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

Abstract

The invention relates to a composition for oral anthelmintic use, a method for preparing and using such a composition

Description

Title of the invention: Composition for oral anthelmintic use, preparation method and use of such a composition Field of 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 use 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. 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 mainly on the use of chemical anthelmintics. However, their intensive and sometimes inappropriate use has led to the development of resistance to anthelmintics.

[0006] Therefore, there is an urgent need to find alternative solutions for sustainable helminth control.

[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] To combat parasitic infections, it is known to use thymol, carvacrol, gamma-terpinene or terpinenol essential oils. Objectives of the invention

[0009] The invention aims in particular to provide an anthelmintic treatment which is effective both against nematode 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 and in particular is more effective than thymol or carvacrol.

[0010] Another objective of the invention is to provide such a technique which is simple to implement and inexpensive. Statement of the invention

[0011] The subject of the present invention is a composition for oral anthelmintic use, said composition comprising eleganolone and / or a linear diterpene of bifurcanol type.

[0012] The inventors have in fact discovered that eleganolone and / or bifurcanol or a linear diterpene of bifurcanol type alone or combined together prove to have high efficacy against zooparasitic organisms of the nematodoid type.

[0013] In a preferred embodiment of the invention, said eleganolone or said bifurcanol-type diterpene is present within an extract of Bifurcaria bifurcata incorporated into said composition.

[0014] In a particular embodiment of the invention, said extract of Bifurcaria bifurcata is an aqueous extract of Bifurcaria bifurcata, an extract obtained by extraction of compounds of Bifurcaria bifurcata in a solvent or an extract obtained by extraction of compounds of Bifurcaria bifurcata in an oil.

[0015] According to one aspect of the invention, said composition is effective against zooparasitic organisms of the nematode type of the suborder Rhabditina and / or Spirurina and in particular organisms of the order Ascarididae or Trichostrongylidae and even more particularly against organisms of the species Haemonchus contortus, Ascaridia suum, Parascaris equorum and Ascaridia galli.

[0016] The invention also relates to a process for preparing an anthelmintic intended to be administered orally comprising:

[0017] - preparing a composition comprising eleganolone and / or a linear diterpene bifurcanol type

[0018] - encapsulating said composition in yeast particles or incorporating it into a gelatinized starch matrix. List of figures

[0019] Other characteristics and advantages of the invention will appear more clearly on reading the following description of embodiments of the invention, given as simple illustrative and non-limiting examples, and the appended figures among which:

[0020] [Fig.l] is a comparison of the effectiveness of different extracts of Bifurcaria bifurcata from France (frozen) and Portugal (dried) to inhibit the development of Heligmosomoides bakeri larvae. Controls, with water and with 1% DMSO, were carried out. The aqueous extracts, the different organic extracts (heptane, EtOAc and BuOH) and the residual aqueous fraction were tested at 1 g / L. The extracts came either from the batch of frozen algae harvested in France (in August 2019), or from the batch of dried algae harvested in Portugal (in July 2020). The results pro 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.

[0021] [Fig.2] is a comparison of the effectiveness between different extracts of Bifurcaria bifurcata from Portugal with two different preservation processes (drying or freezing) on ​​the inhibition of Heligmosomoides bakeri larvae development. A control with water was performed. The aqueous extracts and the different butanol (BuOH) extracts were tested at 1 g / L. The extracts are from a batch of algae that was harvested in November 2021 in Portugal. These algae were either dried (green) or frozen (purple) before extraction. The results come from at least two 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.

[0022] [Fig.3] presents the activity of EtOAc extracts from biomass of Bifurcaria bifurcata from different batches on the development of Heligmosomoides bakeri larvae. The EtOAc organic fractions were carried out directly on the algal biomass from different batches of B. bifurcata. Three of them come from algae harvested in France at different times (March 2022 in brown, June 2021 in turquoise and August 2019 in blue) and frozen before extraction. Two others come from algae harvested in Portugal in November 2021 and either dried (green) or frozen (purple) before extraction. The percentage inhibition of the development of H. bakeri larvae, from the L1 / L2 stage to the L3 stage, is observed in the presence of water (negative control) and these different EtOAc extracts at 0.5 g / L.

[0023] [Fig.4] are 1H (a) and 13C (b) NMR spectra of the heptane fraction of Bifurcaria bifurcata. Two molecules were identified using the NMR spectra obtained for hydrogen atoms (1H, corresponds to profile a) and carbon atoms (13C, corresponds to profile b), namely eleganolone and bifurcanol, two linear diterpenes. The positions of the hydrogen and carbon atoms are numbered (in orange for eleganolone and in green for bifurcanol), as well as the corresponding peaks on the NMR spectra obtained.

[0024] [Fig.5] illustrates the pharmacological effect of Bifurcaria bifurcata on the receptor Nicotine-sensitive acetylcholine from Ascaris suum. Study of the pharmacological effect of an aqueous extract of B. bifurcata as an agonist (a,c) or as an antagonist (b,d) on the nicotinic receptor formed by the ACR-16 subunit of A. suum in the Xenopus laevis egg expression system. (a,b) Representation of the currents recorded using the Two-Electrode Voltage-Clamp technique following the application of 100 pM acetylcholine, 1 g / L and 5 g / L of aqueous extract of B. bifurcata alone or in co-application with ACh. The application times of the products are indicated by the horizontal bars (black for ACh and green for B. bifurcata). (c,d) Box plots representing the currents induced by the application of 100 pM ACh, 1 g / L or 5 g / L of the aqueous extract of B. bifurcata alone or in co-application with 100 pM ACh. The currents are normalized with the current amplitude obtained at the first application of 100 pM ACh.

[0025] [Fig.6] presents the results of a study of the antagonistic effect of different organic extracts of Bifurcaria bifurcata on the N-AChR of Ascaris suum

[0026] Box plots representing the normalized current amplitudes following co-application of organic extracts of B. bifurcata at 0.01 g / L with 100 pM of ACh on the ACR-16 receptor of Ascaris suum. The organic extracts used are as follows: Bif AE corresponds to an ethyl acetate extract obtained after extraction with heptane, Bif AEP corresponds to an extraction with ethyl acetate as the first solvent and Bif H corresponds to an extraction with heptane as the first solvent. The currents are normalized relative to the current induced by the first application of ACh (100 pM).

[0027] [Fig.7] illustrates the pharmacological effect of Bifurcaria bifurcata on the nicotinic acid-sensitive receptor (N-AChR) and the Morantel-sensitive receptor (M-AChR) of Parascaris equorum. Study of the agonist and antagonist effect of a B. bifurcata extract on the acetylcholine receptors of P. equorum, the N-AChR consisting of the ACR-16 subunit (a) and the M-AChR consisting of the ACR-26 and ACR-27 subunits (b). Box plots representing the normalized current amplitudes following the application of an aqueous extract of B. bifurcata at 1 g / L and 5 g / L or a co-application of B. bifurcata at 1 g / L and 5 g / L with 100 pM ACh. Currents are normalized to the current induced by the first application of ACh (100 pM) Detailed description of the invention Differential activity of organic fractions

[0028] Organic extractions carried out from aqueous extracts

[0029] Having identified variability in the effectiveness of these different batches, we were interested in two very distinct batches in order to compare the activity profile of several organic fractions. This was done with the aim of providing additional information on the type of molecules involved in the biological activity.

[0030] For this we selected batches of algae collected in France and frozen as well as algae collected in Portugal and dried, for which the aqueous extracts presented a difference in activity. The place of harvest and the preservation process are different. However, these algae were both harvested in summer (July for the Portuguese algae and August for the French). Three organic extractions were therefore carried out from the aqueous extracts successively, with heptane, then with ethyl acetate (EtOAc) and finally butanol (BuOH). The residual aqueous fraction was also recovered and tested.

[0031] These different extracts were tested via a TDL as a first intention ([Fig.l]). A negative control with water was carried out, as well as a control with 1% DMSO corresponding to the DMSO concentration of the organic extracts. These two control conditions did not show any inhibition of larval development. All extracts were tested at 1 g / L. For the French and Portuguese algae, the results showed that despite the difference in activity observed between the aqueous extracts, the heptane and EtOAc fractions completely inhibited larval development. Surprisingly, while the BuOH extract of the French algae showed high efficiency (99% inhibition of larval development), its Portuguese counterpart only induced a 13% inhibition, thus laying the foundations for a potential explanation of the difference in activity between the aqueous extracts.It is important to note that the residual aqueous fractions did not show any residual activity, indicating that the active molecules are completely extracted by the organic solvents.

[0032] The anthelmintic activity of the organic fractions was then further investigated using the post-hatching larval mortality test (Table 1). The organic extracts were tested at different concentrations. While the BuOH extracts and residual aqueous fractions showed a low impact on larval mortality, even at 3 g / L, the EtOAc extracts were effective at 1 g / L and the heptane extracts had total activity at 0.5 g / L. These results indicate that the active molecules are predominantly present in the heptane and EtOAc extracts. It is interesting to note that no significant difference was observed between the activities of the organic extracts of French and Portuguese algae. On the other hand, contrary to the TDL results, a low activity of the BuOH fraction of the French algae was observed on larval mortality.These results suggest that the molecules involved in the inhibition of larval development could be different from those involved in larval mortality.

[0033] [Table 1] Comparison of the nematicidal activity of different extracts of Bifurcaria bifurcata from France (frozen) and Portugal (dried) on freshly hatched larvae of Heligmosomoides bakeri Type of extract Çotiœntralian (g / Ly Zunc geogràphiifiii Mo» t.iîiti-i'< i sm p-value Fmtee w Portugat Cwirôk S + û 14. 5 uaci ii, > r < ' r fcximt atjiseiis. .3 Pci >.a, i 03109.74 .-<1 .1M i r.-me: >> ino -*.0 w Hupïàiïc ir; 1*01012.15 - i 99.3 + 03 1 Uiftmarki , ... , , fir iiu\' ii > 73.2* >7.90 Poilut.ii ' a - 0 2e fk W.'. Po}IU4.dv < JM* 4.07 {' i' Puf sec j fi * fyx)

[0034] The results come from three independent experiments, with n=6 per sample each time.

[0035] Values ​​represent mean mortality (%) ± standard deviation.

[0036] nd: not determined

[0037] To further explore the latest results obtained, and to determine which factor between the harvesting location or the algae preservation process most impacts the in vitro biological activity, we worked on two batches of algae harvested at the same time in Portugal (November) but with different pre-treatments (drying or freezing). A TDL was carried out on the aqueous extracts of these two batches at 1 g / L as well as on the BuOH fractions at 1 g / L which correspond to the organic fractions where a difference in activity had been observed previously ([Fig.2]). The results obtained show a strong difference in activity between these two extracts, whether for the aqueous extracts or the BuOH extracts. Indeed, the extract from frozen Portuguese algae shows a strong inhibition of larval development (88% for the aqueous extract and 85% for the BuOH fraction) unlike the batch of dried algae (<10% for both extracts).These results demonstrate that the algae preservation process plays a major role in the availability or alteration of active molecules and therefore in the biological activity of the extracts. Based on the previous results, this also demonstrates that the molecules found in the BuOH fraction are the most sensitive to the preservation process used.

[0038] Organic extractions made from algae biomass

[0039] After obtaining these first results from organic fractions from aqueous extracts, we wanted to carry out organic extractions directly on the algae biomasses. This process allows, among other things, to concentrate the active compounds in the organic fractions without going through a first aqueous extraction step. These extractions were carried out by Dr. Maria Matard-Mann and Karel Davalle-Corre (Olmix). The batches concerned are those of algae harvested in France and frozen (in August 2019 and in March and June 2021) as well as batches of Portuguese seaweed harvested in 2021 (dried and frozen). The results presented here only concern the EtOAc fraction tested via a 0.5 g / L TDL ([Fig.3]).

[0040] The results obtained with the TDL demonstrate that there is no difference in activity between the batches studied and that all completely inhibit the development of the larvae at the concentration of 0.5 g / L. These results demonstrate here that an extraction carried out directly from the algae biomass allows a better extraction of the active compounds which are very present in this EtOAc fraction, and this independently of the batches of algae which are nevertheless very varied.

[0041] Exploration of the modes of action involved Characterization of the active molecules

[0042] In order to characterize the active molecules in the organic fractions of B. bifurcata, the different samples were sent to a specialized NMR (Nuclear Magnetic Resonance) platform at the University of Western Brittany (UBO). Due to problems concerning the solubilization of the extracts, only the heptane fraction could be analyzed at present. Analysis of the NMR spectra (1H and 13C) made it possible to identify two different molecules present in this extract: eleganolone and bifurcanol ([Fig.4]). These two compounds are linear diterpenes already described in the literature.

[0043] The report states: "The analyses of the algae extracts provided have made it possible to highlight the presence of a major compound which would correspond to a linear diterpene, eleganolone. A partial structure of another significantly present compound has also been established and has led to the hypothetical presence of another linear diterpene, bifurcanol (or bifurcanol-like). The main difficulty in identifying these compounds lies in the fact that we are dealing with a mixture. In order to confirm the identity of the proposed compounds, mainly in the case of bifurcanol, it would be necessary to carry out simple and rapid fractionations to isolate them and determine their structure without ambiguity. This would also allow access to the structures of other, minority compounds, whose presence has been noted in the extracts."

[0044] Exploration of modes of action by electrophysiology in Xenopus eggs

[0045] Following the identification of diterpenes in an extract of B. bifurcata^ we were interested in the mechanisms and mode of action that could be involved in the observed biological activities. It has been shown in the literature that certain terpenes target cholinergic receptors. Based on this work we investigated what the effects of B. bifurcata extracts could be on different receptors known to be the target of anthelmintics in two species of gastrointestinal nematodes, A. suum, a parasite of pigs, and P. equorum. a horse parasite. These receptors are hetero- or homopentamers, therefore composed of 5 different or identical subunits. Several families of receptors were therefore chosen, among them nicotine-sensitive cholinergic receptors, composed of the ACR-16 subunit of A. suum or P. equorum, and a Morantel-sensitive cholinergic receptor composed of the ACR-26 and ACR-27 subunits of P. equorum. These receptors were expressed on the surface of Xenopus laevis eggs and electrophysiological measurements were carried out to determine the potential agonist or antagonist effect of aqueous and organic extracts of B. bifurcata applied to these receptors. Cholinergic N-AChR receptor of Ascaris suum

[0046] The first receptor studied is a cholinergic receptor of the parasite A. suum composed of 5 ACR-16 subunits forming a nicotine-sensitive receptor. It was expressed on the surface of Xenopus eggs and currents were measured using the Two-Electrode Voltage-Clamp method in order to determine the response of the receptor following the application of the natural ligand (100 pM of ACh) or following the application of aqueous extracts of B. bifurcata (at 1 g / L and 5 g / L) alone or in co-application with ACh ([Fig.5]).

[0047] Firstly, two successive applications of ACh at 100pM are carried out in order to control the replicability of the effect of the natural ligand on the receptor. The results observed here demonstrate that the receptor is correctly expressed on the surface of Xenopus eggs and that the effect is stable because the current amplitudes are comparable.

[0048] Application of the aqueous extract of B. bifurcata alone demonstrates that there is no direct agonist effect on the N-AChR receptor of Ascaris suum. Indeed, no current amplitude is observed following the application of 5 g / L or 1 g / L of extract.

[0049] Conversely, when the B. bifurcata extract is applied at the same time as 100 pM of ACh, a decrease in the current amplitude normally obtained with ACh alone is observed. Indeed, when co-applying ACh with 5 g / L of B. bifurcata, the current obtained is reduced by approximately 90% compared to ACh alone, and with 1 g / L of B. bifurcata, the current is reduced by approximately 50%. These results demonstrate a dose-dependent antagonistic effect of the aqueous extract of B. bifurcata.

[0050] It is important to note here that after the different applications of the extracts of interest the receptors recover correctly because the current amplitudes obtained with the third application of ACh are comparable to that of the first application of ACh.

[0051] To complement the first results obtained, organic fractions of B. bifurcata were also tested on the N-AChR receptor of Ascaris suum ([Fig.6]). The first applications of ACh alone demonstrate stability of the amplitudes of current obtained between the two applications. The fractions tested come from extracts obtained with the heptane solvent (Bif H) first and then successively with the ethyl acetate solvent (Bif AE). The third fraction was obtained using EtOAc directly without prior extraction with heptane (Bif AEP). The extracts were tested at 0.01 g / L in co-application with 100 pM of ACh. The results obtained demonstrate that with the co-application of the Bif AE extract and ACh, a slight decrease in current is observed (approximately 10%) compared to ACh alone. The Bif AEP extract shows a decrease of approximately 25% in the current obtained compared to ACh alone. This result highlights a better efficiency of the fraction extracted with EtOAc directly and not after a first extraction with heptane.Finally, the co-applied heptane fraction induced a stronger antagonistic effect than the ethyl acetate fractions, inducing a decrease of approximately 75% of the current compared to ACh alone. A complete recovery of the receptors was observed with the last application of ACh alone. The results obtained demonstrate that the organic fractions are significantly more effective than the aqueous fractions in inducing an antagonistic effect on these cholinergic receptors, especially concerning the heptane fraction.

[0052] Cholinergic N-AChR and M-AChR receptors of Parascaris equorum

[0053] The second receptors studied are cholinergic receptors of P. equorum, one sensitive to nicotine (N-AChR) and composed of the ACR-16 subunit, the other sensitive to Morantel (M-AChR) and composed of the ACR-26 and ACR-27 subunits. These receptors were also expressed on the surface of Xenopus eggs and the currents were measured using the Two-Electrode Voltage-Clamp method in order to determine the response of the receptor following the application of the natural ligand (100 pM of ACh) or following the application of aqueous extracts of B. bifurcata (at 1 g / L and 5 g / L) alone or in co-application with ACh ([Fig.7]).

[0054] The results obtained on these two receptors are very similar. They indicate that the application of aqueous extracts of B. bifurcata does not have a direct agonist effect. A slight effect is nevertheless observed for the 5 g / L extract on the Peq-M-AChR receptor with a current intensity of approximately 10% compared to the current intensity obtained with the natural ligand.

[0055] Regarding the antagonistic effects observed, during the co-application of 100 pM of ACh and 5 g / L of B. bifurcata, a decrease in current intensity of approximately 40% is observed on both receptors Peq-N-AChR and Peq-M-AChR. For the concentration of B. bifurcata at Ig / L in co-application with the AChR, a decrease of approximately 10% is observed for both receptors. These results demonstrate that there is also a dose-dependent antagonistic effect of the aqueous extract of B. bifurcata on the receptors of P. equorum. The results observed initially on a cholinergic receptor of the parasite A. suum, are confirmed here in another species of animal parasite of veterinary importance. Results and discussion

[0056] In this study, we were interested in the anthelmintic potential of macroalgae. Screening a panel of macroalgae

[0057] The choice of macroalgae studied was determined based on several criteria. We wanted to screen species within the three major families (red, brown and green) based on bibliographic data indicating biological activities, favoring species with antiparasitic activity. However, none of the selected species had been described as having anthelmintic activity on GI parasites and the antiparasitic activities mentioned mainly concerned protozoan parasites. The algae studied (for screening) were collected in Roscoff in Brittany (France). The potential industrial exploitation of the species studied was also taken into account by favoring species that have a distribution large enough to be exploited on a large scale or for which cultivation is possible.

[0058] The screening of the anthelmintic activity of these algae was carried out using two types of aqueous extracts: a so-called "cold" extract (extraction at room temperature) and a "hot" extract (extraction at 60°C). The choice to work initially on aqueous extracts was made for economic reasons and reasons of simplicity. We hypothesized that if an aqueous extract demonstrates anthelmintic activity, this means that the active molecules are present in large quantities and accessible. The effectiveness of a hot extract makes it possible to determine the stability of the active molecules, or even, in the case where the biological activity is better, to improve the extraction of certain molecules.

[0059] The different extracts were therefore tested using a TDL, which is a reference test in parasitology. The study model mainly used during this thesis work is the murine parasite H. bakeri. This parasite species is closely related from a phylogenetical point of view to other digestive trichostrongyles of veterinary importance, such as H. contortus or Teladorsagia circumcicta. As a result, it is commonly used for screening anthelmintic molecules in the pharmaceutical industry.

[0060] The results obtained demonstrate that among the 12 algae tested, 3 brown algae and the "hot" extract of a red algae induce a total inhibition of larval development at 5g / L and the observed effect is dose-dependent. Generally speaking in the literature, the algae described as having anthelmintic properties are brown and red algae. The red algae Digenea simplex and Chondria armata are the best known with their active compounds kainic acid and domoic acid, respectively. These results were also able to illustrate that heated extracts still exhibit biological activity, demonstrating that the active compounds are not affected by temperatures up to 60°C. The results obtained following screening on the murine parasite H. bakeri therefore suggest significant stability of the anthelmintic compounds present in the most effective algae. The results obtained with the red algae extract also indicate that the heating phase during extraction can allow for better extraction of the active molecules, as was observed during the TDL extract screening experiment where the hot extract is more effective than the cold extract (100% inhibition of development versus 83%). The most effective algae species in vitro

[0061] According to the results obtained via TDL, three species of brown algae are particularly effective in inhibiting the development of larvae. These algae are P. canaliculata, B. bifurcata and S. muticum.

[0062] While these algae have the ability to inhibit larval development with IC50s of the same order of magnitude, they nevertheless exhibit a variable in vitro activity profile. Indeed, S. muticum does not exhibit any activity other than inhibiting larval development, while B. bifurcata is capable of acting on different stages of the parasite. Finally, P. canaliculata also acts on the adult stages like B. bifurcata but in a completely different way by inhibiting egg laying by females but without impacting worm survival. These results indicate that these three species of brown algae probably possess active compounds specific to them and do not act on the same targets. These results are interesting and could be used in future experiments by testing a "cocktail" composed of extracts of these three algae together.In addition, tests can be carried out to highlight the potential synergistic effect between algae extracts and reference anthelmintics.

[0063] Only one source of the B. bifurcata algae has been identified and exploited (via QuintaQuanta in Portugal). In order to also characterize the S. muticum and P. canaliculata algae, and with a view to industrial exploitation, it would also be necessary to identify a source to have access to these two species in large quantities. Concerning S. muticum, the Japanese sargassum, its high bioavailability makes it an excellent candidate for industrial exploitation and would then make it possible to exploit a species considered invasive on the coasts of the British Isles, in continental Europe and in North America. The P. canaliculata algae, for its part, is widely used in the cosmetics sector and several marketed products contain it. Even if it is not considered an invasive species, the bioavailability The instability of this algae does not seem to be an obstacle to industrial exploitation.

[0064] Why is Bifurcaria bifurcata the best candidate for animal parasite control? Different in vitro activity phenotypes

[0065] Among the twelve species of algae tested on the murine parasite H. bakeri, B. bifurcata is certainly one of the most interesting. Besides being a very good candidate for inhibiting larval development (with the best IC50 at 0.7 g / L), it also has an effect on egg hatching (28% inhibition at 5 g / L) and direct nematicidal activity on larvae after hatching (67% efficacy at 5 g / L) and on adult worms (70% efficacy up to 0.5 g / L in 6 days).

[0066] It may be interesting to compare these results with anthelmintic activities observed from terrestrial plant extracts. For example, regarding the inhibition of larval development, the aqueous extract of B. bifurcata has an IC50 of approximately 0.7 g / L. This activity is higher than what has been previously reported with several aqueous extracts from plant species including Glycyrrhiza glabra, Fumaria parviflora, Urtica dioica and Myrtus communis on GI parasites of small ruminants. Regarding the larvicidal activity, which is approximately 67% at 5 g / 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 presented in the literature. Indeed, a mortality of 100% of adults of H. bakeri in 24 ha was observed with an aqueous extract of B. bifurcata at 5 g / L, whereas for example, on H.contortus, Eguale et al. noted a mortality rate of only 29% in 24 h with an aqueous plant extract at 8 g / L and Lone et al. used much higher concentrations (25 g / L and 50 g / L) to observe an effect on adult mortality in less than 24 h. The results obtained from aqueous extracts of B. bifurcata indicate a very good anthelmintic activity of the alga and this on multiple stages of development of the murine parasite.

[0067] However, such comparisons should be taken with caution because we cannot exclude that despite their close phylogenetic relationship, H. bakeri and H. contortus may also show different sensitivity to natural compounds. Therefore, we also tested the B. bifurcata extract on other parasite species.

[0068] Transposition to other species of veterinary importance and control of resistance

[0069] We also worked on sheep parasites H. contortus from an anthelmintic-sensitive strain (Weybridge) and a multi-resistant strain (Kokstad). The aqueous extract of B. bifurcata was therefore also tested on these two parasite strains and nematicidal activity on adults and larvae of H. contortus was observed. These results are all the more interesting because this activity is observed on the sensitive strain, but also, and even more effectively, on the multi-resistant strain. These observations are of major importance in the current context of resistance of GI parasites to anthelmintics. Indeed, since the first cases of resistance were identified in the early 1960s, resistance phenomena have become recurrent throughout the world. It is now no longer rare to find sheep or goat farms that exhibit resistance to all anthelmintic treatments available on the market. It is very interesting to see that it is possible to transpose the results obtained on the murine model to other parasite species of veterinary importance. The prospects for the sheep parasite would be to characterize more precisely the in vitro activity of B.bifurcata with additional tests (egg hatching, larval development, larval mobility, etc.).

[0070] What factors can impact the biological activity of B. bifurcata?

[0071] Impact of the environment (season and / or geography)

[0072] The first results obtained with the aqueous extracts of B. bifurcata encouraged us to continue exploring the activity of this algae. For this we needed a larger quantity of raw material, to consider future in vivo experiments. This is why we called on the company Quin-taQuanta located in Portugal to supply us with B. bifurcata. However, as algae are products of the environment, it was important to validate the biological activity of new batches from different harvesting locations.

[0073] The Portuguese seaweed extracts were therefore compared to the French seaweed extracts. The results showed that for two identical aqueous extracts (in terms of preservation and extraction) but with the only difference being the geographical origin (France or Portugal), a difference in effectiveness is observed. Whether via the TDL (IC50 of 0.48 g / L for the Portuguese extract versus 0.66 g / L for the French extract, with a significant difference p<0.001) or the larval mortality test (97% mortality for the Portuguese extract versus only 2% mortality for the French extract at Ig / L), the results indicate a better effectiveness of the algae collected in Portugal. It has already been shown that the composition of secondary metabolites found in B. bifurcata can vary depending on the harvesting location within the French Atlantic coast.These variations may be due to chemical adaptations of the algae to varying biological or physical environmental conditions. It would then be interesting to compare these two extracts from a biochemical point of view in order to identify which compounds are involved in these differences in activity.

[0074] The seaweed harvest season can also play a role in the composition and biological activity. To verify this, Olmix carried out several harvests of B. bifurcata in Brittany at different times of the year: in June 2021, December 2021 and March 2022. These harvests complemented the first harvest of B. bifurcata which took place in August 2019. The results obtained indeed demonstrate a difference in activity, whether on the TDL or on the mortality of the larvae after hatching. In general, the results tend to demonstrate that the batch of B. bifurcata collected in December 2021 is the least effective, while that harvested in June 2021 seems to have a better effectiveness.

[0075] Variations in biological activity have already been observed for the green alga Ulva rigida collected in Tunisia during the 12 months of the year. The results demonstrated that the antimicrobial and antioxidant activity of the alga was stronger in samples collected in spring. Regarding B. bifurcata, a study also demonstrated the variability of the "antifouling" activity of this alga depending on the months of harvest, with better biological activity of the algae harvested from April to August and a peak of activity observed in June. This trend seems to be consistent with our results. However, due to global variations in ocean temperature and the multitude of algae in the world that react differently to environmental conditions (whether salinity, temperature or pollution for example), it is quite difficult to establish a general rule applicable to all species of algae.

[0076] This is why new harvests will have to be carried out during another year in order to validate these initial results and determine whether, for this species in particular, it is possible to identify a more favorable season for harvesting. With a view to industrial exploitation from the algae collected in Portugal, it would be wise to carry out these experiments on Portuguese algae. Impact of conservation processes

[0077] Beyond the impacts that environmental factors can have on the biological activity of algae, treatment processes can also play a role. The first B. bifurcata algae sent to us from Portugal had been dried after harvest. Therefore, when we observed a difference in activity between this batch of algae and the French algae (which had been frozen and harvested in a different month), it was difficult to determine whether this decrease in in vitro biological activity was due to the geographical area, the harvest season or the preservation process of B. bifurcata after harvest. Organic extractions were carried out on these two batches of algae with 3 different solvents (heptane, EtOAc and BuOH) in order to observe a differential activity between these fractions. The TDL results showed that the heptane and EtOAc fractions were very effective in inhibiting larval development, regardless of the batch of algae.The major difference was observed for the BuOH fraction. That of frozen and French algae presents a . high activity at 1 g / L while that from dried and Portuguese algae does not show activity at the same concentration.

[0078] In order to highlight the impact of post-harvest treatment of the algae on biological activity, we then compared similar batches with the only change being the post-harvest treatment of the algae. A harvest of B. bifurcata took place in Portugal in November 2021, and one part was frozen and the other dried. The activity of these two batches of algae was also compared with a TDL on H. bakeri from aqueous extracts and BuOH organic fractions. The results obtained demonstrate that post-harvest treatment of the algae affects biological activity. A strong decrease in the activity of extracts from dried algae was observed compared to extracts from frozen algae, both for aqueous extracts (8% activity versus 88% respectively) and BuOH fractions (9% versus 85% respectively).

[0079] In comparison and as an example, LeLann et al. measured the amount of phenolic compounds and the antioxidant activity of S. muticum and B. bifurcata as a function of different treatments: none (fresh algae), freezing, freeze-drying, oven-drying and greenhouse drying. The results obtained demonstrate that the drying processes reduce the amount of phenolic compounds and also the antioxidant activity in vitro. It is not uncommon for certain molecules, such as phenolic compounds, to be degraded by drying samples at temperatures around 40°C. Cruces et al. observed the same phenomenon with a brown algae and demonstrated that the method chosen to preserve the algae can have consequences on the extraction yield of natural compounds (in this case, phlorotannins) and on the biological activity.They also showed that drying is the worst process because it leads to a significant decrease in the antioxidant activity of the extracts.

[0080] In our case, the active molecules of the BuOH fraction also seem particularly sensitive to the algae drying process but not all active molecules seem to be affected in the same way, explaining why the heptane and EtOAc extracts show strong activity regardless of the process used. It would be relevant to verify whether these results are repeatable between batches of dried and frozen algae harvested in France.

[0081] In order to complete the analysis of the impact of the preservation processes, we wanted to ensure that the preservation of the algae at +4°C before freezing had no impact on the biological activity. And indeed the samples not preserved at +4°C and the samples preserved at +4°C for 24 h and 48 h do not show any difference in activity to inhibit the development of the larvae, whether at 5 g / L or 1 g / L.

[0082] What are the active molecules and modes of action involved in the biological activity of B. bifurcata?

[0083] Investigations into the active molecules involved

[0084] As described previously, we highlighted a difference in activity between the two batches of B. bifurcata from France (frozen) and Portugal (dried). The aqueous extracts from the Portuguese batch having a lower activity compared to the extracts from the French batch, this suggests that the active molecules are still present but in smaller quantities. In order to further investigate this hypothesis, we carried out different organic extractions (heptane, EtOAc and BuOH) and characterized their respective biological activities. The results showed that the fractions carrying the main biological activity were those obtained with heptane and EtOAc, regardless of their origin (France or Portugal) and the process used before the aqueous extractions (freezing or drying). This confirmed the presence of active molecules in the Portuguese batch of algae.It is worth noting that active molecules with antiparasitic activity against the protozoa Trypanosoma and Plasmodium have been previously identified in B. bifurcata and were also extracted using EtOAc. Our results are also in agreement with a previous study carried out on the brown algae Saccharina latissima and Laminaria digitata, showing that the organic fraction extracted with the alkane solvent (corresponding to hexane) also exhibited larvicidal activity against two species of GI parasites of sheep (Tela-dorsagia circumcinctd) and pig (A. suum).

[0085] Importantly, the results of the experiments involving the different organic fractions during the TDL also highlighted an interesting feature regarding the BuOH extract. Indeed, we observed a major difference in activity between the BuOH fraction of the French alga and that of the Portuguese alga (99% and 13% inhibition of larval development respectively at 1 g / L) suggesting that the anthelmintic properties of B. bifurcata could be based on different families of molecules. This confirms the particular interest of using aqueous extracts of B. bifurcata as a potential source of anthelmintics because they combine a range of active compounds potentially 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 the heptane, EtOAc and BuOH extracts of B. bifurcata. To verify this it will be necessary first to calculate the IC50 of these different fractions on the inhibition of larval development and then compare the IC50 with combinations of different fractions between them.

[0086] NMR analysis of the heptane fraction (which is very active in vitro) identified two linear diterpenes: eleganolone and bifurcanol. Regarding the molecules involved in the biological activity of B. bifurcata, many studies have focused on this alga and described a large number of its compounds. It is known to produce a large quantity of terpenoids, the structure of which is composed of several isoprene units (C5H8). Among these terpenoids, Biard et al. were the first to report the presence of linear diterpenes in B. bifurcata, including eleganolone as the major compound (2% of dry weight). Le Lann et al. also highlighted this major compound in this same algal species. They identified 4 different chemical profiles in extracts collected in different seasons and in different geographical areas.The presence of eleganolone does not appear to be affected by seasonal changes or environmental factors, but it can greatly affect other compounds. This terpene has already been described as having anti-protozoal activity against Trypanosoma and Plasmodium. Bifurcanol, on the other hand, is less well characterized in B. bifurcata but has already been isolated in a study demonstrating its antimitotic activity. This study also details the composition of B. bifurcata according to 3 different geographical areas and during the year. The results indicate a small variation in the diterpene ratio during the seasons but bifurcanol is not found in the same proportions depending on the harvest location. A review provides a very detailed account of the different linear diterpenes extracted from B.bifurcata, their extraction methods or even the pre-treatment of algae, as well as the biological activities reported to these different diterpenes, thus offering new perspectives for their use as anthelmintics.

Claims

Claims

1. A composition for oral anthelmintic use, said composition comprising eleganolone and / or a linear diterpene of bifurcanol type.

2. Composition according to claim 1, characterized in that said eleganolone or said diterpene of bifurcanol type is present within an extract of Bifurcaria bifurcata incorporated into said composition.

3. Composition according to claim 2, characterized in that said extract of Bifurcaria bifurcata is an aqueous extract of Bifurcaria bifurcata, an extract obtained by extraction of compounds of Bifurcaria bifurcata in a solvent or an extract obtained by extraction of compounds of Bifurcaria bifurcata in an oil.

4. Composition according to any one of claims 1 to 4, characterized in that it is effective against zooparasitic organisms of the nematode type of the suborder Rhabditina and / or Spirurina and in particular organisms of the order Ascarididae or Trichostrongylidae and even more particularly against organisms of the species Haemonchus contortus, Ascaridia suum, Parascaris equorum and Ascaridia galli.

5. A process for preparing an anthelmintic for oral administration comprising - preparing a composition comprising eleganolone and / or a linear diterpene of bifurcanol type - encapsulating said composition in yeast particles or incorporating it into a gelatinized starch matrix.

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