Composition for oral anthelmintic use based on fucales, method of preparation and use of such a composition

Abstract

The invention relates to a composition for pharmaceutical, veterinary or food use containing at least one alga and / or at least one extract of an alga for oral anthelmintic use, said alga belonging to the order Fucales, the use of such a composition in the preparation of a medicinal product for treating, controlling and preventing endoparasitic infections in a warm-blooded animal or a human being and a method of preparing a medicinal product from such a composition.

Description

Title of the invention: Composition for oral anthelmintic use based on fucales, method for preparing and use of such a composition Scope of the invention

[0001] The field of the invention is that of antiparasitic treatments, in particular against gastrointestinal nematodes.

[0002] More specifically, the invention relates to a composition for pharmaceutical, veterinary or food use for oral anthelmintic administration, its use as a medicinal product and a method for preparing such a composition. Prior art

[0003] Gastrointestinal nematodes are a major concern for human and animal health worldwide.

[0004] In livestock, these parasites affect the health of the animals by causing loss of appetite, diarrhea, anemia and, in severe cases, death.

[0005] Parasitism also impairs productivity and leads to 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.

[0006] Therefore, it is urgent to find alternative solutions for sustainable control of helminths.

[0007] In order to prevent the emergence of resistance to anthelmintic drugs, an increasing number of studies are now focusing on naturally occurring compounds that can control gastrointestinal nematode populations.

[0008] Recent studies have shown the strong potential of algae through their anti-inflammatory, antimicrobial, anticancer and also anti-parasitic activities.

[0009] It has been known in Japan for many centuries that the red algae Digenea simplex acts as a powerful vermifuge for humans and is effective for the treatment of ascariasis.

[0010] One drawback of the red alga Digenea simplex is its limited distribution area. Furthermore, there is no effective culture system for Digenea simplex. Finally, the effectiveness of Digenea simplex has only been demonstrated against Ascaris lumbricoides.

[0011] De 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, also reports results of the antiparasitic potential of certain organic extracts of algae on gastrointestinal nematodes of goats.

[0012] There is therefore a need to be able to offer alternative compositions containing in particular algae which are effective against different species of gastrointestinal nematodes which affect other mammals.

[0013] It is also known from the article by 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 exhibit anthelmintic potential against the plant-parasitic nematode Meloidogyne incognita. This study showed that different species of macroalgae have activity in inhibiting egg hatching and juvenile larval mortality.

[0014] Besides 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. Objectives of the invention

[0015] The invention aims in particular to provide an anthelmintic treatment which is effective both against nematodoid parasites of the suborders Rhabditina and / or Spirurina, sensitive or resistant to anthelmintics known in veterinary or human medicine, and which also preserves the health of the infected host and the environment.

[0016] Another objective of the invention is to provide such a technique which is simple to implement and inexpensive. Description of the invention

[0017] The present invention relates to a composition for pharmaceutical, veterinary or food use containing at least one alga and / or at least one extract of an alga for anthelmintic use by oral route, said alga belonging to the order Fucales.

[0018] The inventors have indeed discovered, in a surprising and unexpected way, that algae and / or algae extracts of the order Fucales prove to be effective against zooparasitic organisms of the nematodoid type of the suborders Rhabditina and / or Spirurina 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.

[0019] In the context 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.

[0020] 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.

[0021] In a particular embodiment of the invention, said seaweed belongs to the Sargassaceae or Fucaceae family.

[0022] According to a particular embodiment of the invention, said alga is of the genus

[0023] Sargassum, Bifurcaria, Cystoseira, Pelvetia, Fucus, or Ascophyllum.

[0024] According to one particular aspect, the said alga is of the subgenus Bactrophycus.

[0025] In a particular embodiment of the invention, said alga is of the species Bifurcaria bifurcata, Sargassum muticum or Pelvetia canaliculata.

[0026] 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.

[0027] Advantageously, a composition such as described above is effective against zooparasitic organisms of the nematodoid type of the suborder Rhabditina or Spirurina and in particular against organisms of the order Ascarididae or Tricha strongylidae 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.

[0028] 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.

[0029] It should be noted that dried seaweed can be obtained by air drying, for example in the sun, or in an oven or by freeze-drying.

[0030] In another embodiment of the invention, the algae included in the composition or the algae from which the algae extract is taken may be fresh algae.

[0031] Preferably, a composition such as described above comprises at least 0.1% by mass of said algae relative to the total mass of said composition by wet weight.

[0032] Preferably, a composition such as described above comprises at least 0.01% by mass of said seaweed extract relative to the total mass of said composition by wet weight.

[0033] In an advantageous embodiment of the invention, said seaweed was collected on the foreshore or at sea during the months of March to August and in that said seaweed extract is obtained from seaweed collected on the foreshore or at sea during the months of March to August.

[0034] This collection period is particularly preferred for algae collected or cultivated in the northern hemisphere.

[0035] In an advantageous embodiment of the invention, a composition such as described above is effective on isolates of organisms of the suborder Rhabditina, and in particular on isolates of organisms of the order Tricha strongylidae, 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 levamsiole and / or Tetrahydropyrimidines, such as pyrantel.

[0036] The invention also relates to the use of a composition as described above in the preparation of a drug to treat, control and prevent endoparasitic infections in a warm-blooded animal or a human being.

[0037] The invention further relates to a method for preparing a medicinal product for pharmaceutical or veterinary use comprising the following steps:

[0038] - prepare an extract of an alga of the order Fucales, preferably of the family of 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;

[0039] - incorporation of said extract into a liquid or a paste so as to form said medicine. List of figures

[0040] Other features and advantages of the invention will become more apparent upon reading the following description of an embodiment of the invention, given by way of simple illustrative and non-limiting example, and the accompanying figures, among which:

[0041] [Fig. 1] is a graph representing the percentage inhibition of H. bakeri development, from stage L1-L2 to stage L3, in the presence of water (control), ivermectin (IpM) and different extracts of Bifurcaria bifurcata (cold extraction (1) and hot extraction (2)) at 5g / L. The results are from at least three independent experiments, with n=6 per sample each time. Statistical analyses were performed using a Kruskal-Wallis test with multiple comparisons test, ***p<0.001 compared to the control;

[0042] [Fig.2] represents a dose-response curve for extract 1 of B. bifurcata for Inhibition of development up to the L3 larval stage. The solid line represents the adjusted logistic regression curve and the shaded area indicates the 95% confidence interval;

[0043] [Fig.3] represents a dose-response curve for extract 2 of B. bifurcata for inhibition of development up to the L3 larval stage. The solid line represents the adjusted logistic regression curve and the shaded area indicates the 95% confidence interval;

[0044] [Fig.4] is a graph representing the percentage of inhibition of development of *H. polygyrus bakeri*, from the L1-L2 to the L3 stage, in the presence of water (Control), or of different batches of *B. bifurcata* at 5 g / L. Algae from France were collected in 2019 (FR-1) and 2021 (FR-2, FR-3, FR-4) and were frozen either directly (FR-2), after 24 h at +4°C (FR-3), or after 48 h at +4°C (FR-1 and FR-3). The results come from at least three independent experiments, with n=6 per sample in each case. Statistical analyses were performed using a Kruskal-Wallis test and a Wilcoxon multiple comparison test with ns, not significant.

[0045] [Fig. 5] illustrates the effect of water (control), thiabendazole (TBZ) at 0.5 pg / mL, and B. bifurcata extract at 5 g / L on H. polygyrus bakeri on the inhibition of egg hatching after 48 h. The results are from at least three independent experiments, with n=6 per sample. Statistical analyses were performed using a Kruskal-Wallis test with a test of

[0046] [Fig. 6] illustrates the effect of water (control), thiabendazole (TBZ) at 0.5 pg / mL and The effect of a 5 g / L extract of B. bifurcata on H. polygyrus bakeri on the mortality rate of larvae after hatching was observed. Results were obtained 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 (p<0.001).

[0047] [Fig.7] is a graph representing adult survival curves of H. Polygyrus bakeri were exposed to different concentrations of aqueous extract of Bifurcaria bifurcata for 6 days. Survival curves were obtained using the Kaplan-Meier method, and the shaded area indicates the 95% confidence interval.

[0048] [Fig.8] represents dose-response curves of H. larval development. Polygyrus bakeri, from the L1-L2 to the L3 stage, for different batches of B. bifurcata collected in France and Portugal. Both batches were made from algae collected in France and frozen before extraction (blue), and from dried algae collected in Portugal (red). The different IC50 values ​​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

[0049] [Fig.9] is a comparison, in graphical form, of the efficiency of different extracts of B. bifurcata from France (frozen) and Portugal (dried) for Inhibiting the development of H. polygyrus bakeri larvae was a controlled experiment using water. Aqueous extracts, various organic extracts (heptane, ethyl acetate, and butanol), and the residual aqueous fraction were tested at Ig / L. The extracts were obtained either from the batch of frozen algae harvested in France (blue) or from the batch of dried algae harvested in Portugal (red). The results are from at least three independent experiments, with n=6 per sample in each case. Statistical analyses were performed using a Kruskal-Wallis test with multiple comparisons, with p<0.001.

[0050] [Fig. 10] illustrates the results, obtained with the alga Bifurcaria bifurcata, of the hatching and post-hatching mortality test, of larval development tests and of adult worm survival tests on the parasite H. contortus.

[0051] [Fig. 11] is a graph summarizing the results of the L3 larval development test of the species H. contortus for different species of algae at a concentration of 0.5 or 5 g / L.

[0052] [Fig. 12] illustrates the results with a dose of 5 g / L of Bifurcaria bifurcata seaweed extract of the post-egg hatching larval mortality test for the susceptible Weybridge strain of H. contortus and for the resistant Kokstadt strain of H. contortus

[0053] [Fig. 13] are dose-hatching curves for the susceptible Weybridge strain and for the resistant Kokstadt strain

[0054] [Fig. 14] are results of a migration test (MTA) on Ascaridia galli larvae obtained for algae extracts obtained from a batch of B. bifurcata algae from France and a batch of B. bifurcata algae from Portugal.

[0055] [Fig. 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 larval mortality after hatching, for algae harvested in France or Portugal depending on the seasonality or method of preparation (drying and / or freezing).

[0056] [Fig. 16] is a dose-response curve of the larval development of H. Bakeri larvae at the L3 stage as a function of the dosage of Pelvetia caniculata extract.

[0057] [Fig. 17] is the result of the L3 larval development test of the species H. contortus for extracts of Pelvetia caniculata at 5g / L (codes AM228-19 and AM229-19).

[0058] [Fig. 18] illustrates results of an oviposition test showing the effect of Pelvetia caniculata extract at different dosages on the oviposition of adult H. bakeri worms.

[0059] [Fig. 19] is a graph summarizing the number of abnormal egg-layings (less than 50 eggs per worm) obtained by an egg-laying test of adult H. bakeri worms for the negative control (water), for water with a NaCl concentration of 3.55 g / L, which corresponds to the salinity of an algae extract at 5g / L, and for an extract of Pelvetia caniculata at 5g / L.

[0060] [Fig.20] is the result of the L3 larval development test of H. Bakeri larvae for Sargassum muticum extracts at 5g / L (codes AM244-19 and AM245-19).

[0061] [Fig.21] is the result of the L3 larval development test of H. larvae. contortus for extracts of Sargassum muticum at 5g / L (codes AM244-19 and AM245-19).

[0062] [Fig. 22] is a dose-response curve of the larval development of H. bakeri larvae at the L3 stage as a function of the dosage of Sargassum muticum extract. Detailed description of the invention

[0063] Subsequently, by way of example, results of in vitro tests relating to the anthelmintic properties of the algal species Bifurcaria bifurcata, the species Sargassum muticum (family Sargassaceae) and the species Pelvetia caniculata (family Fucaceae) are presented.

[0064] It should be noted at the outset that the species Bifurcaria bifurcata, the species Sargassum muticum and the species Pelvetia caniculata have the advantage of being available in large quantities.

[0065] • Test results concerning Bifurcaria bifurcata

[0066] Collection of Bifurcaria bifurcata algae and preparation of aqueous extracts

[0067] The brown algae B. bifurcata (FR-1) were harvested in Roscoff (Brittany, France, 48°43'53.6"N 3°59'15.5"W) in 2019. The algae were washed with 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 perform 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 remaining pellet was resuspended in a volume of water equal to its weight and incubated for one hour at 60°C in a water bath. After centrifugation at 10,000 g for 20 minutes, the supernatant was collected (extract 2). The two extracts obtained were lyophilized separately for optimal preservation.

[0068] In order to study the potential variation in anthelmintic activity related to geographical origin and processing, batches of B. bifurcata were also collected in Peniche (Leiria, Portugal, 39°19'33"N 9°21'35.4"W) in 2020. QuintaQuanta / Mermaid Fragrance was the biomass supplier. The algae from Portugal were sent dried or frozen. They were not washed before drying, so desalting was carried out during the rehydration phase with reverse osmosis water. Extraction was then performed in the same way as for the frozen algae.

[0069] 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

[0070] The lyophilized algal material (3 g) was solubilized in 30 ml of MilliQ water prior to 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 rotary vacuum evaporator (Hei-VAP Core, Heidolph) at 33°C, resulting in 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 to create a stock solution stored at -20°C and then diluted according to the required dose. Parasitic material

[0071] 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 Animal Experimentation Ethics Committee (CEA VdL No. 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 minutes of centrifugation at 600 g, the pellet was resuspended in a 360 g / L NaCl solution.A second centrifugation of 5 minutes at 900g was carried out, the supernatant was discarded and the eggs rinsed with water before being collected and counted.

[0072] To collect the adult worms, the mice were sacrificed by cervical dislocation and the small intestine was retrieved. The intestine was opened along its entire length with dissecting scissors. It was then placed in a filter at the top of a glass funnel connected to a collection tube. The worms migrated through the preheated (37 °C) DMEM culture medium from the filter to the collection tube as described above.

[0073] Hatching test and post-hatching mortality test

[0074] One hundred eggs (60pL) were placed in glass tubes in the presence of the algae extract (30pL). The tubes were placed at 21°C for 48 hours to induce hatching. eggs. The test consists of counting the number of unhatched eggs and the number of larvae released after the eggs hatch. The egg hatching rate is calculated (larvae / (larvae + eggs)) and the results are corrected for the negative control to obtain the percentage of egg hatching inhibition, as follows: ^07 SI .T1-, . , / hatching ratio of eggs exposed to the extract LUU / jJ inhibition of 1 egg hatching = 100 - ——----—-----—“77 x 100 hatching ratio of negative control eggs f

[0076] Among the hatched larvae, the number of live and dead larvae was also counted to obtain the post-hatching larval mortality percentage, which is calculated as follows:

[0077] Percentage of larval mortality after hatching = (laps^live larvae) x 100 Larval Development Test (LDA)

[0078] The 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 dispensed into glass tubes (80 µl per tube). The tubes were placed at 21°C for 48 h to allow the eggs to hatch properly. After 48 h, 20 µl of Earl's medium were added, along with the algae extracts to be tested (50 µl). The tubes were placed back in the incubator at 21°C for 7 days to allow for proper larval development. The test then consists of counting the number of young L1 / L2 larvae relative to the number of fully developed L3 larvae. The larval development ratio was calculated (L3 / (L3 + L1 / L2)) and the results were corrected for the negative control to obtain the percentage of larval development inhibition, as follows: v cx~\ , x , , , . „ „„ / development ratio of larvae exposed to the extract ,

[0079] inhibition of larval development = 100 - x 100) Survival test

[0080] Adult worms were placed in 48-well plates containing 120 pL of DMEM medium supplemented with antibiotics (Penicillin 100 g / mL and Streptomycin 100 pg / mL) and 60 pL of seaweed extracts to obtain the different final concentrations (5 g / L, 1 g / L, 0.5 g / L, and 0.1 g / L). The worms were kept at 37°C, and the number of survivors was counted daily during the experiment. Worms were considered dead when they did not respond to contact stimulation (prick with a worm pick). Statistical analyses

[0081] R Studio 4.2.1 was used for the statistical analysis of the mean, median, calculation of the CI50 by nonlinear regression, and survival analysis using the Kaplan-Meier method. Depending on the data distribution and the homogeneity of variances, the Krus-kal-Wallis test with multiple comparisons (Dunn's test and Wilcoxon test) was used. Results

[0082] The larval development test (LDA) reveals the anthelmintic activity of aqueous extracts of B. bifurcata on H. polygyrus bakeri

[0083] The inhibition of the larval development of H. polygyrus bakeri larvae by aqueous extracts of B. bifurcata was studied using water as a negative control and ivermectin 1 pM (reference anthelmintic) as a positive control.

[0084] In water, development from the L1 / L2 to L3 stage is approximately 97%, whereas the application of 1 pM ivermectin completely inhibits larval development. As an additional control, we also investigated the potential effect of salt (NaCl) to differentiate between the putative activity of the bioactive compounds and the salt content of the algae. At 5 g / L, the NaCl solution had no effect on larval development (similar to the control in water only).

[0085] To account for a potential effect of extraction temperature on biological activity, two aqueous extracts (cold and hot extractions) were tested ([Fig. 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 the "cold" extract 1 ([Fig. 2]) and 0.97 g / L for the "hot" extract 2 ([Fig. 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 ([Fig. 4]). In general, these results demonstrate that the B. bifurcata algal species screened with LDA exhibits strong, reproducible and dose-dependent vermifuge activity on the larval stages of H. polygyrus.

[0086] Subsequently, the "cold" and "hot" extracts both exhibiting significant anthelmintic activity, the cold extracts of B. bifurcata which exhibit the lowest IC50 were tested.

[0087] Action of B. bifurcata on the inhibition of egg hatching and nematicid activity on adult worms of H. polygyrus bakeri

[0088] In order to further study the anthelmintic activity of B. bifurcata on other life stages of the parasite, additional tests were carried out on eggs and adult worms.

[0089] Initially, egg hatching tests (48 hours) were performed using water as a negative control and thiabenazole (the reference deworming drug for egg hatching tests) as a positive control ([Fig. 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).

[0090] Strikingly, strong nematicidal activity was also observed on freshly hatched larvae with 67% (p<0.001) mortality ([Fig.6]). This suggests B. bifurcata exhibits dual anthelmintic activity, acting on egg hatching and directly nematicidal activity on newly 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).

[0091] Secondly, the direct nematicidal activity of B. bifurcata was also investigated on adult worms via a survival test. The test lasted 6 days, during which the survival rate of adult worms was 100% in the culture medium. The aqueous extract of B. bifurcata was applied at four different concentrations (from 5 g / L to 0.1 g / L; [Fig. 7]). At the highest concentration, all worms died within 24 hours. At the 1 g / L concentration, we observed a low survival rate of 10% after three days. Finally, nematicidal activity was still detectable at a concentration of 0.5 g / L, with a survival rate of 30% after 6 days. These results strongly support the 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.

[0092] Taken together, these results clearly demonstrate that aqueous extracts of B. bifurcata algae have anthelmintic activities against the murine parasite H. polygyrus bakeri.

[0093] Impact of the extraction process and / or geographical origin on the vermifuge activity of B. bifurcata.

[0094] When working with algal biomass, it was important to study the reproducibility of the result as a function of geographical origin and extraction process. Indeed, these two factors are well known to influence the phytochemical composition of the extracts and subsequently their 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). In order to monitor the potential impact of geographical factors and combined processes, dose-response curves were generated for the two types of extracts ([Fig. 8]). While our results confirm dose-dependent anthelmintic activities of the two batches of B.bifurcata, they also highlight a variability in efficiency (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.

[0095] In order to further explore the phytochemical family potentially responsible for the variability in the anthelmintic activity of B. bifurcata, we carried out Organic extractions were performed on aqueous extracts at Ig / L obtained from batches of algae from France (frozen) and Portugal (dried). At 1 g / L, the aqueous extract from France remained highly active, while its counterparts from Portugal were almost ineffective, thus increasing our ability to detect the corresponding chemical component responsible for the differential anthelmintic activity. Fractionation of the aqueous extracts was carried out sequentially with three distinct solvents (heptane, EtOAc, and BuOH), and their respective anthelmintic properties were monitored using LDA as previously described ([Fig. 9]). The organic extracts (heptane, EtOAc, and BuOH) and the residual aqueous fraction were tested at Ig / L. For both the French and Portuguese algae, the heptane and ethyl acetate extracts induced complete inhibition of larval development.Surprisingly, while the butanolic extract of the French algae exhibited high efficacy on larval development (99%), its Portuguese counterpart induced only 13% inhibition, thus laying the groundwork for a potential explanation of their differential activity. Importantly, the residual aqueous fractions showed no remaining activity, indicating that the active molecules are completely recovered by the organic solvents.

[0096] The anthelmintic activity of the organic fractions was then further investigated using a post-hatching larval mortality test. The organic extracts were tested at different concentrations. While the BuOH extracts and the residual aqueous fractions showed a weak impact on larval mortality even at 3 g / L, the ethyl acetate extracts were effective at 1 g / L and the heptane extracts had full activity at 0.5 g / L. These results indicate that the active molecules are mainly present in the heptane extract but also in the ethyl acetate extract. Interestingly, no significant difference was observed between the activities of the organic extracts from the French and Portuguese algae. Contrary to the LDA results, there was only weak activity of the BuOH fraction from the French algae with regard to larval mortality.These results suggest that the molecules involved in inhibiting larval development may differ from those involved in larval mortality.

[0097] The nematicidal activity of various extracts of B. bifurcata from France (frozen) and Portugal (dried) on freshly hatched H. polygyrus bakeri larvae is reported in the summary table below. The results shown in this table are from three independent experiments, with n = 6 per sample in each case. The values ​​reported are the mean mortality (%) ± standard deviation.

[0098] [Tables 1] Extract Concentration (g / L) Geographic Location / Process Mortality (% ±SD) p-value France (frozen) vs Portugal (dried) Control - 0 - 0.065 + 0.34 - Bifurcaria bifurcata Aqueous Extract 3 France (frozen) 12 + 7.15 3.1105 Portugal (dried) 0.318 + 0.74 Heptane 0.5 France (frozen) 100 + 0.00 1 Portugal (dried) 99.8 + 0.51 EtOAc 1 France (frozen) 73.2 + 17.90 1 Portugal (dried) 55.3 + 31.20 BuOH 3 France (frozen) 2.49 + 3.42 1 Portugal (dried) 3.54 + 4, 07 Residual aqueous fraction 3 France (frozen) 0 + 0.00 Not determined

[0099] Conclusions on the effect of B. bifurcata on H. polygyrus bakeri

[0100] Taken together, the results obtained demonstrate that aqueous extracts of the alga B. bifurcata contain bioactive compounds with in vitro vermifuge activity against the different life stages of the murine parasite H. polygyrus bakeri. It was observed that the aqueous extract of B. bifurcata is sufficiently potent to inhibit larval development in the murine parasite H. polygyrus bakeri with 100% efficacy at a concentration of 5 g / L. This algal extract also showed effects on egg hatching (28% inhibition at 5 g / L) and direct nematicidal activity against larvae (67% efficacy at 5 g / L) and adult worms (70% efficacy down to 0.5 g / L in 6 days).

[0101] The results obtained with B. bifurcata were also compared to the reported antihelminthic efficacies of plant extracts. For example, the larval development test showed very high activity of the alga B. bifurcata with an IC50 of approximately 0.8 g / L. This activity is higher than that reported for the plant species Glycyrrhiza glabra, Fumaria parviflora, Urtica dioica, and Myrtus communis against gastrointestinal parasites of small ruminants. Regarding larvicidal activity, which is approximately 67% at 5 g / L, it is comparable to that reported when plant extracts were evaluated as anthelmintics. Finally, the nematicidal activity observed against adult worms is greater than that known from the prior art. Indeed, we observe 100% mortality of adult H. polygyrus bakeri in 24h 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 24h 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 1." 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 must be made with caution because we cannot rule out that despite their close phylogenetic relationship, H. polygyrus bakeri and H. contortus could also exhibit differential sensitivity to natural compounds.

[0102] 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., E. Tuhy and K. Chojnacka. "Seaweed extract by microwave assisted extraction as plant growth biostimulant." Open Chemistry 13 (2015): that temperature can impact the stability of certain molecules such as algal polyphenols, one hypothesis is that the active molecules are either present in large quantities or are unaffected by temperature, thus highlighting the significant stability of the soluble anthelmintic compounds of B. bifurcata.

[0103] Furthermore, a difference in anthelmintic activity was observed between B. bifurcata from France (frozen) and Portugal (dried). The aqueous fractions of the Portuguese seaweed batches had lower activity compared to their French counterparts. However, the Portuguese batches retained approximately 30% inhibition of larval development, which suggests that the active molecules were still present but in smaller quantities. This hypothesis was also confirmed by comparing the mass balances of the fractionated algae from the two batches. Indeed, the BuOH fraction represented 3.1% of the total mass for the French algae, while it represented only 2.6% for the Portuguese batch (Table SI). To further investigate this hypothesis, various organic extractions (heptane, ethyl acetate, and butanol) were performed, and their respective biological activities were characterized (see Fig. 15). The results showed that the fractions with the main biological activity were those obtained with heptane and EtOAc, regardless of their origin and the process used prior to the aqueous extractions (i.e., frozen or dried). This confirmed the presence of active molecules in the Portuguese algae batch. It is worth noting that some of the active molecules exhibit antiparasitic activity against the protozoa Trypanosoma and Plasmodium previously identified in B.bifurcata were also extracted using EtOAc. These results are consistent with the study by Bonde et al. (Bonde, CS, L. Bomancin, Y. Lu, HT Simonsen, M. Martinez-Valladares, M. Pena-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 on two species of gastrointestinal parasites of sheep (Teladorsagia circumcinctd) 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 was observed between the BuOH fraction of French and Portuguese algae (99% and 13% inhibition of larval development, respectively), suggesting that the anthelmintic properties of B. bifurcata may be based on different families of molecules. This confirms the particular interest in using aqueous extracts of B. bifurcata as anthelmintic sources, as 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 potential synergistic effect between the different active compounds present in both the heptane and the BuOH extract of B. bifurcata. Results on Haemonchus contortus

[0104] Results obtained with an extract of the alga Bifurcaria bifurcata (identified by the code AM242-19 in the following figures) on the sheep gastrointestinal parasite H. contortus are subsequently presented.

[0105] 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 (0.5 g / L, Ig / L and 5g / L) of B. bifurcata extract.

[0106] A dose of 5g / L of B. bifurcata extract shows inhibition of larval development and nematicid activity on H. contortus larvae and to a lesser extent on adult H. contortus worms (IC50 of 4.39 g / L).

[0107] Figure 11 shows 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 11 shows that the extract of the alga Bifurcaria bifurcata (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 caniculata (AM 228-19 and AM 229-19) and Sargassum muticum (AM 244-19 and AM 245-19).

[0108] Figure 12 illustrates the results of the post-egg hatching larval mortality test with a 5 g / L dose of a B. bifurcata extract for the Weybridge susceptible strain of H. contortus and for the Kokstadt resistant strain of H. contortus. The dose-hatching curves for this Weybridge susceptible strain (IC50 = 0.05 pg / mL) and for the Kokstadt resistant strain (IC50 = 2.31 pg / mL) are shown in Figure 13. Results on Ascaridia galli

[0109] Bifurcaria bifurcata seaweed extract at 5g / L showed an effect on the poultry gastrointestinal parasite Ascaridia galli on both adult mortality and egg development.

[0110] The results also showed that the activity on Ascaridia galli larvae of the extracts of the alga Bifurcaria bifurcata was substantially similar regardless of the geographical origin of Bifurcaria bifurcata, as can be seen in [Fig. 14] where are represented the results obtained in vitro of a migration test (MTA) for extracts of the alga Bifurcaria bifurcata obtained from two batches of algae, one from France, the other from Portugal. [YES] • Test results concerning Pelvetia caniculata Pelvetia caniculata activity

[0112] The anthelmintic activity of aqueous extracts of the alga Pelvetia caniculata was also tested.

[0113] As can be seen from the dose / larval development curve in [Fig. 16], the extract of Pelvetia caniculata (designated under the code AM228-19) has an effect on larval development at the L3 stage of H. Bakeri larvae, with an estimated IC50 at 1.03 g / L. We also observed with the extract of Pelvetia caniculata at 5g / L an almost total inhibition of larval development at the L3 stage of H. contortus larvae at the L3 stage (see [Fig. 17]).

[0114] It was also observed via an egg-laying test by adult H. bakeri worms (see [Fig. 18]) that the extract of Pelvetia caniculata at 8g / L significantly reduces the quantity of eggs produced by adult larvae and that the number of abnormal egg-layings (counting less than 50 eggs) of H. bakeri larvae increases by about 50% with the extract of Pelvetia caniculata at 5g / L compared to the number of abnormal egg-layings observed with the negative control (see [Fig. 19]).

[0115] • Results of tests concerning Sargassum muticum

[0116] The anthelmintic activity of aqueous extracts of the seaweed Sargassum muticum was also tested.

[0117] Two extracts of Sargassum muticum at 5g / L (extracts designated under the codes AM244-19 and AM245-19) showed almost total inhibition of larval development at the L3 stage of H. bakeri larvae (see [Fig.20]) and H. contortus larvae (see [Fig.21]).

[0118] The dose / larval development curve of H. 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 [Fig.22]).

Claims

Demands

1. A composition for pharmaceutical, veterinary or food use containing at least one alga and / or at least one extract of an alga for use in the treatment of helminthiasis, in a form suitable for oral administration, against organisms of the suborder Rhabditina and / or Spirurina resistant to anthelmintics belonging to the macrocyclic lactones group, such as ivermectin, moxidectin and eprinomectin, the Benzimidazoles group, such as Thiabendazole, the Imidazothiazoles group, such as levamisole and / or the Tetrahydropyrimidines group, such as pyrantel, said alga belonging to the order Fucales.

2. Composition according to claim 1, characterized in that said seaweed belongs to the Sargassaceae or Fucaceae family.

3. Composition according to claim 1, characterized in that said seaweed 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 seaweed 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, characterized in that it is effective against organisms of the order Ascarididae or Trichostrongylidae and in particular against organisms of the species Haemonchus contortus, Heligmosomoides polygyrus and Ascaridia galli.

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

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

10. Composition according to any one of claims 1 to 8, characterized in that it comprises at least 0.01% by mass of said seaweed extract relative to the total mass of said composition, by wet weight.

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

12. Composition according to any one of claims 1 to 11, characterized in that it is effective against isolates of organisms of the order Trichostrongylidae resistant to anthelmintics belonging to the macrocyclic lactones group, such as ivermectin, moxidectin and eprinomectin, the Benzimidazoles group, such as thiabendazole, the Imidazothiazoles group, such as levamsiole and / or the Tetrahydropyrimidines group, such as pyrantel.

13. A method for preparing a medicinal product for pharmaceutical or veterinary use effective in the treatment of helminthiasis by oral administration against organisms of the suborder Rhabditina and / or Spirurina resistant to anthelmintics belonging to the macrocyclic lactone group, such as ivermectin, moxidectin, and eprinomectin; the benzimidazole group, such as thiabendazole; the imidazothiazole group, such as levamisole; and / or the tetrahydropyrimidine group, such as pyrantel, comprising the following steps: - preparing an extract of an alga of the order Fucales, preferably of the family Sargassaceae or Fucaceae, more preferably of the genera Sargassum, Bifurcaria, Cystoseira, Pelvetia, Fucus, or Ascophyllum, even more preferably of the species Bifurcaria bifurcata, Sargassum muticum, or Pelvetia canaliculata - incorporation of said extract into a liquid or paste so as to form said medicinal product.

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