WO2008155393A1 - Use of oxygenated heterocycles chosen from xanthones and biflavonoids for the preparation of a composition intended to act as an anti-coccidial agent - Google Patents

Abstract

The present invention relates to the use of oxygenated heterocycles chosen from among xanthones and biflavonoids for the preparation of a composition intended to act as an anti-coccidial agent, wherein the coccids are selected from the group made up of Eimeria, Isospora, Toxoplasma, Besnoitia and Neospora. In particular, the composition is for use in the prevention or treatment of coccidiosis in humans or animals. Advantageously, such a composition is for oral administration. The present invention also relates to a new compound of formula (Ia), laurentinone A.

Description

 "Use of oxygenated heterocycles selected from xanthones and biflavonoids for the preparation of a composition intended to act as an anti-coccidial agent"

The present invention relates to the use of oxygenated heterocycles chosen from xanthones and biflavonoids for the preparation of a composition intended to act as an anti-coccidial agent, the coccidia being advantageously chosen from the group consisting of Eimeria, Isospora, Toxoplasma, Besnoitia and Neospora. In particular, the composition is intended for the prevention or treatment of coccidiosis in humans or animals. Advantageously, such a composition is intended to be administered orally. The present invention also relates to a novel compound of formula (Ia), laurentinone A.

The extreme variety of microorganisms, the variety of their cycles, and the need, in most cases, to resort to the specific host for their study, make microbiology a complex world, which partially explains the weakness of our current knowledge and the need for new strategies for therapeutics. Added to the many human diseases, the cost of losses due to infectious diseases in the veterinary field is considerable. In addition to high mortality in the farms, the infectious agents in question induce a growth retardation of the infected animals. To these costs must be added the necessary and costly implementation of prevention strategies. According to recent data, preventive food additives, anti-infectives and antiparasitics account for 14.3%, 17% and 27.5% respectively of the veterinary medicine market.

If our means of treatment have eradicated some infectious diseases, the main ones have not disappeared and the appearance of chemoresistance leads to the search for new molecules. This is all the more true as the main molecules must be withdrawn from the market in accordance with the new European and international legislation in terms of human toxicity.

Animal coccidiosis in particular is a serious problem in animal pathology, whether for poultry, cattle or rabbit. They are due to protozoa of the Phylum Apicomplexa, class Sporozoa. In the poultry industry, coccidiosis causes considerable economic losses (US $ 800 million per year in 2002). A dozen species parasitize the chicken. The most common are: Eimeria tenella (bloody diarrhea), Eimeria necatrix (intestinal bloody diarrhea) and Eimeria acervulina, as well as Eimeria maxima (chronic intestinal coccidiosis). The number of species for the turkey is seven, the most important of which are: Eimeria meleagrimitis (located in the jejunum) and Eimeria adenoeides (located in the lower ileum, caeca and rectum).

For these birds, the most sensitive animals are those that are growing and young adults. Infected animals, consuming less food and water, are diarrhea, and may become emaciated and dehydrated. The rate of egg production is reduced in layers. Caecal coccidiosis can produce bloody droppings and anemia, often followed by death. Intestinal coccidiosis can be confused with the hemorrhagic syndrome of anemia and other enteric diseases.

In cattle, eimerioses are caused by about fifteen species, of which the most strongly pathogenic are Eimeria bovis and Eimeria zuernii, and more occasionally Eimeria auburnensis. These are intestinal parasites (small intestine, cecum, colon), causing severe diarrhea that are haemorrhagic when the species E. zuernii is involved. Young calves aged 1 to 6 months are the most sensitive animals; however, older individuals (1 to 2 years old) may be affected. Annual losses are in the range of US $ 100 million in the US.

Coccidiosis in rabbit (rabbit) are also important. The species responsible are at the intestinal level: Eimeria intestinalis and Eimeria flavescens, and at the hepatic level: Eimeria stiedai. Diarrhea causes weight loss and mortality.

It is difficult, if not impossible, to prevent coccidiosis by hygiene alone. It is necessary to add a static coccidio drug to the diet that prevents the growth of coccidia in the digestive tract and their multiplication. Coccidiosis remains an important problem in poultry farming, despite the effective molecules present on the market (chemoresistance problem) and the launch on the market of an attenuated vaccine (Paracox). The problem is increasing due to the evolution of EU legislation on the use of additives and consumer pressure. Toxoplasma, on the other hand, is an obligate intracellular parasite responsible for abortion in ewes and women, as well as very serious problems for the fetus or the immunocompromised patient such as AIDS (deadly cerebral toxoplasmosis). The infestation is carried out by ingestion of oocysts produced by the cat or that of the meat (sheep, beef). It should be noted that in Western developed countries, 50 to 60% of humans are parasitized by toxoplasm in the form of microcysts in the muscles and brain. However, behavioral disorders have been shown both in rodents (experimentally) and in humans (analysis of psychological and psychiatric records, Nature).

Many molecules have been studied, but most often treat only symptoms due to the multiplication form or are relatively cytotoxic. It is therefore necessary to look for molecules with anti-cystic effect being non-cytotoxic.

It is important to note that the physiology and metabolism of coccidia belonging to the genera Toxoplasma and Eimeria are similar.

Beyond the preservation of animal health, the major challenge of veterinary medicine is the guarantee of men's safety. This is to prevent any transmission of microbes or drug residues by animal foods, while preventing the spread of zoonotic diseases that are harmful to human health.

Thanks to veterinary drugs, the man managed to stop or even eradicate serious diseases such as tuberculosis and foot-and-mouth disease. New products also help protect livestock against infections of all kinds. However, despite many successes, the need for new innovative medicines or food additives in veterinary medicine remains important.

The Applicant has surprisingly found that oxygenated heterocycles selected from xanthones and biflavonoids can be used as anti-coccidial agents, particularly in the prevention or treatment of coccidiosis-like parasitic diseases, such as eimeriosis. or toxoplasmosis, in humans or animals. These oxygenated heterocycles are natural compounds that can be extracted from plants, and which have the advantage of being non-cytotoxic.

In particular, xanthones are secondary metabolites found in higher plants, lichens and fungi.

Xanthones C and O glycosides have been demonstrated (Hostettmann et al., Phytochemistry, 1977, 481). Plants belonging to the family Guttiferaceae are known to develop oxygenated xanthones, and more particularly isoprenylated xanthones (Banerjii 1994). A series of isoprenyl xanthones isolated from Moraceae have been characterized and their activities highlighted (hypotensive, antirhinoviral, and antitumor effect (Hano et al 1990, Planta Medica, 478).

Xanthones have been studied from a chemotaxonomic point of view, but also for their biological and pharmacological properties (Cardona et al, 1990, Phytochemistry, 3003). It has been shown that some of these xanthones have antiviral, cardiotonic, antimicrobial, or anti-hepatotoxic properties (Banerji et al 1994). In addition, xanthones and their derivatives are bronchodilators in the treatment of asthma (Balasubramanian 1988).

However, xanthones have never been described to date as anti-coccidial agents, to prevent or treat coccidiosis, especially caused by the parasitic microorganisms Eimeria, Isospora, Toxoplasma, Besnoitia and Neospora, especially in the field veterinary.

The subject of the present invention is therefore the use of oxygenated heterocycles chosen from xanthones and biflavonoids for the preparation of a composition intended to act as an anti-coccidial agent in humans or animals, the coccidia being chosen from the group consisting of Eimeria, Isospora, Toxoplasma, Besnoitia and Neospora. In a particularly advantageous manner according to the invention, the oxygenated heterocycles chosen from xanthones and biflavonoids are used as anti-coccidial agents in animals, in the veterinary field, in particular in cattle.

By the term "xanthone" is meant in the sense of the present invention, the compounds of formula:

By the term "biflavonoid" is meant in the sense of the present invention, the compounds of formula:

or

In a particular embodiment of the present invention, the oxygenated heterocycles are xanthones corresponding to the general formula (I)

(I) in which each of the radicals R 1 to R ₆ independently represents a hydrogen atom, a hydroxyl (OH) radical, a C ₁ -C ₆ alkoxy radical, a linear or branched, advantageously C 1 to C ₆ , alkyl radical, or a linear or branched alkene radical, advantageously C 1 to C ₆ , or R 1 and R ₂ together form a ring, such as a pyranic or furan derivative, optionally substituted with at least one C 1 to C ₆ alkyl group.

According to an exemplary embodiment of the present invention, each of the radicals R 1 to R ₆ independently represents a hydrogen atom, a hydroxyl radical, a C ₁ -C ₆ alkoxy radical, such as a methoxy, or a linear alkene radical or branched, advantageously C ₁ -C ₆ , typically C ₁ -C ₅ .

Among the compounds of formula (I) of the present invention, there may be mentioned the following preferred compounds:

R ₁ , R ₂ , R 3 and R ₅ represent a hydrogen atom; R ₄ represents a hydroxy radical; and R ₆ represents an alkoxy radical, advantageously a methoxy radical.

R 1 and R ₆ represent a branched, preferably C ₁ -C ₅ , alkene radical, such as a prenyl group; R ₂ represents a hydroxy radical; R 3 and R ₄ represent a hydrogen atom; and R ₅ represents an alkoxy radical, advantageously a methoxy radical.

R ₁ , R ₄ and R ₆ represent a branched, preferably C ₁ -C ₅ , alkene radical, such as a prenyl group; R3 represents a hydrogen atom; and R ₂ and R 5 represent a hydroxy radical.

According to another embodiment of the present invention, R 1 and R ₂ together form a ring such as a pyranic or furan derivative, advantageously substituted by at least one alkyl group, typically C 1 to C ₆ .

In this embodiment, R ₃ advantageously represents a linear or branched alkene radical, advantageously C 1 to C ₆ , typically C 1 to C ₅ ; and R ₄ , R ₅ and R ₆ each advantageously represent independently a hydrogen atom or a hydroxy radical.

Among the compounds of formula (I) of the present invention, there may be mentioned the following preferred compound: R 1 and R ₂ together form a pyran ring, advantageously substituted by at least one methyl group, in particular substituted with a dimethyl gem group, typically at the 2-position; R ₃ represents a branched alkene radical, such as a 1,1-dimethyl-2-propene group; R ₄ represents a hydroxy radical; and R 5 and R ₆ represent a hydrogen atom.

Advantageously, the compound (I) has the following formula:

In another particular embodiment of the present invention, the oxygenated heterocycles are biflavonoids corresponding to the general formula (II)

(H) in which the radical R represents a hydrogen atom or a hydroxyl radical.

The xanthones and biflavonoids according to the present invention are advantageously natural compounds, extracted from plants, typically plants of African origin. In a particularly advantageous manner according to the present invention, the oxygenated heterocycles of the xanthones and biflavonoids type are extracted from plants selected from the group consisting of Vismia laurentii, Pentadesma grandifolia, Allanblackia monticola, Allanblackia floribunda, and Allanblackia gabonensis.

In the context of the present invention, it is advantageous to use as anti-coccidial agents xanthones or biflavonoids as defined above, or else extracts containing such compounds.

The xanthones and biflavonoids used according to the present invention may also have been chemically synthesized.

Advantageously, the composition according to the present invention is intended for the prevention or treatment of coccidiosis, such as eimeriosis or toxoplasmosis, in humans or animals. Particularly advantageously, the composition according to the invention is used for the prevention or treatment of coccidiosis in animals, in the veterinary field, in particular in cattle. In particular, this composition can be used to prevent or treat parasitic diseases of livestock, for example cattle, rabbits, pigs, or birds, for example poultry such as chickens or turkeys.

Advantageously, the composition of the present invention is intended to be administered orally.

The composition according to the present invention may in particular be in the form of a tablet, capsule, powder, granule, solution or oral suspension.

Advantageously, the composition of the present invention is a pharmaceutical composition, a food composition such as a food, a food or drink, a food supplement or a food additive.

The composition according to the present invention may contain acceptable excipients as well as conventional additives.

The subject of the present invention is also a novel compound corresponding to the following formula (Ia), laurentinone A:

The following figures and examples are given without limitation and illustrate the present invention.

Figure 1 represents the in vitro human cell line cytotoxicity study of the products EVL9 (concentration: 0.01 mg / mL and 0.005 mg / mL) and PG2 (concentration: 0.01 mg / mL and 0.005 mg / mL ).

Figure 2 represents the study of the in vitro cytotoxicity on human cell line of AM5 products (concentration: 5 μg / mL), AFR6 (concentration: 5 μg / mL and 10 μg / mL), AFR 7 (concentration: 5 μg / mL and 10 μg / mL), AFR8 (concentration: 5 μg / mL and 10 μg / mL), EAFR (concentration: 10 μg / mL), EFAM (concentration: 5 μg / mL and 10 μg / mL), and ERAM (concentration: 5 μg / mL and 10 μg / mL).

Figure 3 shows the antiparasitic activity of the ERPG products (concentration: 0.02 mg / mL and 0.01 mg / mL), and ETVL (concentration: 0.02 mg / mL, 0.01 mg / mL and 0.002 mg / mL), on infested cells.

Figure 4 represents the antiparasitic activity of PG2 (concentration: 0.005 mg / mL, 0.01 mg / mL) on infested cells.

Figure 5 shows the antiparasitic activity of EVL9 (concentration: 0.005 mg / mL and 0.01 mg / mL) in infested cells.

Figure 6 represents the antiparasitic activity of AM5 (concentration: 5 μg / mL), AFR6 (concentration: 5 μg / mL), AFR7 (concentration: 5 μg / mL and 10 μg / mL), AFR8 (concentration: 5 μg / mL and 10 μg / mL), EAFR (concentration: 10 μg / mL), EFAM (concentration: 5 μg / mL and 10 μg / mL), and ERAM (concentration: 5 μg / mL and 10 μg / mL), on infested cells. Figure 7 shows the growth percentages of chickens for coccidiostatic products, such as EFAM, EAGC, AM5, AFR8, and PG2, over a 26-day period. Percentages are calculated relative to uninoculated and untreated control (this control = 100% chicken growth). For the untreated inoculated control, there was 55% mortality.

Figure 8 shows the growth percentages of chickens for coccidiostatic products, such as EFAM, ERAM, ERPG, and ETVL, over a period of 48 days. Percentages are calculated relative to uninoculated and untreated control (this control = 100% chicken growth). For the untreated inoculated control, there was 55% mortality.

Figure 9 shows the mortality rate in coccidiostatic trials.

Examples:

Example 1: Preparation of compounds of formula (I): l, 5,6-trihydroxy-8- methoxyxanthone: laurentinone A (EVL9) Ri, R _2, R ₃ and R ₅ = H; R ₄ = OH, and R ₆ = OCH ₃

Laurentinone A was extracted from the plant Vismia laurentii.

Vismia laurentii is a 1.5 to 3 m shrub up to 15 m tall, found in the African forest, in general and more specifically in Senegal, Liberia, Ghana, Nigeria, Ivory Coast, Sierra Leone and in Cameroon.

Extraction:

Stems of Vismia laurentii, harvested in Cameroon, were cut, dried and crushed. The powder obtained, 3 kg, was extracted by maceration at room temperature for 48 hours in methanol (10 L). After filtration, the resulting solution was concentrated by evaporation under reduced pressure to give 150 g of a brown viscous residue which constitutes the ETVL extract. The latter is characterized on the basis of thin layer chromatography of silica gel in hexane-ethyl acetate 75:25, and revealed with UV and with sulfuric acid. Isolation:

This ETVL extract was subjected to flash chromatography on silica gel (Merck 60, 70-230 Mesh) and eluted with a hexane-AcOEt mixture of increasing polarity. 50 fractions of 250 ml each were collected and pooled on the basis of C. C. M., thus leading to 5 series called S1-S5.

Treatment of the series:

The S2 series (9 g), obtained from flash chromatography by elution with hex-AcOEt (3/1), was in turn subjected to silica gel column chromatography (70-230 mesh ) using the Hexane-AcOEt mixture as the elution gradient. A total of 105 fractions, each of 150 ml capacity, were collected and pooled on the basis of C.CM.

Fractions F53-63, obtained from fraction S2 by elution with Hexane-AcOEt (4/1), were pooled and subjected again to column chromatography on silica gel, eluted with a Hexane-HCl mixture. AcOEt of increasing polarity. 40 fractions of 15 ml each were collected. From fraction F28-33, eluted with Hexane-AcOEt (85/15), compound EVL9 precipitated at room temperature as a yellow powder (18 mg).

Structure of EVL9 or laurentinone A:

Laurentinone A was obtained in the form of an orange-yellow powder. It melts in the hexane-ethyl acetate mixture, and gives a solution in methanol a positive reaction to ferric chloride, indicating the presence within it of phenolic hydroxyls.

The empirical formula C _M H ₁₀ O _{O 6} was deduced from its positive resolution high resolution mass spectrum ESI-TOF, on which the peak of pseudomolecular ion [M + H] ⁺ at m / z 275 was observed, 0586, corresponding to the formula C ₁₄ H ₁₁ O ₆ and containing 10 degrees of unsaturation.

The UV spectrum of this compound taken from methanol has five maxima of absorption at λmax 204, 237, 266, 331 and 410, characteristic of the tetraoxygenated xanthones (Terreaux et al., 1995, Imuna et al, 1996). The presence in laurentinone A of phenolic hydroxyls, a carbonyl and a large number of sp ² carbons is corroborated by the existence on its IR spectrum of characteristic valence bands at υ3453 cm ^-1. (OH free), υ3115 cm ^-1 (chelated OH), υ664 cm ^-1 (conjugated carbonyl and chelate), υ 1,617, 1584 cm ^-1 (aromatic C = C).

Its ¹³ C NMR spectrum (75.45 MHz, DMSO-d ₆ , see Table 1) shows 14 signals due to the 14 carbon atoms present in the molecule. The analysis of these signals by means of the APT and HSQC techniques makes it possible to highlight: 9 negative phase signals, all corresponding to quaternary carbons, among which a carbonyl signal at δ 180.7, and six hybrid oxygenated carbons sp ² resonant between δ 142.6 and 160.9; 5 positive phase signals corresponding to the primary and tertiary carbons, including four other signals, all sp ² hybrids, and resonant at δ95.3; 106.9; 109.5 and 136.3 being due to tertiary carbons, and a sp hybrid with the methoxyl group and resonant at δ55.9.

The ¹ H NMR spectrum (300 MHz, DMSO-d ₆ , see Table 1), supplemented by the COSY 45 spectrum of this compound, reveals the presence of: a phenolic hydroxyl chelated at δ12.95 (1H, s) , attributable to the hydroxyl at position 1; two signals exchangeable all with heavy water at δ 10.25 and 9.75, attributable to the phenolic hydroxyls; an ABM system of three aromatic protons consisting of a triplet of a proton at δ 7.65 having a coupling constant J = 8.4 Hz and two doublets resolved by a proton each at δ 7.00 and 6 respectively; 85, with coupling constants J = 8.4 and 1.9 Hz; a singlet of an aromatic proton at δ 7.10; and a singlet of three protons at δ 3.90, attributable to the protons of a methoxyl group. Table 1: ¹³ C NMR and ¹ H NMR spectral data of laurentinone A

Type Multiplicity ° C ¹ H [Hi, J (Hz)]

1,160.9 s -

2 109.5 s 6.85 (dd, 8.4, 1.9)

3,136.30 s 7.65 (t, 8.4)

4 106.9 d 7.00 (dd, 8.4, 1.9)

4a 155.6 s -

5a 142.6 s -

5 133.3 d -

6 142.8 s -

7 95.3 s 7.10 (s)

8 146.2 s -

8a 111.3 s -

9 180.7 s -

9a 107.6 s -

1-OH 12.95 (s)

5-OH - - 9.75 (s)

6-OH 10.25 (s)

The NOESY spectrum of EVL9 clearly shows that the ABM system is located on cycle A of the xanthonic ring. The methoxyl group is located at the 8-position in the peri-position relative to the carbonyl (δ _H 3,90 / δ _x 55,9) (Terreaux et al, 1995). This is confirmed by the HMBC spectrum.

On the NOESY spectrum of laurentinone A, it is observed that the single proton of ring B is in position 7 and the two phenolic hydroxyls in position 5 and 6.

On the basis of all these data, the structure of EVL9 or 1,5,6-trihydroxy-8-methoxyxanthone was attributed to laurentinone A. This structure is described for the first time in the plant kingdom and in the literature.

laurentinone A

Example 2 Preparation of compounds of formula (I): 1,3,6,7-tetrahydroxy-2,5,8-tri (3-methylbut-2-enyl) xanthone: garcinone E (PG 2) R ₁ , R ₄ and Re = prenyl group; R3 = H; and R ₂ and R 5 = OH

Garcinone E was extracted from the plant Pentadesma grandifolia.

Pentadesma grandifolia is a tree up to 20 meters in height and 80 centimeters in diameter, with straight bole, cylindrical, without buttresses at the base, found in Africa, and particularly in Cameroon at altitudes between 800 and 1600 m.

Extraction and purification:

Bark of Pentadesma grandifolia, harvested in Cameroon, was cut into small pieces, dried, and crushed. The powder obtained, 4 kg, is macerated in 8L of methylene chloride-methanol (1: 1) cold for 48H.

Evaporation of the solvent under reduced pressure yielded 156 g of a viscous residue of yellowish color which constitutes the ERPG extract, characterized by its C.C.C. in ethyl acetate-methanol 75: 25, and revealed with UV and sulfuric acid.

Isolation:

A portion of this ERPG extract, 90 g, was chromatographed in a silica gel column using hexane-ethyl acetate mixture of increasing polarity as eluent. The 100 mL fractions are collected and pooled on the basis of TLC. This yielded nine series indexed from B1 to B9. The B5 series, consisting of fractions 85-106 eluted with hexane-ethyl acetate (7: 3), was rechromatographed in a column of silica gel, eluted with hexane-ethyl acetate (7, 5: 2.5). The fractions of 100 ml are collected. Fractions 34-65, PG2 crystallizes as yellow-orange crystals and is soluble in acetone.

Characterization of the PG2 compound:

The PG2 compound was obtained as yellow-orange crystals and melted at 134-136 ° C. It gives a positive reaction to the ferric chloride test, characteristic of phenolic hydroxyls.

Its high-resolution, positive-mode ESI-TOF mass spectrum gives a quasi-molecular ion at m / z 465.2244 (C28H33O6), corresponding to the protonated molecule [M + H] ⁺ , as well as two m / z 488 and 504 respectively corresponding to adducts [M + Na] ⁺ and [M + K] ⁺ . It is deduced that the compound PG2 has a molar mass of 464, corresponding to the empirical formula C ₂ S H ₃₂ O _O (13 degrees of unsaturation).

The infrared spectrum (IR) of this compound, recorded on a KBr tablet, exhibits, among other things, vibration bands of valencies characteristic of a free hydroxyl (υ 3460 cm ^-1 ), a chelated hydroxyl (υ 3250 cm ^-1 ) and a conjugated carbonyl (υ 1647 cm ^-1 ) Absorption bands corresponding to the valence vibration frequencies of C = C double bonds of aromatics and olefins (υ 1650) are also observed on this spectrum. cm ^"1 ), as well as those due to the CO bonds of the ethers (υ 1245, 1118 cm ^-1 ).

Its ¹³ C NMR spectrum (75.45 MHz, ₆ -acetone, see Table 2) shows 28 signals due to the 28 carbon atoms present in the molecule. The spectrum analysis makes it possible to detect 18 negative phase signals, corresponding to the secondary and quaternary carbons, among which a carbonyl signal at δ 183.2, and at least six hybrid oxygenated carbons sp ² resonant between δ 144.1 and 162.9; 10 positive phase signals corresponding to the primary and tertiary carbons, including six methyls resonating at δ 17.5, 17.7; 18.1; 25.7; 25.8; 25.9; the other four signals, all sp ² hybrids and resonant at δ93.2; 123.8; 124.9 and 125.2, being due to tertiary carbons. All this information, combined with the presence on the ¹ H NMR spectrum (300.1 MHz, acetone-dβ, see Table 2) of a proton signal at δ13.90, corresponding to a chelated hydroxyl, shows that PG ₂ is a xanthone.

On the ¹ H NMR spectrum, there is also: a signal integrating two protons exchangeable with heavy water at δ9.43 and a third at 2.80 attributable to three hydroxyl groups, a singlet of a proton at 6 , 38, attributable to an aromatic proton, and finally three sets of signals attributable to the γ, γ-dimethylallyl moieties and consisting of three δ5,28 (m, H-2 ') olefinic protons; 5.26 (m, H-2 ") and 5.25 (m, H-2 '"); three pairs of allyl protons at δ3.46 (d, J = 7.12 Hz, CH ₂ - 1 '); 3.56 (d, J = 7.19 Hz, CH ₂ -I ") and 4.20 (d, J = 7.35 Hz, CH ₂ -I '") and six methyl groups at δ 1, 68 (s). , CH ₃ -5 ', 5 ", 5'"); l, 80 (s, CH ₃ -4 '"); 1.82 (s, CH ₃ -4') and 1.86 (s, CH ₃ -4 ").

On the xanthonic skeleton, the first prenyl group occupies the C-8 position, and is in the peri position, because of the removal of these allylic protons HI "from the cone of paramagnetic anisotropy of the carbonyl, since it resonates at δ4.20 instead of δ3.2-3.40 The second group γ, γ-dimethylallyl, is in position C-2 on the basis of the long-distance correlations C, H, observed on its HMBC spectrum. effect, on this spectrum, the protons of the allyl CH ₂ in position - 1 '(δ3,46) give, inter alia, ³ J correlation tasks with the carbon resonant at δl61,5 and the carbon C-2 resonant at This position is further confirmed by the correlations observed on the NOESY spectrum between the H-1 'allyl protons and the proton of the chelated hydroxyl at position 1.

The aromatic resonant proton at δ6.38 is therefore localized in position C-4 and has both the correlation tasks ² J, ³ J with the carbons C-4a (155,2) and C-3 (163,0). the third prenyl group is in position 5.

On the basis of all these physical and spectral data, the structure of 1,3,6,7-tetrahydroxy-2,5,8-tri (3-methylbut-2-enyl) xanthone, also called garcinone E, was assigned to PG2. This compound has been described for the first time by Sakai et al. in 1993. Table 2: 13 H NMR and 1 H NMR spectral data of garcinone E

EXAMPLE 3 Preparation of Compounds of Formula (I) or (II) Extracted, Isolated and Purified from Allanblackia monticola and Allanblackia floribunda Plants, and Preparation of extracts from Allanblackia gabonensis Plant

Compound (I), wherein R 1 and R ₆ = prenyl group; R ₂ = OH; R ₃ and R ₄ = H; and R ₅ = OCH ₃ , was extracted from the Allanblackia monticola plant. This is α-mangostine (AM5). Compound (I), wherein R 1 and R ₂ together form a pyran ring, substituted by a dimethyl gem group; R3 is 1,1-dimethyl-2-propene; R ₄ = OH; and R 5 and R ₆ = H, was extracted from the plant Allanblackia floribunda. This is the macluraxanthone (AFR6).

Compound (II), in which R = H, was extracted from the plant Allanblackia floribunda. This is volkensiflavone (AFR7).

Compound (II), in which R = OH, was extracted from the plant Allanblackia floribunda. This is the morelloflavone (AFR8).

Allanblackia monticola is a tree about 20m tall and 60 cm in diameter. The barks are brown with yellow internal indentations and red outer indentations. The flowers are yellow and give off a characteristic odor around 11 o'clock in the morning. The fruits, more or less ovoid or elongated by about 12 cm, contain in addition many seeds (about 10 to 12 seeds per fruit).

Allanblackia floribunda is a tree up to 30 m tall and 80 cm in diameter with a base wheelbase, a straight, cylindrical bole, short, horizontal branches, very numerous forming a plume of dense foliage. The bark is brown, thin, scaly; the brittle slice, reddish on the outside, yellow on the inside, exudes a yellow latex. The evergreen leaves are opposite, simple, entire (12-25 x 5-8 cm) leathery, shiny above with very many parallel lateral veins, with petioles 1 to 1.5 cm long. The flowers are large (5 cm in diameter) arranged in clusters. They are white, pink or red type 5 unisexual pedicels 2.5 to 8 cm long. The fruits are elongated berries (20 to 50 x 8 to 15 cm), light brown speckled with chestnuts with 5 ribs more or less marked, hanging at the end of a peduncle leaving exuding an abundant yellow latex. Seeds (2.5 x 2 cm) numerous (40 to 100 per fruit) and brown are surrounded by a gelatinous pulp and pinkish. It contains an edible fat (boandjo butter by Yokadouma) (Vivien and Faure, 1995).

Allanblackia gabonensis is one of the Allanblackia species found in tropical Africa (Cameroon, Zaire) between 500 and 1750 m altitude.

These are barely flared trees, but sometimes with buttresses straight. Its branches are horizontal and drooping. The bark is reddish and scaly. The reddish slice exudes a yellow latex. This species has small leaves (6 to 12 cm long and 2.5 to 5 cm wide) obovate, cuspid, opposite and red hanging. It has thick pedicels 2 to 6 cm long, large white, red or pink and unisexual flowers. Its fruits, globular or ovoid, light brown, chestnut-brown, contain 10 to 12 seeds per fruit.

Bark of the trunk, roots, leaves and fruits of AHanblackia monticola, as well as roots of AHanblackia floribunda, and fruits of AHanblackia gabonensis, were harvested in Cameroon.

Extraction: Obtaining extracts EAFR, EAM, EFAM, ERAM, and EAGC

The bark of the trunk, roots and leaves of AHanblackia monticola were cut into small pieces, dried and then crushed. This resulted in 3 kg of trunk bark, 2.5 kg of leaves and 2 kg of roots, respectively. Extraction by maceration in the methylene chloride-methanol mixture (1/1) of the various powders made it possible to obtain, after evaporation under reduced pressure, 250 g of the trunk bark extract (EAM), 225 g of leaf extract (EFAM), and 115g of root extract (ERAM).

The roots of Allanblackia floribunda were cut into small pieces, dried and then crushed. The resulting powder, 5 kg, is extracted by maceration successively with CH ₂ Cl ₂ -MeOH (1: 1) for 24 hours and with pure MeOH for 4 hours. Each of the extracts is concentrated to dryness under reduced pressure using a rotary evaporator, and gives respectively 137 g of CH ₂ Cl ₂ -MeOH and 45 g of pure MeOH, which are combined on the basis of TLC. analytic to lead to a total extract (EAFR).

The fruits of Allanblackia gabonensis were cleared of their seeds, and the hulls were cut, dried and crushed. The powder (250 g) thus obtained was extracted by cold maceration in CH ₂ Cl ₂ -MeOH (1: 1) for 48 hours. After concentrating to dryness of the solvent under reduced pressure, 18 g of EAGC extract with CH ₂ Cl ₂ -MeOH were obtained. Isolation and purification of AM 5

150 g of the 250 g of raw extract of the Allanblackia monticola trunk bark are fractionated on a flash column containing 200 g of 70-230 mesh size silica. The elution is made by means of a hexane-ethyl acetate mixture of increasing polarity. The 300 ml fractions are collected and pooled on the basis of TLC. This allowed to obtain 4 indexed series A, B, C and D.

Series B, from fractions 18-39 which were eluted with hexane-ethyl acetate 7.5 / 2.5, consisted of a viscous mass of 25 g.

It is chromatographed on a silica column (particle size 70-230 mesh) and eluted with hexane-ethyl acetate mixture of increasing polarity. 95 fractions of 150 ml each were collected and pooled on the basis of TLC. Fractions 71-80, collected on the basis of thin-layer chromatography, contain 4 products, after several column chromatographies using 9/1 hexane-acetate as eluent. Thus, two compounds could be isolated in pure form, among which 15 mg of a yellow powder dissolved in acetone, indexed AM ₂ , and 300 mg of yellow crystals, indexed AM5.

Compound AM5 (α-mangostine): This compound crystallizes in the form of a yellow powder; Mp = 178-180 ⁰ C; Molecular weight 410 g / mol; Gross formula: C ₂₄ H ₂₆ O ₆ .

Isolation and purification of the AFR6, AFR7 and AFR8 compounds 150 g of the 182 g of the total extract (EAFR) of Allanblackia floribunda were subjected to flash chromatography containing 500 g of silica with a particle size of 70-230 mesh in order to and fractionated with methylene chloride, hexane-ethyl acetate 50%, pure ethyl acetate and finally 10% ethyl acetate-methanol. 20 fractions of 400 ml each were collected and pooled on the basis of TLC.

Fractions 50 to 53, eluted with hexane-ethyl acetate (9/1), precipitate in the form of a yellow powder which, after washing and filtration, gives a pure AFR ₆ indexed compound.

Series B, consisting of fractions 6 to 13 which were eluted with Hexane-AcOEt 50% and 100% pure AcOEt, consists of a viscous mass of 40 g. This series is rechromatographed in a column of silica gel, and eluted with Hexane AcOEt system of increasing polarity. The 150 ml fractions are collected and pooled under the base of the TLC.

Fractions 37 to 45 crystallized after 3 days in methylene chloride as yellow crystals which, after washing and filtration, gave an AFR ₇ -acetate-soluble compound.

Fractions 50-62 also crystallize in methylene chloride as yellow crystals which, after washing and filtration, provide an AFR ₈ indexed compound.

Macluraxanthone (AFRβ)

"Crystallizes as yellow crystals" Positive Ferric Chloride Test

- P.F. 17FC

"Gross formula C23H22O6

Volkensiflavone (AFR ₇ )

• Crystallizes as yellow crystals in methylene chloride "Positive Ferric Chloride Test

- PF 201.6-202.8 ⁰ C

• Raw formula C30H20O10

Morelloflavone (AFR ₈ )

• Crystallizes as a yellow powder in methylene chloride. Positive Ferric Chloride Test

Mp 244.6-245.8 ° C

• Gross formula C30H20O11

AFR7 (R = H) AFR8 (R = OH)

Example 4 Study of the biological activity of in vitro products

Example 4.1. Evaluation of the sulforhodamine B cytotoxicity of compounds (I) or (II) and extracts containing such compounds according to the present invention

Sulforhodamine B (SRB) is a protein-binding dye whose adsorption can be measured spectrophotometrically at 550 nm (pink color).

For this test, human fibroblast MRC5 cells (Biomérieux) were cultured at 37 ° C. in a 96-well plate (Costar), at 2.10 ⁴ cells per well, in 200 μl of MEM medium (Gibco), to which 5% of fetal calf serum were added. After 24h culture, the medium is changed and the extracts, which we want to test the toxicity, are added after being taken up in the appropriate volume of appropriate solvent (dimethylsulfoxide or water). The concentrations indicated in the figures of the present application are the final concentrations in the wells. We leave 48h in culture. The cells are then fixed by adding 50 μl of cold TCA (trichloroacetic acid) per well to the surface of the culture medium, and the plate is allowed to stand for 5 minutes at room temperature and then for 2 hours at 4 ° C.

The supernatant is then removed, and the wells are washed 5 times with 100 μl of water (the plate being emptied by turning). The plate is then allowed to dry. It can be kept dry until staining. 100 μl of sulforhodamine B (0.4% in 1% acetic acid) are added to each well. The coloration lasts 20 minutes. Sulforhodamine is then removed, and the wells rinsed 5 times with 1% acetic acid. The cell-bound staining is then solubilized for 5 minutes by adding 100 μl of 10 mM Tris Base (pH 10.5).

The optical density is read at 550 nm on a microplate reader (Bio-Rad model 550).

The result of the analysis of the cytotoxicity of the compounds EVL 9 and PG 2 appears in FIG. 1. This figure thus shows that the products EVL9 (laurentinone A) and PG2 (garcinone E), at the concentrations 0.01 mg / ml and 0.005 mg / mL, are not cytotoxic. The 20% DMSO used as a control is toxic, since more than 60% of cells are killed.

The results of the analysis of the cytotoxicity of the products AM5 (α-mangostin), AFR6 (macluraxanthone), AFR7 (volkensiflavone), and AFR8 (morelloflavone), as well as extracts containing such compounds: EAFR, EFAM, ERAM, appear in Figure 2.

It thus appears that the various products tested, namely the compounds AM5, AFR6, AFR7, AFR8, and extracts containing such compounds do not exhibit significant cytotoxicity at the concentrations used.

Example 4.2. Evaluation of the antiparasitic (anticoccidial) activity of compounds (I) or (II) and extracts containing such compounds

In order to evaluate the antiparasitic (anticoccidial) activity of the compounds and / or extracts according to the present invention, the ELISA test was used on the coccidia Toxoplasma gondii (virulent strain RH) by measurement of the β-galactosidase activity.

The test is carried out on the specific recognition of an antibody of the surface parasitic proteins of Toxoplasma gondii (T gondii), which makes it possible to evaluate the multiplication of the parasite.

The T. gondii strain used corresponds to the recombinant RH strain, a gene encoding β-galactosidase having been introduced into the genome of the parasite. MRC5 cells (human fibroblasts), confluent in 96-well plates, are infested with 2.10 ⁴ tachyzoites (200 μL) for 1 hour. After washing the wells (once with supplemented MEM), the extracts to be tested are then added at the right concentration in a volume of 200 μL.

Two controls are made: healthy cells on the one hand, and untreated infected cells on the other hand.

After lysis of the cells (48 to 72 hours), the plate is centrifuged at 200 g for 5 minutes at room temperature. Lysis is carried out with 50 μl of lysis buffer for 1 hour at 50 ^° C.

The lysis buffer contains: - 100 mM HEPES pH 8

1 mM MgSO ₄ - Triton X 100 1%

- DTT 5 mM

The lysis is checked under a light microscope. 160 μL of reaction buffer are added and the mixture is left for 5 minutes at 37 ° C.

The substrate of the enzyme is added to the wells: 40 μl of 6.25 mM chlorophenolred-β-D-galactopyranoside in solution in phosphate buffer pH 7.3.

The 0.1 M phosphate buffer pH 7.3 contains:

- Na ₂ HPO ₄ 1 M

- NaH ₂ PO ₄ 1 M pH is adjusted to 7.3.

The reaction buffer contains:

- Phosphate buffer 100 mM pH 7.3

β-Mercaptoethanol 102 mM

MgCl ₂ 9 Mm.

The reaction is carried out at 37 ° C. until a red color is obtained. Optical Density (OD) at 540 nm (greater sensitivity at 570 nm) / 420 nm is measured. This reflects the amount of β-galactosidase present in the well, which reveals the amount of parasites present in the well.

The result of the analysis of the anticoccidial activity of ERPG and ETVL extracts appears in Figure 3. The concentrations used are: 0.02 mg / mL, 0.01 mg / mL and 0.002 mg / mL. They were determined on the basis of the results of the cytotoxicity study. These are non-cytotoxic concentrations.

Thus, the effect of the ERPG extract at 0.02 mg / mL is as follows: 96% +/- 0%; 0.01 mg / mL: 74% +/- 1%; and 0.002 mg / mL: 56% +/- 3%.

The effect of the ETVL extract at 0.02 mg / mL is as follows: 95% +/- 0%; 0.01 mg / mL: 95% +/- 0%; and 0.002 mg / mL: 68% +/- 4%.

Figure 3 shows that the products are effective at the highest used concentrations.

The results of the anticoccidial activities of PG2 (garcinone E) and EVL9 (laurentinone A) products appear respectively in Figures 4 and 5.

For this study, PG2 and EVL9 products are solubilized in DMSO. The effect of DMSO solutions has been analyzed beforehand. It appears that the antiparasitic effects of plant products observed are due to the action of molecules, and not to the action of DMSO. The concentrations used for EVL9 and PG2 are: 0.01 mg / mL and 0.005 mg / mL.

It appears in these figures that the effect of the EVL9 molecule at 0.005 mg / ml is as follows: 73% +/- 4%; at 0.01 mg / mL: 86% +/- 8%.

And, the effect of the PG2 molecule at 0.005 mg / mL is as follows: 86% +/- 2%; at 0.01 mg / mL: 91% +/- 7%.

Figures 4 and 5 thus show that the products are effective at the concentrations used.

Thus, the two compounds have a strong anti-parasitic (anticoccidial) activity against T.gondii: on average 85% of development inhibition for the two concentrations tested (3 trials). ERPG and ETVL extracts also have high antiparasitic (anticoccidial) activity against T. gondii.

The results of the anticoccidial activities of the AM5, AFR6, AFR7, AFR8, and EAFR, EFAM, and ERAM extracts are shown in Figure 6. The concentrations used are: AM5: 5 μg / mL, AFR6: 5 μg / mL and 10 μg / mL, AFR7: 5 μg / mL and 10 μg / mL, AFR8: 5 μg / mL and 10 μg / mL, EAFR: 10 μg / mL, EFAM: 5 μg / mL and 10 μg / mL, ERAM: 5 μg / mL and 10 μg / mL.

Figure 6 shows that the products are effective at the concentrations used.

It thus appears that the various products and extracts have an average anticoccidial activity of between 80 and 92%.

Example 5: Study of the biological activity of the products in vivo.

The study of the biological activity of the products in vivo was carried out on batches of chicks to which were administered solutions prepared with the following products (200mL / day / chicken for 9 days):

Extracts tested:

ERPG: 0.02 mg / mL and 0.04 mg / mL

ETVL: 0.02 mg / mL and 0.04 mg / mL EFAM: 0.02 mg / mL

ERAM: 0.02 mg / mL and 0.04 mg / mL EAGC: 0.02 mg / mL and 0.03 mg / mL

Molecules tested:

AM5: 0.005 mg / mL

AFR8: 0.005 mg / mL PG2: 0.005 mg / mL

Conduct of the experiment

JO: arrival of the chicks

D12: banding, weighing, histogram of weights, rejection of extremes.

252 chickens weighing an average of 275 grams were so retained. These chickens were distributed by batch of 6 chickens in 42 cages, 18 chickens per batch (3 repetitions per batch). Each batch received the solutions prepared according to the concentrations recommended above on the basis of 200 ml / day / chicken for 9 days. The 2 control lots received only water.

J19: The chickens were weighed, and 13 lots out of 14 were infested by oocysts of sporulated coccidia. A control lot was not infested.

J25: Control of the excretion of oocysts of coccidia.

After control of oocyst excretion, the chickens were weighed, and 3 chickens were sacrificed per cage and autopsied. Lesional scores were noted.

J27: Control of excretion of oocysts.

In addition to the parasito logical criteria (ectocyst excretion, lesional scores), zootechnical criteria were also studied, namely the weight gain, the GMQ and the consumption index.

Note: All the dead chickens during the test after the infestation, were autopsied. For all these chickens, the cause of death is coccidiosis. They all had bloody diarrhea with lesional scores of +4 in the intestines.

The experiment was conducted over two periods: 26 and 48 days.

The results of the coccidio static effects of the extracts and molecules according to the invention appear in FIGS. 7 and 8.

For Figure 7, growth percentages of chickens are calculated relative to the uninoculated and untreated control (100% chicken growth) over a 26 day period. For the untreated inoculated control, there was 55% mortality.

For Figure 8, the growth percentages of the chickens are calculated relative to the uninoculated and untreated control (100% chicken growth) over a period of 48 days. For the untreated inoculated control, there was 55% mortality.

These tests showed that the different biological products tested gave satisfactory results.

Thus, for the various extracts tested, despite a more or less significant excretion of oocysts, the animals kept a normal and increasing appetite, which was de facto reflected on their body weight, and therefore their growth. Their performances are not very far from those of uninoculated and untreated controls (see Figures 7 and 8).

On the other hand, inoculated and untreated controls died of coccidiosis (bloody diarrhea, intestinal lesions), or had a considerable decrease in their food consumption (see Figures 7 and 8).

The mortality rate of chickens during coccidiostatic tests appears in Figure 9. No mortality was attributed to the tested products, which confirms their non-toxic nature.

This test makes it possible to conclude that the tested products are non-toxic for chickens and exhibit coccidiostatic activity.

Claims

1. Use of oxygenated heterocycles selected from xanthones and biflavonoids for the preparation of a composition intended to act as an anti-coccidial agent, the coccidia being selected from the group consisting of Eimeria, Isospora, Toxoplasma, Besnoitia and Neospora.

2. Use according to claim 1, characterized in that the composition is intended to act as an anti-coccidial agent in animals, particularly livestock.

3. Use according to claim 1 or 2, characterized in that the oxygenated heterocycles are xanthones corresponding to the general formula (I)

(I) wherein each of Ri to R ₆ independently represents a hydrogen atom, a hydroxyl radical, an alkoxy radical preferably Ci to C ₆ alkyl radicals, linear or branched, preferably C, to C _6, or a linear or branched alkene radical, advantageously C 1 to C ₆ , or R 1 and R ₂ together form a ring, such as a pyranic or furan derivative, optionally substituted with at least one C 1 to C ₆ alkyl group.

4. Use according to claim 3, characterized in that each of the radicals R 1 to R ₆ independently represents a hydrogen atom, a hydroxyl radical, an alkoxy radical such as a methoxy, or a linear or branched alkene radical, advantageously Ci to C ₅ .

5. Use according to claim 4, characterized in that R ₁ , R ₂ , R 3 and R 5 represent a hydrogen atom;

R ₄ represents a hydroxy radical; and R ₆ represents a methoxy radical.

6. Use according to claim 4, characterized in that R 1 and R ₆ represent a branched alkene radical, advantageously a prenyl group; R ₂ represents a hydroxy radical;

R ₃ and R ₄ represent a hydrogen atom; and

R5 represents a methoxy radical.

7. Use according to claim 4, characterized in that R ₁ , R ₄ and R ₆ represent a branched alkene radical, advantageously a prenyl group;

R3 represents a hydrogen atom; and R ₂ and R ₅ represent a hydroxy radical.

8. Use according to claim 3, characterized in that R 1 and R ₂ together form a ring such as a pyranic or furan derivative, preferably substituted by at least one alkyl group, typically C 1 to C ₆ ;

R3 represents a linear or branched alkene radical, advantageously C 1 to C ₅ , and

R ₄ , R ₅ and R ₆ each independently represent a hydrogen atom or a hydroxy radical.

9. Use according to claim 8, characterized in that R 1 and R ₂ together form a pyranic ring, preferably substituted by at least one methyl group, in particular substituted with a dimethyl group;

R3 represents a branched alkene radical, such as a 1,1-dimethyl-2-propene group;

R ₄ represents a hydroxy radical; and

R 5 and R ₆ represent a hydrogen atom.

10. Use according to claim 1 or 2, characterized in that the oxygenated heterocycles are biflavonoids corresponding to the general formula (II)

(H) in which the radical R represents a hydrogen atom or a hydroxyl radical.

11. Use according to any one of the preceding claims, characterized in that the oxygenated heterocycles are extracted from plants selected from the group consisting of Vismia laurentii, Pentadesma grandifolia, Allanblackia monticola, Allanblackia floribunda, and Allanblackia gabonensis.

12. Use according to any one of the preceding claims, characterized in that the composition is intended for the prevention or treatment of coccidiosis in humans or animals, in particular parasitic diseases of cattle, for example cattle, rabbits, pigs, or birds, for example poultry such as chickens or turkeys.

13. Use according to any one of the preceding claims, characterized in that the composition is intended to be administered orally.

14. Use according to any one of the preceding claims, characterized in that the composition is a pharmaceutical composition, a food composition such as a food or drink, or a food supplement.

15. Compound corresponding to the following formula (Ia):