Fucales-based composition for use in the oral treatment of helminthiasis, and method for obtaining a corresponding preparation

Abstract

The invention relates to a preparation for pharmaceutical, veterinary or food use containing at least one alga and / or at least one extract from an alga belonging to the Fucales order for use as an anthelmintic in the oral treatment of helminthiasis caused by organisms in the Rhabditina and / or Spirurina suborder which are resistant to anthelmintics belonging to the groups of macrocyclic lactones, benzimidazoles, imidazothiazoles and / or tetrahydropyrimidines, to the use of such a preparation to treat, control and prevent helminth infections in a warm-blooded animal or a human being, and to a method for obtaining such a preparation.

Description

[0001]Composition for use in the treatment of oral helminthiasis based on fucales, and method for obtaining a corresponding preparation 1. Field of the invention The field of the invention is that of antiparasitic treatments, in particular against gastrointestinal nematodes. More specifically, the invention relates to a composition for pharmaceutical, veterinary or food use for use in the treatment of oral helminthiasis, and a method for obtaining a corresponding preparation. 2. State of the Art Gastrointestinal nematodes are a major concern for human and animal health worldwide. In livestock, these parasites affect the health of animals by causing inappetence, diarrhea, anemia and, in severe cases,death. Parasitism also harms productivity and causes significant economic losses in animal production. In the absence of effective alternative strategies, the control of parasitic nematodes affecting human or animal health relies primarily on the use of chemical anthelmintics. However, their intensive and sometimes inappropriate use has led to the development of resistance to anthelmintics. Therefore, there is an urgent need to find alternative solutions for sustainable helminth control. To prevent the emergence of resistance to anthelmintic drugs, a growing number of studies are now focusing on naturally occurring compounds that can control gastrointestinal nematode populations. Recent studies have shown the strong potential of algae through their anti-inflammatory, antimicrobial,anticancer but also antiparasitic. It has been known in Japan for many centuries that the red alga Digenea simplex acts as a powerful dewormer for humans and is effective for the treatment of ascariasis. A disadvantage of the red alga Digenea simplex is that its distribution area is limited. Furthermore, there is no effective culture system for Digenea simplex. Finally, the effectiveness of Digenea simplex has only been demonstrated against Ascaris lumbricoides. We also know of Santos, FO, APM Cerqueira, A. Branco, MJM Batatinha and MB Botura. "Anthelmintic activity of plants against gastrointestinal nematodes of goats: A review." Parasitology 146 (2019): 1233-46,results of the antiparasitic potential of certain organic extracts of algae on gastrointestinal nematodes of goats. There is therefore a need to be able to propose alternative compositions containing algae in particular that are effective against different species of gastrointestinal nematodes that affect other mammals. We also know from the article Nour El-Deen, A. and A. Issa. "Nematicidal properties of some algal aqueous extracts against root-knot nematode, meloidogyne incognita in vitro." Egyptian Journal of Agronematology 15 (2016): 67-78, that aqueous extracts of macroalgae have an anthelmintic potential on the plant-parasitic nematode Meloidogyne incognita. This study showed that different species of macroalgae have an activity on the inhibition of egg hatching and the mortality of juvenile larvae. In addition to the fact that these results concern a plant parasite, and not a gastrointestinal nematode,it should be noted that the effective concentrations reported in this study are well above 5g / L. 3. Objectives of the invention The invention aims in particular to provide an anthelmintic treatment which is effective both against nematode parasites of the suborders Rhabditina and / or Spirurina, also known as nematode parasites of the orders Strongylida and Rhabditida of the classification of the National Museum of Natural History, sensitive or resistant to anthelmintics known in veterinary or human medicine, and which also preserves the health of the infected host and the environment. Another objective of the invention is to provide such a technique which is simple to implement and inexpensive. 4. Statement of the invention The subject of the present invention is a composition for pharmaceutical, veterinary or food use containing at least one alga and / or at least one extract of an alga, said alga belonging to the order Fucales,for use in the oral treatment of helminthiasis caused by organisms of the suborder Rhabditina and / or Spirurina, and in particular caused by organisms of the family Ascarididae, Heligosomatidae or Trichostrongylidae, and even more particularly caused by isolates of organisms of the species Haemonchus contortus, Heligmosomoides polygyrus and Ascaridia galli, said organisms being resistant to anthelmintics belonging to the groups of macrocyclic lactones, such as ivermectin, moxidectin and eprinomectin, benzimidazoles, such as thiabendazole, imidazothiazoles, such as levamisole and / or tetrahydropyrimidines, such as pyrantel. The inventors have in fact discovered, surprisingly and unexpectedly,that the algae and / or algae extracts of the order Fucales prove effective against zooparasitic organisms of the nematodoid type of the suborders Rhabditina and / or Spirurina sensitive or resistant to known anthelmintics based on macrocyclic lactone, benzimidazole, imidazothiazole and / or tetrahydropyrimidine at the egg stage, the larval stage and / or the adult stage and in particular that they can inhibit the hatching of eggs, kill larvae, inhibit the development of larvae and / or affect the survival of adult individuals. It should be noted that for the purposes of the invention, the nematode suborders Rhabditina and Spirurina refer to the most commonly used classification, and correspond to the suborders covered by the orders Strongylida and Rhabditida of the classification newly proposed by the National Museum of Natural History. For the purposes of the invention,the term "algae extract" means an algae juice obtained by pressing fresh algae, possibly with the addition of additives, or a substance obtained from fresh, dried or frozen algae by any known process of assisted or unassisted extraction, solid-liquid separation, fractionation, or concentration. It should also be noted that the invention is not limited to a composition comprising a single species of algae, several species of algae of the order Fucales being able to be incorporated into the composition. In a particular embodiment of the invention, said algae belongs to the Sargassaceae or Fucaceae family. According to a particular embodiment of the invention, said algae is of the genus Sargassum, Bifurcaria, Cystoseira, Pelvetia, Fucus, or Ascophyllum. According to a particular aspect, said algae of the genus Sargassum is of the subgenus Bactrophycus. In a particular embodiment of the invention,said alga is of the species Bifurcaria bifurcata, Sargassum muticum or Pelvetia canaliculata. According to a particular aspect of the invention, said extract is an aqueous extract of an alga or an organic extract obtained from an aqueous extract of an alga or an organic extract obtained by direct extraction of fresh or dried alga. Advantageously, a composition as described above is effective against zooparasitic organisms of the nematodoid type of the suborder Rhabditina or Spirurina and in particular organisms of the order Ascarididae, Heligosomatidae or Trichostrongylidae and even more particularly against organisms of the species Haemonchus contortus, Heligmosomoides polygyrus and Ascaridia galli, so that it inhibits the hatching of eggs, causes the death of larvae, inhibits development at the larval stage and / or acts on the survival of adult individuals. According to a particular aspect of the invention,said algae is in dried or frozen form and said algae extract is obtained from dried or frozen algae. It should be noted that the dried algae may be obtained by drying in the open air, for example in the sun, or in an oven or by freeze-drying. In another embodiment of the invention, the algae used in the composition or the algae from which the algae extract is derived may be fresh algae. Preferably, a composition as described above comprises at least 0.1% by mass of said algae relative to the total mass of said composition by wet weight. Preferably, a composition as described above comprises at least 0.01% by mass of said algae extract relative to the total mass of said composition by wet weight. In an advantageous embodiment of the invention,said algae was collected on the foreshore or at sea during the months of March to August and in that said algae extract is obtained from algae collected on the foreshore or at sea during the months of March to August. This collection period is particularly preferred for algae collected or cultivated in the northern hemisphere. According to a particular aspect of the invention, a composition as described above is effective on isolates of organisms of the suborder Rhabditina, and in particular on isolates of organisms of the order Trichostrongylidae, said organisms being sensitive or resistant to anthelmintics belonging to the groups of macrocyclic lactones, such as ivermectin, moxidectin and eprinomectin, benzimidazoles, such as thiabendazole, imidazothiazoles, such as levamisole and / or tetrahydropyrimidines, such as pyrantel. The invention also relates to a composition for pharmaceutical use,veterinary or food product containing at least one alga and / or at least one extract of an alga, said alga belonging to the order Fucales, for use in the oral treatment of helminthiasis caused by organisms of the suborder Rhabditina and / or Spirurina, and in particular caused by organisms of the family Ascarididae, Heligosomatidae or Trichostrongylidae, and even more particularly caused by isolates of organisms of the species Haemonchus contortus, Heligmosomoides polygyrus and Ascaridia galli, said organisms being sensitive to anthelmintics belonging to the groups of macrocyclic lactones, such as ivermectin, moxidectin and eprinomectin, benzimidazoles, such as thiabendazole, imidazothiazoles, such as levamisole and / or tetrahydropyrimidines,such as pyrantel. The invention also relates to the use of a preparation for oral administration comprising a composition as described above for controlling and preventing helminthiases caused by organisms of the suborder Rhabditina and / or Spirurina, and in particular caused by organisms of the family Ascarididae, Heligosomatidae or Trichostrongylidae, and even more particularly caused by isolates of organisms of the species Haemonchus contortus, Heligmosomoides polygyrus and Ascaridia galli, said organisms being sensitive or resistant to anthelmintics belonging to the groups of macrocyclic lactones, such as ivermectin, moxidectin and eprinomectin, benzimidazoles, such as thiabendazole, imidazothiazoles, such as levamisole and / or tetrahydropyrimidines, such as pyrantel,in a warm-blooded animal or a human being. The invention also relates to a method for obtaining a preparation for pharmaceutical, food or veterinary use for the oral treatment of helminthiasis caused by organisms of the suborder Rhabditina and / or Spirurina, and in particular caused by organisms of the family Ascarididae, Heligosomatidae or Trichostrongylidae, and even more particularly caused by isolates of organisms of the species Haemonchus contortus, Heligmosomoides polygyrus and Ascaridia galli, said organisms being sensitive or resistant to anthelmintics belonging to the groups of macrocyclic lactones, such as ivermectin, moxidectin and eprinomectin, benzimidazoles, such as thiabendazole, imidazothiazoles, such as levamisole and / or tetrahydropyrimidines, such as pyrantel,said method of obtaining comprising the following steps: - preparing an extract of an alga of the order of fucales, preferably of the family Sargassaceae or Fucaceae, more preferably of the genus Sargassum, Bifurcaria, Cystoseira, Pelvetia, Fucus, or Ascophyllum, even more preferably of the species Bifurcaria bifurcata, Sargassum muticum or Pelvetia canaliculata; - incorporating said extract into a liquid, a paste, or a powder so as to form said preparation. 5. List of figures Other characteristics and advantages of the invention will appear more clearly on reading the following description of an embodiment of the invention, given as a simple illustrative and non-limiting example, and the appended figures among which: - figure 1 is a graph representing the percentage of inhibition of the development of H. polygyrus bakeri, from the L1-L2 stage to the L3 stage, in the presence of water (control),of ivermectin (1µM) and different extracts of Bifurcaria bifurcata (cold extraction (1) and hot extraction (2)) at 5g / L. The results come from at least three independent experiments, with n=6 per sample each time. Statistical analyses were performed using a Kruskal-Wallis test with multiple comparison test, ***p<0,001 versus control; - Figure 2 represents a dose-response curve for B. bifurcata extract 1 for development inhibition up to the L3 larval stage. The solid line represents the fitted logistic regression curve and the shaded area indicates the 95% confidence interval; - Figure 3 represents a dose-response curve for B. bifurcata extract 2 for development inhibition up to the L3 larval stage. The solid line represents the fitted logistic regression curve and the shaded area indicates the 95% confidence interval; - Figure 4 is a graph showing the percentage inhibition of H. polygyrus bakeri development, from the L1-L2 stage to the L3 stage, in the presence of water (Control), or different batches of B. bifurcata at 5g / L. The algae from France were collected in 2019 (FR-1) and 2021 (FR-2, FR-3, FR-4) and were frozen either directly (FR-2) or after 24 hours at +4°C (FR-3),or after 48 h at +4°C (FR-1 and FR-3). Results are from at least three independent experiments, with n=6 per sample each time. Statistical analyses are performed with a Kruskal-Wallis test and a Wilcoxon multiple comparison test with ns, not significant; - Figure 5 illustrates the effect of water (control), thiabendazole (TBZ) at 0.5µg / mL and B. bifurcata extract at 5g / L on H. polygyrus bakeri on the inhibition of egg hatching after 48h. Results are from at least three independent experiments, with n=6 per sample. Statistical analyses were performed using a Kruskal-Wallis test with a multiple comparison test; - Figure 6 illustrates the effect of water (control), thiabendazole (TBZ) at 0.5µg / mL and B. bifurcata extract at 5g / L on H. polygyrus bakeri on the mortality rate of larvae after hatching. The results come from at least three independent experiments,with n=6 per sample. Statistical analyses were performed using a Kruskal-Wallis test with a Wilcoxon multiple comparison test with ***p<0.001; - Figure 7 is a graph representing survival curves of H. polygyrus bakeri adults exposed to different concentrations of aqueous extract of Bifurcaria bifurcata for 6 days. The survival curves were obtained using the Kaplan-Meier method and the shaded area indicates the 95% confidence interval; - Figure 8 represents dose-response curves of H. polygyrus bakeri larval development, from the L1-L2 stage to the L3 stage, for different batches of B. bifurcata collected in France and Portugal. Both batches come from algae collected in France and frozen before extraction,and dried seaweed collected in Portugal. The different IC50 were estimated from these curves. The solid line represents the fitted log-logistic regression curve and the shaded area indicates the 95% confidence interval. ***p<0.001; - Figure 9 is a comparison, in graphic form, of the effectiveness of different extracts of B. bifurcata from France (frozen) and Portugal (dried) to inhibit the development of H. polygyrus bakeri larvae. A control with water was carried out. The aqueous extracts, the different organic extracts (heptane, ethyl acetate and butanol) and the residual aqueous fraction were tested at 1g / L. The extracts came from either the batch of frozen seaweed harvested in France or the batch of dried seaweed harvested in Portugal. The results come from at least three independent experiments,with n=6 per sample each time. Statistical analyses were performed using a Kruskal-Wallis test with multiple comparison test, with ***p<0.001; - Figure 10 illustrates the results, obtained with the alga Bifurcaria bifurcata, of the hatching and post-hatching mortality test, larval development tests and adult worm survival tests on the parasite H. contortus; - Figure 11 is a graph summarizing the results of the L3 larval development test of the species H. contortus for different algal species at a concentration of 0,5 or 5 g / L; - Figure 12 illustrates the results with a dose of 5 g / L of Bifurcaria bifurcata algal extract of the larval mortality test after egg hatching for the sensitive Weybridge strain of H. contortus and for the resistant Kokstadt strain of H. contortus; - Figure 13 shows dose-hatch curves for the sensitive Weybridge strain and for the resistant Kokstadt strain; - Figure 14 illustrates results of a migration test (MTA) on Ascaridia galli larvae obtained for algal extracts obtained from a batch of B. bifurcata algae from France and a batch of B. bifurcata algae from Portugal; - Figure 15 illustrates the biological activity of the organic fractions Heptane and EtOAc carried out on Bifurcaria bifurcata a) in terms of inhibition of larval development and b) in terms of mortality of larvae after hatching,for algae harvested in France or Portugal depending on the seasonality or the method of preparation (drying and / or freezing); - Figure 16 is a dose-response curve of the larval development of H. polygyrus bakeri larvae at the L3 stage as a function of the dosage of Pelvetia canaliculata extract; - Figure 17 is the result of the L3 larval development test of the species H. contortus for Pelvetia caniculata extracts at 5g / L (codes AM228-19 and AM229-19); - Figure 18 illustrates the results of an oviposition test showing the effect of Pelvetia canaliculata extract at different dosages on the oviposition of adult H. polygyrus bakeri worms; - Figure 19 is a graph summarizing the number of abnormal spawnings (less than 50 eggs per worm) obtained by an spawning test of adult worms of H. polygyrus bakeri for the negative control (water), for water with a NaCl concentration of 3.55g / L, which corresponds to the salinity of an algal extract at 5g / L,and for a Pelvetia canaliculata extract at 5g / L; - Figure 20 is the result of the L3 larval development test of H. polygyrus bakeri larvae for Sargassum muticum extracts at 5g / L (codes AM244-19 and AM245-19); - Figure 21 is the result of the L3 larval development test of H. contortus larvae for Sargassum muticum extracts at 5g / L (codes AM244-19 and AM245-19); - Figure 22 is a dose-response curve of the larval development of H. polygyrus bakeri larvae at the L3 stage as a function of the Sargassum muticum extract dosage. 6. Detailed Description of the Invention Hereinafter, results of in vitro tests relating to the anthelmintic properties of the algal species Bifurcaria bifurcata, of the species Sargassum muticum (family Sargassaceae) and of the species Pelvetia canaliculata (family Fucaceae) are presented as examples. It will be noted as a preliminary point that the species Bifurcaria bifurcata,the species Sargassum muticum and the species Pelvetia canaliculata have the advantage of being available in large quantities. • Test results for Bifurcaria bifurcata Collection of Bifurcaria bifurcata algae and preparation of aqueous extracts The brown algae B. bifurcata (FR-1) were collected in Roscoff (Brittany, France, 48°43'53.6''N 3°59'15.5''W) in 2019. The algae were washed in seawater and stored for 24 to 48 hours at 4°C before processing. The cleaned algae were desalinated in reverse osmosis water and stored at -20°C. To carry out the aqueous extractions,The algae were thawed at room temperature and ground using an ULTRA-TURRAX (registered trademark). The ground algae were incubated for 1 hour at room temperature and then centrifuged for 20 minutes at 10,000 g to recover the supernatant (extract 1). The residual pellet was resuspended in a volume of water equivalent to the weight of the pellet and incubated for one hour at 60 ° C in a water bath. After centrifugation at 10,000 g for 20 minutes, the supernatant was recovered (extract 2). The two extracts obtained were lyophilized separately for optimal conservation. To investigate the potential variation in anthelmintic activity related to geographical origin and process, batches of B. bifurcata were also collected in Peniche (Leiria, Portugal, 39°19'33''N 9°21'35.4''W) in 2020. Quinta Quanta / Mermaid Fragrance was the biomass supplier. The seaweed from Portugal was sent dried or frozen. It was not washed before drying,Desalting is therefore carried out during the rehydration phase with osmosis water. The extraction was then carried out in the same way as for frozen algae. All extracts were resuspended at a concentration of 15 g / L in mineral water (Volvic brand), aliquoted and stored at -20°C until use. Preparation of organic extracts The freeze-dried algal material (3 g) was solubilized in 30 ml of MilliQ water before extraction, then extracted sequentially three times with each solvent (n-heptane, ethyl acetate (EtOAC) and finally n-butanol (BuOH): (1:1, v / v)). All solvents were of analytical grade and were purchased from Carlo Erba France. These three different extracts were filtered through filter paper and evaporated to dryness, along with the residual water fraction, in a vacuum rotary evaporator (Hei-VAP Core, Heidolph) at 33°C, leading to an average recovery of 6 mg (Heptane), 34 mg (EtOAc),147 mg (BuOH) and 1.7 g (Water) of resulting dry extracts. The dried extracts were dissolved in DMSO at 100 mg / ml for a stock solution stored at -20°C and then diluted according to the required dose. Parasitic material Eggs, larval stages and adult worms of H. polygyrus bakeri were obtained from experimentally infected mice (C57BL / 6JRj) under controlled conditions. Experimental infestations, mouse maintenance and euthanasia were conducted in strict compliance with national ethical guidelines and approved by the Comité d'Ethique en Experimentation Animale (CEA VdL N°19) under protocol number APAFIS# 21504. Mice were orally inoculated with 200 L3s of H. polygyrus bakeri. Ten days after infection, fecal samples were collected for egg extraction. The collected feces were pooled,mixed with water in a mortar and filtered through different 100 µm and 35 µm filters. The eggs were collected in the 35 µm filter and mixed with kaolin to agglomerate them. After 5 min of centrifugation at 600g, the pellet was resuspended in a 360g / L NaCl solution. A second 5 min centrifugation at 900g was performed, the supernatant was discarded and the eggs were rinsed with water before being collected and counted. To collect adult worms,Mice were sacrificed by cervical dislocation and the small intestine was retrieved. This was opened along its entire length with a pair of dissecting scissors. The intestine was then placed in a filter at the top of a glass funnel connected to a collection tube. Worms migrate through pre-warmed DMEM culture medium (37°C) from the filter to the collection tube as previously described. Hatching test and post-hatching mortality test One hundred eggs (60µL) were placed in glass tubes in the presence of the seaweed extract (30µL). The tubes were placed at 21°C for 48h to induce egg hatching. The test consists of counting the number of unhatched eggs and the number of larvae released after egg hatching. The egg hatch rate is calculated (larvae / (larvae + eggs)) and the results corrected for the negative control to obtain the percentage inhibition of egg hatching, as follows: ′ ^^ ^^ ^^ ^^ ^^ ^^, ′^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ to ^^′ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ℎ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = 100 − ( ′ × 100) ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ô ^^ ^^ ^^é ^^ ^^ ^^ ^^ Among the hatched larvae, the number of live and dead larvae was also counted to obtain the percentage of post-hatching larval mortality, which is calculated as follows: 100 Larval Development Test LDA was performed by taking 7.8 ml of egg suspension, which contained approximately 1666 eggs / mL, and adding 2 ml of an E. coli suspension and 200 µl of amphotericin B. The resulting mixture was then distributed into glass tubes (80 µl per tube). The tubes were placed at 21°C for 48 hours to allow the eggs to hatch properly. At 48 hours, 20 µL of Earl's medium was added along with the algal extracts to be tested (50 µL). The tubes were placed back in the incubator at 21°C for 7 days to allow proper development of the larvae. The test then consists of counting the number of young L1 / L2 larvae compared to the number of fully developed larvae at the L3 stage.The larval development ratio was calculated (L3 / (L3 + L1 / L2)) and the results corrected for the negative control to obtain the percentage inhibition of larval development, as follows: ^^ ^^ℎ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = 100. Survival Test Adult worms were placed in 48-well plates containing 120µL of DMEM medium supplemented with antibiotics (Penicillin 100U / mL and Streptomycin 100µg / mL) and 60µL of seaweed extracts to obtain the different final concentrations (5g / L, 1g / L, 0.5g / L and 0.1g / L). Worms were placed at 37°C and the number of survivors was counted every day during the experiment. Worms were considered dead when they did not respond to contact stimulation (pricking with a worm pick). Statistical Analysis R Studio 4.2.1 was used for statistical analysis of mean, median, IC50 calculation by nonlinear regression and survival analysis using the Kaplan-Meier method. Depending on the data distribution and homogeneity of variances, the Krus-kal-Wallis test with multiple comparison test (Dunn test and Wilcoxon test) was used.Results Larval development assay (LDA) reveals anthelmintic activity of aqueous extracts of B. bifurcata on H. polygyrus bakeri The inhibition of larval development of H. polygyrus bakeri larvae by aqueous extracts of B. bifurcata was investigated using water as a negative control and 1 µM ivermectin (reference anthelmintic) as a positive control. In water, the development from the L1 / L2 stage to the L3 stage was approximately 97%, whereas the application of 1 µM ivermectin completely inhibited larval development. As an additional control, we also investigated the potential effect of salt (NaCl) to distinguish between the putative activity of bioactive compounds and the salt content of the algae. At 5g / L, the NaCl solution had no effect on larval development (similar to the control in water only).To account for a potential effect of extraction temperature on biological activity, two aqueous extracts (cold and hot extractions) were tested (Figure 1). Both 5 g / L extracts of B. bifurcata induced complete inhibition of larval development. Their respective IC50 values ​​were 0.68 g / L for “cold” extract 1 (Figure 2) and 0.97 g / L for “hot” extract 2 (Figure 3). It is important to note that the anthelmintic activity of B. bifurcata at 5 g / L was confirmed on four batches of FR-1 to FR-4 algae collected in 2019 and 2021 that were frozen either directly, after 24 h at +4°C, or after 48 h at +4°C (Figure 4). Overall, these results demonstrate that the LDA-screened algal species B. bifurcata exhibits strong, reproducible, and dose-dependent anthelmintic activity on the larval stages of H. polygyrus.Subsequently, since both the "cold" and "hot" extracts showed significant anthelmintic activity, the cold extracts of B. bifurcata, which showed the lowest IC50, were tested. Action of B. bifurcata on inhibition of egg hatching and nematicidal activity on adult worms of H. polygyrus bakeri To further investigate the anthelmintic activity of B. bifurcata on other life stages of the parasite, additional tests were carried out on eggs and adult worms. First, egg hatch tests (48 hours) were carried out using water as a negative control and thiabendazole (reference anthelmintic drug for egg hatch tests) as a positive control (Figure 5). Application of aqueous extract of B. bifurcata (5 g / L) induced a 28% inhibition of egg hatching compared to the negative control (p < 0.001).Strikingly, strong nematicidal activity was also observed on freshly hatched larvae with 67% (p<0.001) mortality (Figure 6). This suggests that B. bifurcata has dual anthelmintic activity, i.e., on egg hatching and direct nematicidal activity on freshly hatched larvae. It should be noted that the same tests were performed using longer incubation periods (72h and 96h) and the same results were obtained (data not shown). Second, the direct nematicidal activity of B. bifurcata was also investigated on adult worms via a survival test. The test lasted for 6 days, during which the survival rate of adult worms was 100% in the culture medium. The aqueous extract of B. bifurcata algae was applied at four different concentrations (from 5 g / L to 0.1 g / L; Figure 7). At the highest concentration, all worms died within 24 hours.For the 1g / L concentration, we observed a low survival rate of 10% after three days. Finally, nematicidal activity was still detectable at a concentration of 0.5g / L with a survival rate of 30% after 6 days. These results strongly support a direct nematicidal activity of B. bifurcata on adult H. polygyrus bakeri worms. It should be noted that the test was not carried out over a longer period of time because the survival rate of the worms in the control condition began to decrease. Taken together, these results clearly demonstrate that aqueous extracts of B. bifurcata algae have anthelmintic activities against the murine parasite H. polygyrus bakeri. Impact of the extraction process and / or geographical origin on the anthelmintic activity of B. bifurcata. When working with algal biomasses, it was important to study the reproducibility of the result depending on the geographical origin and extraction process.Indeed, these two factors are well known to influence the phytochemical composition of the extracts and then the biological activity. Thus, we used LDA assays to compare the anthelmintic activity of B. bifurcata from France (frozen before aqueous extraction) with batches of B. bifurcata harvested on the Portuguese coast (dried before aqueous extraction). To monitor the potential impact of combined geographical and process factors, dose-response curves were generated for both types of extracts (Figure 8). While our results confirm dose-dependent anthelmintic activities of the two batches of B. bifurcata, they also highlight a variability in efficacy (e.g. IC50 = 0.7g / L for the French frozen batch vs. IC50 = 1.5g / L for the Portuguese dried batch) which could reflect either the geographical origin of the algae and / or the impact of the drying process before aqueous extraction.To further explore the phytochemical family potentially responsible for the variability in B. bifurcata anthelmintic activity, we performed organic extractions on the 1g / L aqueous extracts obtained from batches of seaweed from France (frozen) and Portugal (dried). At 1g / L, the French aqueous extract is still highly active, whereas its Portuguese counterparts are almost ineffective, thus increasing our ability to detect the corresponding chemical component responsible for the differential anthelmintic activity. Fractionation of the aqueous extracts was performed sequentially with three distinct solvents (heptane, EtOAc, and BuOH), and their respective anthelmintic property was monitored using LDA as previously described (Figure 9). The organic extracts (heptane, EtOAc, and BuOH) and the residual aqueous fraction were tested at 1g / L.For both French and Portuguese seaweeds, heptane and ethyl acetate extracts induced complete inhibition of larval development. Surprisingly, while the butanolic extract of the French seaweed showed high efficacy on larval development (99%), its Portuguese counterpart induced only 13% inhibition, thus laying the groundwork for a potential explanation for their differential activity. Importantly, the residual aqueous fractions showed no remaining activity, indicating that the active molecules are completely recovered by organic solvents. The anthelmintic activity of the organic fractions was then further investigated using a post-hatching larval mortality assay. The organic extracts were tested at different concentrations.While BuOH extracts and residual aqueous fractions showed little impact on larval mortality even at 3g / L, ethyl acetate extracts were effective at 1g / L and heptane extracts had full activity at 0.5g / L. These results indicate that the active molecules are mainly present in the heptane extract but also in the ethyl acetate extract. Interestingly, 5 no significant difference was observed between the activities of organic extracts from French and Portuguese seaweeds. In contrast to the LDA results, there was only a low activity of the BuOH fraction of French seaweeds with respect to larval mortality. These results suggest that the molecules involved in the inhibition of larval development might differ from those 10 involved in larval mortality. The nematicidal activity of different extracts of B.bifurcata from France (frozen) and Portugal (dried) on freshly hatched larvae of H. polygyrus bakeri is reported in the summary table below. The results shown in this table are from three independent experiments, with n = 6 per 15 samples each time. The values ​​reported are the mean mortality (%) ± standard deviation. Location. E xtrait Geographical Concentration Mortality France (frozen) / (g / L) vs process Portugal (dried) Control - 0 - 0.065 ± 0.34 - France 12 ± 7.15 (frozen) Aqueous extract 3 3.1.10 -5 0.318 ± 0.74 (dried) Bifurcaria bifurcata France 100 ± 0.00 (frozen) Heptane 0.5 1 Portugal 99.8 ± 0.51 (dried) France 73.2 ± 17.90 (frozen) EtOAc 1 1 Portugal 55.3 ± 31.20 (dried) France 2.49 ± 3.42 (frozen) BuOH 3 1 Portugal 3.54 ± 4.07 (dried) Residual aqueous fraction France 3 0 ± 0.00 Not determined (frozen) Conclusions on the effect of B. bifurcata on H. polygyrus bakeri Taken together, the results obtained demonstrate that aqueous extracts of the alga B. bifurcata contain bioactive compounds with in vitro anthelmintic activity on the different life stages of the murine parasite H. polygyrus bakeri. It was thus observed that the aqueous extract of B. bifurcata is sufficiently potent to inhibit larval development on the murine parasite H. polygyrus bakeri with an efficiency of 100% at a concentration of 5g / L.This seaweed extract also showed effects on egg hatching (28% inhibition at 5g / L) and direct nematicidal activity on larvae (67% efficacy at 5g / L) and adult worms (70% efficacy up to 0.5g / L in 6 days). The results obtained with B. bifurcata were also compared with the anthelmintic efficacies reported for plant extracts. For example, the larval development test showed a very high activity of the seaweed B. bifurcata with an IC50 of approximately 0.8g / L. This activity is higher than what has been reported for the plant species Glycyrrhiza glabra, Fumaria parviflora, Urtica dioica and Myrtus communis on gastrointestinal parasites of small ruminants. Regarding the larvicidal activity, which is about 67% at 5g / L, it is comparable to what has been reported when plant extracts have been evaluated as anthelmintics.Finally, the nematicidal activity observed on adult worms is higher than what is known from the prior art. Indeed, 100% mortality of H. polygyrus bakeri adults is observed in 24 hours with an aqueous extract of B. bifurcata at 5g / L, whereas for example, on Haemonchus contortus, Eguale et al. (Eguale, T., G. Tilahun, A. Debella, A. Feleke and E. Makonnen. "Haemonchus contortus: In vitro and in vivo anthelmintic activity of aqueous and hydro-alcoholic extracts of hedera helix." Experimental Parasitology 116 (2007): 340-45) noted a mortality rate of only 29% in 24 hours with an aqueous plant extract at 8g / L and Lone et al. (Lone, B., M. Chishti, F. Bhat, H. Tak, S. Bandh and A. Khan. "Anthelmintic activities of aqueous and methanol extracts of prunella vulgaris l." Nat Prod Chem Res 5 (2017): 269) used much higher concentrations (25g / L and 50g / L) to observe an effect on adult mortality in less than 24h.However, such a comparison should be taken with caution because we cannot exclude that despite their close phylogenetic relationship, H. polygyrus bakeri and H. contortus might also show differential sensitivity to natural compounds. It was also observed that the heated extracts still exhibited biological activity, thus demonstrating that the active compounds are not affected by temperatures up to 60°C. Because it has already been reported in Michalak, I., Ł. Tuhy and K. Chojnacka. "Seaweed extract by microwave assisted extraction as plant growth biostimulant." Open Chemistry 13 (2015): that temperature can have an impact on the stability of certain molecules such as algal polyphenols, one hypothesis is that the active molecules are either present in large quantities or are not affected by temperature, thus highlighting a significant stability of the soluble anthelmintic compounds of B. bifurcata. Furthermore, a difference in anthelmintic activity was highlighted between B. bifurcata from France (frozen) and Portugal (dried). The aqueous fractions of the algae batches from Portugal have lower activity compared to their counterparts from France. However, the Portuguese batches retained approximately 30% inhibition of larval development, which tends to indicate that the active molecules were still present but in lower quantities. This hypothesis was also confirmed by comparing the mass balances of the fractionation of the two algae batches.Indeed, the BuOH fraction represented 3.1% of the overall mass for French algae while it represents only 2.6% of the Portuguese one (Table S1). In order to further investigate this hypothesis, different organic extractions (heptane, ethyl acetate and butanol) were carried out and their respective biological activity was characterized (see Figure 15). The results showed that the fractions carrying the main biological activity were those obtained with heptane and EtOAc, independently of their origin and the process used before the aqueous extractions (i.e., frozen or dried). This confirmed the presence of active molecules in the Portuguese batch of algae. It should be noted that active molecules harboring antiparasitic activity against the protozoa Trypanosoma and Plasmodium previously identified in B. bifurcata were also extracted using EtOAc. These results are in agreement with the study of Bonde et al. (Bonde, CS, L. Bornancin, Y. Lu, HT Simonsen, M. Martínez-Valladares, M. Peña-Espinoza, H. Mejer, AR Williams and SM Thamsborg. "Bio-guided fractionation and molecular networking reveal fatty acids to be principal anti-parasitic compounds in nordic seaweeds." Frontiers in Pharmacology 12 (2021): 674520) carried out on the brown algae Saccharina latissima and Laminaria digitata, showing that the organic fraction extracted with another alkane solvent (i.e., hexane) also exhibited larvicidal activity against two species of gastrointestinal parasites of sheep (Teladorsagia circumcincta) and pigs (Ascaris suum). Furthermore, the results of the different organic fractions on the larval development test also highlighted a specific action of the butanol extract.Indeed, a major difference in activity between the BuOH fraction of the French and Portuguese alga was observed (99% and 13% inhibition of larval development, respectively), suggesting that the anthelmintic properties of B. bifurcata may rely on different families of molecules. This confirms the particular interest of using aqueous extracts of B. bifurcata as anthelmintic sources because they combine a range of active compounds leading to distinct modes of action. Bonde et al. showed that the anthelmintic activity of individual algal compounds is moderate compared to the activity of the fractions from which they were obtained. In this regard, it is tempting to speculate on a putative synergistic effect between the different active compounds present in both the heptane and the BuOH extract of B. bifurcata.Results on Haemonchus contortus Results obtained with an extract of the alga Bifurcaria bifurcata (identified by the code AM242-19 in the following figures) on the gastrointestinal parasite of sheep H. contortus are presented below. Figure 10 shows the results obtained for the hatching test for a dose of 5g / L of B. bifurcata extract, using water as a negative control and Thiabendazole as a positive control, and for the larval development test (LDA) and for the adult worm survival test for different concentrations (0.5g / L, 1g / L and 5g / L) of B. bifurcata extract. At a dose of 5g / L of B. bifurcata extract, inhibition of larval development and nematicidal activity were observed on H. contortus larvae and, to a lesser extent, on H. contortus adult worms (IC50 of 4.39g / L). Figure 11 shows the results of the larval development test at the L3 stage of the species H.contortus for different algae species at a concentration of 0.5 or 5 g / L. Figure 11 shows that the Bifurcaria bifurcata algae extract (AM 242-19 and AM 243-19) at 5 g / L completely inhibits the L3 larval development of H. contortus, as do the extracts of Pelvetia canaliculata (AM 228-19 and AM229-19) and Sargassum muticum (AM244-19 and AM 245-19). Figure 12 shows the results of the larval mortality test after egg hatching with a dose of 5 g / L of a B. bifurcata extract for the sensitive Weybridge strain of H. contortus and for the resistant Kokstadt strain of H. contortus. The dose-hatch curves for this susceptible Weybridge strain (IC50=0.05 µg / mL) and for the resistant Kokstadt strain (IC50=2.31 µg / mL) are shown in Figure 13.Results on Ascaridia galli The Bifurcaria bifurcata algal extract at 5g / L showed an effect on the poultry gastrointestinal parasite Ascaridia galli on both adult mortality and egg development. The results also showed that the activity on Ascaridia galli larvae of Bifurcaria bifurcata algal extracts was substantially similar regardless of the geographical origin of Bifurcaria bifurcata, as can be seen in Figure 14, which shows the results obtained in vitro from a migration test (MTA) for Bifurcaria bifurcata algal extracts obtained from two batches of algae, one from France and the other from Portugal. • Test results on Pelvetia canaliculata Activity of Pelvetia canaliculata The anthelmintic activity of aqueous extracts of the alga Pelvetia canaliculata was also tested.It can be seen from the dose / larval development curve in Figure 16 that Pelvetia canaliculata extract (designated under the code AM228-19) has an effect on the larval development at the L3 stage of H. polygyrus bakeri larvae, with an IC50 estimated at 1.03 g / L. A near-total inhibition of larval development at the L3 stage of H. contortus larvae was also observed with Pelvetia canaliculata extract at 5 g / L (see Figure 17). It was also observed via an egg-laying test by adult worms of H. polygyrus bakeri (see figure 18) that the extract of Pelvetia canaliculata at 8g / L significantly reduced the quantity of eggs produced by the adult larvae and that the number of abnormal spawnings (counting less than 50 eggs) of the larvae of H. polygyrus bakeri increased by approximately 50% with the extract of Pelvetia canaliculata at 5g / L compared to the number of abnormal spawnings observed with the negative control (see figure 19).• Test results for Sargassum muticum The anthelmintic activity of aqueous extracts of the alga Sargassum muticum was also tested. With two extracts of Sargassum muticum at 5 g / L (extracts designated under the codes AM244-19 and AM245-19), we observed a near-total inhibition of larval development at the L3 stage of H. polygyrus bakeri larvae (see Figure 20) and H. contortus larvae (see Figure 21). The dose / larval development curve of H. polygyrus bakeri larvae also allowed us to estimate that the IC50 of the Sargassum muticum extract 245-19 was equal to 1.57 g / L (see Figure 22).

Claims

CLAIMS 1. Composition for pharmaceutical, veterinary or food use containing at least one alga and / or at least one extract of an alga, said alga belonging to the order Fucales, for use in the oral treatment of helminthiasis caused by organisms of the suborder Rhabditina and / or Spirurina, and in particular caused by organisms of the family Ascarididae, Heligosomatidae or Trichostrongylidae, and even more particularly caused by isolates of organisms of the species Haemonchus contortus, Heligmosomoides polygyrus and Ascaridia galli, said organisms being resistant to anthelmintics belonging to the groups of macrocyclic lactones, such as ivermectin, moxidectin and eprinomectin, benzimidazoles, such as thiabendazole, imidazothiazoles, such as levamisole and / or tetrahydropyrimidines, such as pyrantel. 2.Composition according to claim 1, characterized in that said alga belongs to the Sargassaceae or Fucaceae family.

3. Composition according to claim 1, characterized in that said alga is of the genus Sargassum, Bifurcaria, Cystoseira, Pelvetia, Fucus, or Ascophyllum.

4. Composition according to claim 3, characterized in that said alga is of the subgenus Bactrophycus.

5. Composition according to claim 1, characterized in that said alga is of the species Bifurcaria bifurcata, Sargassum muticum or Pelvetia canaliculata.

6. Composition according to any one of claims 1 to 5, characterized in that said extract is an aqueous or organic extract of an alga or an organic extract obtained from an aqueous extract of an alga.

7. Composition according to any one of claims 1 to 6, wherein said algae is in fresh, dried or frozen form and said algae extract is obtained from fresh, dried or frozen algae.

8. Composition according to any one of claims 1 to 7, characterized in that it comprises at least 0.1% by mass of said algae relative to the total mass of said composition, by wet weight.

9. Composition according to any one of claims 1 to 7, characterized in that it comprises at least 0.01% by mass of said algae extract relative to the total mass of said composition, by wet weight.

10. Composition according to any one of claims 1 to 9, characterized in that said algae was collected on the foreshore or at sea during the months of March to August and in that said algae extract is obtained from algae collected on the foreshore or at sea during the months of March to August.

11. Process for obtaining a preparation for pharmaceutical, food or veterinary use for the oral treatment of helminthiasis caused by organisms of the suborder Rhabditina and / or Spirurina, and in particular caused by organisms of the family Ascarididae or Trichostrongylidae, and even more particularly caused by isolates of organisms of the species Haemonchus contortus, Heligmosomoides polygyrus and Ascaridia galli,said organisms being resistant to anthelmintics belonging to the groups of macrocyclic lactones, such as ivermectin, moxidectin and eprinomectin, Benzimidazoles, such as thiabendazole, imidazothiazoles, such as levamisole and / or tetrahydropyrimidines, such as pyrantel, said preparation method comprising the following steps: - preparing an extract of an alga of the order of fucales, preferably of the family Sargassaceae or Fucaceae, more preferably of the genus Sargassum, Bifurcaria, Cystoseira, Pelvetia, Fucus, or Ascophyllum, even more preferably of the species Bifurcaria bifurcata, Sargassum muticum or Pelvetia canaliculata; - incorporating said extract into a liquid, a paste or a powder so as to form said preparation.

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