cells contain said contaminant or at least one of its metabolites in its cell content and / or in the region of its membrane. In the case of contamination of living or synthetic cells by absorption, most of the contaminating substance is fixed in and / or on the cell wall. The contamination by adsorption is advantageously carried out by lipophilic contaminating substances. The adsorption in the region of the cell wall can be effected by incubation of the cell material and the contaminating substance previously diluted in a selective solvent in a suitable medium and at a suitable temperature; depending on the duration of incubation and concentration of the cell material used, the entire contaminating substance of the incubation medium or (as is often the case) only a part of this substance is fixed by adsorption in the natural or synthetic cell walls.

If the agent II is a chemical substance, the latter can be any substance to be tested. It may be, in particular, an oxidising substance, an anti-oxidant substance, a composition or combination of products, or a metabolite. Among the substances that can be tested and / or determined are the pigments (especially the flavonoids), the proteins, the enzymes, the peptides, the amino acids, the antibodies and the antigens and, more generally, all the products on which Basis antibodies can be produced. The substances which can be tested and / or determined include, in particular, the products or agents which are active in the organism and / or in the cells.

For application of the determination method according to the invention, the isolated and washed cell material is suspended in a liquid, biological, preferably isotonic, medium. It is at a temperature in the order of 10 to 60 ° C, preferably at 15 to 4O ⁰ C, and more preferably vcn at a temperature of 37 ° C in the presence of a free radical generator at a concentration 10 to 20% p / v Cell material incubated. The best mode of using the method of the present invention is to suspend either a red blood cell or synthetic tar wall fragment containing liposomes and a releasable color marker in a physiological, isotonic serum, which may have been buffered, and at 37 ⁰ C in the presence of 2,2'-azo-bis (2-amidinopropane) dichlorohydrate at a concentration of 10OnM for a final volume of 2 ml (preferably) aqueous-liquid, biological medium or of 2,2'-azo-bis (2,4-dimethylvaleronitrile) in the same proportions for an organic, biological, liquid medium to incubate. The release of the free radicals, starting from the producer of free radicals, is preferably initiated by photons and better by a thermal shock (in a special case, a heating to 37 ⁰ C). At regular intervals (for example every 20 minutes), samples are taken (0.02 ml) until the cell residue has disappeared (this generally corresponds to a duration of 150 to 600 minutes, and in particular 200 to 300 minutes); Each sample is diluted with 1 ml of physiological serum and centrifuged (eg at 3000-900 Og for 10-60 seconds, preferably at 3000-600 Og). Excessive centrifugation, especially above 9000 g, can interfere with the measurement by inducing mechanical lysis )); a remainder portion (0.2 ml) of the supernatant thus recovered is then added to the wells of a microtest plate or a group of microcuvettes for reading the optical density by spectrophotometry (especially at 350-600 nm, regardless of the source of the pigmented cells or the synthetic walls, and especially at 405-410 and 540 nm if the cell material is red blood cells).

In practice, the results of variations in optical density are expressed in% relative to the maximum resolution of the cell material (100%). A theoretical curve oriented at the experimental points makes it possible, in particular, to obtain the time corresponding to a 50% dissolution (this time is the better, the better the red blood cells or the synthetic walls are the oxidative stress in vitro, among those mentioned above Conditions), the slope of the sigmoid peaks and the latency (determined by the tangent of the inflection point of said sigmoid).

The above-described kinetic evaluation can be replaced by a dose-effect type evaluation. The method of determination according to the invention is very simple to use and may take the form of a kit, a bag or a kit of determination kits either for diagnostic purposes or for "screening" studies of different molecules and their metabolites, especially the molecules which have an oxidative or antioxidant activity and on the other hand The method of determination can also be carried out on human or animal plasma to evaluate the influence of said molecules on said plasma, which then constitutes agent II in such determinations is of particular interest for (i) the determination of anti-malarial agents, which are in general substance that reduce the resistance of erythrocytes and liposomes to free radicals, (ii) for the diagnosis of diabetes, as soon as disease in the organism hypo-oxidative e) and (iii) to evaluate the influence of food or food additives, in particular the colorants on the organisms.

This determination method has the advantage that it can be carried out in very short periods, preferably in 5 hours or faster.

When using liposomes as cell material according to the invention, the following liposome preparations are proposed.

Preparation A:

Particles containing a combination of liposomes, a colored or fluorescent marking agent released under the action of free radia, and a binder which ensures the cohesion of each particle.

Preparation B:

A liposome matrix consisting of a combination of liposomes, a colored or fluorescent marking agent which can be released under the action of free radicals, and a binder which ensures cohesion of the matrix.

Preparation C:

Inert substrate to which is adhesively bonded a liposome layer constituting the matrix corresponding to the above-mentioned Preparation B.

Preparation D:

An inert substrate comprising on at least one of its faces at least one region coated with a colored or fluorescent marking agent which can be released under the action of free radicals, said surface and said zone being coated with a layer consisting of a mixture of liposomes and dehydrating agent which ensures the cohesion of said layer; here the liposome layer masks the zone covered with the colored or fluorescent marker.

Preparation E:

An inert porous substrate containing a colored or fluorescent marking agent released under the action of free radicals and supplied by impregnation, said substrate being coated with a liposome layer similar to that of Preparation D, which comprises the substrate masked said marker.

Prsparat F:

Liposome matrix corresponding to preparation D sensitized to the surface by a chromogenic marker known from the field of chromogenic peptidic substrates (see EP-A-0280610); when exposed to free radicals, fragments or debris of liposomes containing said label are separated from the matrix, these fragments or debris of the matrix are isolated and collected by filtration or centrifugation, then said fragments or debris are placed in a suitable biological environment brought back into suspension to develop the dyeing according to a known method (see above-mentioned document EP-A-0280610).

In the above-mentioned Preparations DE, the colored or fluorescent labeling agent which can be released under the action of free radicals is physically masked by the liposome matrix, the thickness of which must be relatively low in order to readily access the said marker upon exposure to the free radicals to have. The preparations AC can either be a purely physical masking of the colored or fluorescent agent for labeling, which can be released under the action of free radicals, or a combination of said marker with the more highly processed liposomes, such as fixation, Immobilization or sequestration.

Finally, a bag, kit or set is recommended for determination containing the cell material (i) of the invention and optionally the free radical generator and / or the liquid biological dilution media.

embodiment

The invention is explained in more detail below with reference to some examples.

Upon reading the following embodiments, further advantages and features of the invention will become apparent. All these elements are by no means limitative but are given by way of example for illustration. Examples 1-9 concern the kinetic determinants and Examples 10-15 dose-effect type determinations.

example 1

In order to quantify the method of the present invention, the influence of the concentration of 2,2'-azo-bis (2-amidinopropane) dichlorohydrate on rat red blood cells was examined.

After removal and washing, the rat erythrocytes are suspended in isotonic physiological serum (hematocrit 15% p / v). These red blood cells are contacted with the 2,2'-azo-bis (2-amidinopropane) dichlorohydrate in doses of OmM (control), 25 mM, 50 mM and 10 mM, the final volume of the aqueous-fluid biological medium being 2 ml , The release of the free radicals is initiated by heat by the reaction medium is heated to 37 ⁰ C. Extracts of 0.02 ml are taken every 20 minutes for 200 to 240 minutes, each sample being diluted in 1 ml of physiological serum and centrifuged (15 seconds, 4000 g). A no residue rising part (0.2 ml) of the

Supernatant is then transferred to the wells of a microtest plate by spectrometry to read the optical density (especially at 540 nm).

The results shown in Figure 1 are expressed in terms of time (in minutes) corresponding to 50% hemolysis (t _M %). The curve (a) refers to the concentration of 10 mM 2.2'-azo-bis (2-amidinopropane dichloride hydrate and indicates a value t _MS = 158 minutes, curve (b) refers to the concentration of 5OmM 2 , 2'-azo-bis (2-amidinopropane) dichlorohydrate and indicates a value t _W s = 176 minutes, curve (c) refers to the concentration of 25mM 2,2'-azo-bis (2-amidinopropane) dichlorhydrate and indicates a value t _M % = 214 minutes and curve (d) refers to the control in the absence of 2,2'-azo-bis (2-amidinopropane) dichlorohydrate.

The results of Figure 1 illustrate the dose-response ratio in cell dissolution relative to the free radical generating molecule.

Example 2

This example relates to the action of an antioxidant agent in the special case of ascorbic acid.

The procedure is as described in Example 1 above using an aqueous-liquid biological medium (physiological serum) containing rat erythrocytes, 10 mM 2.2'-azo-bis (2-amidinopropane) dichlorohydrate and 0 (Absence of the antioxidant) and 0.1 mM ascorbic acid, respectively, wherein the release of the free radicals is initiated 0.5 hours after contacting the ascorbic acid with the red blood cells and the free radical generator.

The results shown in FIG. 2 show that in foals of ascorbic acid (curve [a]) the value t _M % is equal to 158 minutes and that in the presence of 0.1 mM ascorbic acid (curve (b)) the value t _M% is equal to 244 minutes. In other words: the presence of an antioxidant, such. As ascorbic acid, delays the cell dissolution, which is caused by the free radicals.

Example 3

This example relates to the action of an antioxidant agent taken up in the membrane by the rat erythrocytes, namely butylhydroxytoluene [abbreviated to BHT; systematic nomenclature: 2,6-di- (1,1-dimethylethyl) -4-methylphenol.

The procedure according to the experimental conditions described in Example 1, wherein red blood cells of healthy rats (control) erythrocytes previously at 37 ⁰ C for 0.5 h in the presence of BHT and its solvent, the ethanol (for the absorption of BHT in the cell membrane of Blood cells), which are then pre-incubated, which are then resuspended to perform the determination. The red blood cells thus treated are introduced into the physiological serum of Example 1 and combined with 10 mM of 2,2'-azo-bis (amidinopropane) dichlorohydrate, and the resulting reaction medium is then heated to 37 ° C.

The results are shown in Fig. 3, and the control curve (a) gives a value t _M % equal to 163 minutes. The curve (b), which refers to the red blood cells of healthy rats which have been previously pre-incubated with the solvent of the BHT, the ethanol at a concentration of 0.5% p / v 0.5 h at 37 ⁰ C, gives a value t «>% Equals 107 minutes. Curve (c), which refers to the same red cells as curve (b), but which were pre-incubated with BHT and ethanol under the same conditions (the test medium contains 0.03 mM BHT and 0.5% p / v ethanol ) ergibi, a value _{like that? i} t equal to 193 minutes. Comparison of curve (c) with curves (a) and (b) illustrates the protection that BHT gives to cell dissolution. In other words, the antioxidant used here causes a time delay of the free radical induced cell dissolution.

Example 4

This example relates to the comparison of erythrocytes originating from two different experimental subjects.

These red blood cells are resuspended after separation and washing at the same concentration to be exposed to the action of the free radicals derived from a free radical generator which in the particular case is 10OmM 2,2'-azo-bis (2 -amidinopropane) dichlorohydrate.

The results are shown in FIG. The curve (a) represents the hemolysis percentage of blood cells of an adult smoker and gives a value t _M % equal to 84.4 minutes. Curve (b) represents the percent haemolysis of an adult non-smoker and gives a value t _60% equal to 93.3 minutes. The comparison of curves (a) and (b) shows that in the smoker the normal defense against the attack by free radicals is reduced compared to non-smoking subjects.

Example 5

This example relates to the use of plant cells to evaluate the effect of radiation on the oxidative state.

Chamomile cells were divided into two lots, one of the lots is exposed to radiation with a radioactive source of 3.7 χ 10 ^e Bq (100 microcurie), then the irradiated and unirradiated lot are incubated for 7 months in an inert atmosphere (nitrogen or argon). kept. Thereafter, the cells of the two lots are suspended in aqueous-liquid, organic medium and then reacted with a free radical generator, the 2,2'-azo-bis (2-amidinopropane) dihydrochloride 0.5 h at 15 ⁰ C in contact. The release of the free radicals is initiated by heating to 37-4O ⁰ C, then according to the conditions of Example 1, the withdrawals and analyzes are made. The kinetic analysis of the dissolution of the cell material shows that the previously irradiated cells have a lower resistance to the free radicals than the unirradiated cells. The corresponding curves are similar to those in FIG. 3 (the kinetics of the resolution of the irradiated cells or of the non-irradiated cells, approximately the course of the curves a or b or c of the aforementioned FIG. 3).

Examples

A batch of chamomile seeds is irradiated with a radioactive source of 3.7 χ 10 ⁶ Bq, and this lot, like a non-irradiated lot, is stored for 8 months in an inert atmosphere. Then, the irradiated grains of unirradiated seeds are ground, and the resulting crushed particles are brought into contact with the rat erythrocytes suspended in tin physiological serum for 1 hour at 15 ° C. Thereafter, the free radical generator, the 2,2'-azo-bis (2-amidinopiopan) dichlorohydrate introduced and as indicated in Example 1 procedure. Examination of the kinetics of the dissolution of the blood corpuscles in the presence of crushed particles of irradiated or unirradiated chamomile seeds gives curves which are approximately similar to the curves a and b or c of Figure 3, respectively.

It can be seen that the enzymatic molecular material of the seed grains was changed by the radiation and in some cases was partially destroyed.

This Example 6 and Example 5 above illustrate that the method of the invention can be used to evaluate the quality of food of animal and / or vegetable origin to determine if the food to be tested has undergone more or less degradation before its consumption ,

Example 7

The procedure is as described in Example 3, wherein the BHT is replaced by phenothiazine. It is noted that the phenothiazine has an unexpected antioxidant activity in the sense that it retards the free radical initiated dissolution of red blood cells.

Examples

The procedure is as given in Example 7, wherein the erythrocytes are replaced by a synthetic cell material containing liposome according to the above-mentioned preparation C. It is as in Example 7 to observe the antioxidant effect of phenothiazine,

Example 9

It is prepared by extraction of dried flats with water, which has been previously brought to a boil, and lyophilization of the resulting filtrate, a tea lyophilizate, one lot is exposed before the lyophilization of ionizing radiation (5OkGy) and the Kontrollos is not irradiated. Then both lots are stored for 9 months in a vacuum.

Now proceed as in Example 3, wherein the BHT is replaced by the irradiated Teelos or the unirradiated comparison lot. It should be noted that the irradiated lot has less resistance to the free radicals than the control lot.

Example 10-15

The following Examples 10-15 illustrate the measurement of antiradical activity under the conditions of dose-response determination.

Growing aliquots (20 to 30 mM) from the free radical generator [2,2'-azo-bis (2-amidinopropane) dichlorohydrate], dissolved in an aqueous medium (water or physiological serum), are placed in test tubes and then lyophilized , After resolubilization of the stated amounts of free radical generators in a constant volume of physiological experimental serum, which optionally contains a substance to be investigated, the cell material according to the invention is added in a constant amount.

The appropriate test media are incubated at 37 ^° C. for a fixed period of time (2.5 h). Then, the effect of the free radicals is evaluated by the cellulase solution.

In Example 10, crude blood cells of healthy rats were processed and found to be 108.0 ± 20mM / L in the concentration of the free radical generator (CH _MS ) causing 50% dissolution of the red blood cells.

In Example 11, the same red blood cells of healthy rats were used, which were also used in Example 10, the experimental medium also containing 10 mM Mannitol. It is noted that the CH ₆ Ov is 155.6 + 6.1 mM / l, which confirms the antioxidant activity of the mannitol with respect to the CH ₆₀ S of Example 10.

In Example 12, the same red blood cells of healthy rats were used, which were also used in Example 10, wherein the test medium also an oxidizing agent, the bis (dimethylanide) of azo-dicarboxylic acid (it is an oxidizing compound of the thiols) in a Dose of 25OmM. It is noted that the said ρ oxidizing agent renders the red blood cells more susceptible to the action of the free radicals to decrease the erythrocyte glutathione, lowering the CH ₆ Os to a value of 60.8 ± 8.1 mM / l (compared to the value from Example 10).

In Examples 13-15, similar results to those of Examples 10-12 were obtained wherein the red blood cells were replaced by a synthetic cell material containing liposomes corresponding to the above-mentioned Preparation C.

Claims (11)

1.3 the cell dissolution is evaluated by the free radicals with respect to a control sample containing said non-contaminated cell material. 1.2 the release of free radicals is induced by the producer of free radicals, and 1.1 in a suitable, liquid, biological medium, a free radical generator and a cell material are brought together that from the complex of a) human, animal and plant cells b) fragments of said cells and c) synthetic walls and their fragments containing liposomes wherein said cell material has previously been contaminated by a potentially aggressive agent; A method for the determination or evaluation of the antioxidant activities of a living organism or a potentially aggressive agent, said method comprising the use of free radicals as an agent for inducing the dissolution of Zelia, characterized in that 2. The method according to claim 1, characterized in that the cell material is selected from the complex formed by pigmented cells of human, animal or vegetable origin. 3. The method according to claim 1, characterized in that said pigmented cells are erythrocytes of human or animal origin. 4. The method according to claim 1, characterized in that the cell material is a fragment of synthetic cell walls. 5. The method according to claim 4, characterized in that the cell material is a Lipobomenmaterial. A method according to claim 1, characterized in that said free radical generator is selected from the complex consisting of the products which release free radicals corresponding to a kinetics of 0 to 1 kinetics. 7. An ancestor according to claim 1, characterized in that said free radical generator is selected from the complex consisting of the products which release oxidative free radicals. 8. The method according to any one of claims 1,6 and 7, characterized in that said free radical generator is the 2,2'-azobis (2-amidinopropane) dichlorohydrate. 9. The method according to claim 1, characterized in that 50 to 20OmM of the producer treier Radikaie with a Zeümateria! at a concentration of 10 to 20% p / v in aqueous biological medium. 10. The method according to any one of claims 1 and 9, characterized in that the free radicals are generated by incubation at 37 ° C. 11. Determination kit, characterized in that it contains (i) the cell material according to claim 1 and optionally (ii) the free radical generator and / or the liquid biological dilution media.