studyng the effects of Cantharellus cibarius fungi on Opisthorchis felineus trematode and on parasite host – c57Bl/6 inbred mice

look for new drugs for the treatment of opisthorchiasis, with the maximum antiparasitic and minimal side effect. In this work, a potentially anthelmintic effect of the methanol extract of the golden chanterelle mushroom ( Cantharellus cibarius ) was investigated. In in vitro experiments, the significantly reduced mobility and survival rates of juvenile O. felineus specimens with increasing concen-trations (10–1 000 μg/ml) of the C. cibarius extract were shown. In in vivo studies, administration of the C. cibarius extract on the first day after parasitic infection of inbred C57BL/6 mice resulted in a decrease of the number of helminths in the bile ducts of the liver, evaluated 6 weeks after infection. In another series of experiments, administration of the C. cibarius extract for 7 days to mice infected with O. felineus for five weeks had no anthelmintic effect. In both cases, the state of the infected hosts, evaluated by a number of physiological and biochemical parameters (relative weight of organs, blood indices), did not dete-riorate, indicating that there was no adverse effect of the C. cibarius extract. The results obtained suggest that the C. cibarius extract might have anthelmintic properties if applied as parasite larvae excyst.

opisthorchiasis is a dangerous parasitic disease caused by trematodes in the family opisthorchiidae. one of the causes of this infection is the species Opisthorchis felineus, which is common in the Russian Federation and Western europe. The disease has a large number of complications and relatively few effective treatments, so nowadays it is relevant to look for new drugs for the treatment of opisthorchiasis, with the maximum antiparasitic and minimal side effect. In this work, a potentially anthelmintic effect of the methanol extract of the golden chanterelle mushroom (Cantharellus cibarius) was investigated. In in vitro experiments, the significantly reduced mobility and survival rates of juvenile O. felineus specimens with increasing concentrations (10-1 000 μg/ml) of the C. cibarius extract were shown. In in vivo studies, administration of the C. cibarius extract on the first day after parasitic infection of inbred C57BL/6 mice resulted in a decrease of the number of helminths in the bile ducts of the liver, evaluated 6 weeks after infection. In another series of experiments, administration of the C. cibarius extract for 7 days to mice infected with O. felineus for five weeks had no anthelmintic effect. In both cases, the state of the infected hosts, evaluated by a number of physiological and biochemical parameters (relative weight of organs, blood indices), did not deteriorate, indicating that there was no adverse effect of the C. cibarius extract. The results obtained suggest that the C. cibarius extract might have anthelmintic properties if applied as parasite larvae excyst.
Due to the largescale Opisthorchiasis distribution, duration of the disease and severe consequences caused in the infected humans and animals, constant search for drug treatment methods is conducted on laboratory animals. To this purpose, Opisthorchiasis is frequently modeled on golden hamsters -Mesocricetus auratus (Pinlaor et al., 2009;Pakharukova et al., 2015). However, these animals are not natural hosts for this trematodes. Some Opisthorchiasis studies are performed on knockout (Nair et al., 2011) and C57BL/6 inbred mice (Zelentsov, 1974;Avgustinovich et al., 2016;2017a, b, 2018. For O. felineusinfected C57BL/6 mice, a prolonged (up to six weeks) juvenile marita stage is typical, which distinguishes them from hamsters. It gives particular advantages in studying the chronic effect of agents on immature O. felineus maritae.
Praziquantel is the main and most effective agent for human opisthorchiasis treatment. Some authors assume that parasites develop resistance to this agent (Greenberg, 2014). Praziquantel also has side effects (Erko et al., 2012) and considerable cytotoxical influence on liver cells . Therefore, researching new compounds, effective against opisthorchiasis remains important. Fungi of Cantharellus genus, particularly C. cibarius, are considered as potential source of such agents. These fungi demonstrate antinematode properties in nature (Muszyńska et al., 2016). They are broadly use in traditional medicine (Cieniecka Rosłonkiewicz et al., 2007), because the fungus contains substances with potential therapeutic properties against various diseases (Valverde et al., 2015;Nyman et al., 2016). In vitro and in vivo studies revealed high antioxidant and anti inflammatory properties of fungi extracts, more pronounced in alcohol extracts than in aqueous extracts (Vamanu, Nita, 2014). Methanolic fungi extract has an advantage over aqueous extract due to its antimicrobial effect (Kozarski et al., 2015;Muszyńska et al., 2016), while antimicrobial activity of methanolic extract against Escherichia coli is 7 times higher than ethanolic extract (Aina et al., 2012). Singledose or chronic intraperitoneal introduction of C. cibarius methanolic extracts are used for mice, the dose range -200-800 mg/kg (Khalili et al., 2014. No studies of C. cibarius efficacy in treating opisthorchiasis infections have been performed. Thus, the study objective was to determine anthelminthic properties of C. cibarius methanolic extract in two modes of administering to mice of C57BL/6 inbred line: at the stage of introducing O. felineus (the first day of infection) and at the stage of parasites attached to biliary ducts (5 weeks of infection). Fungi extract effect upon O. felineus was also assessed in vitro. In addition, we intended to obtain and compare the chemical compositions of C. cibarius methanolic and ethanolic extracts.

Materials and methods
Animals. Mature male mice of C57BL/6 line were obtained from the Center for Genetic Resources of Laboratory Animals (RFMEFI61914X0005 and RFMEFI61914X0010) of the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences (Novosibirsk, Russia). The animals were kept in groups (3-6 animals) in standard cages 36 × 23 × 10 cm with light conditions -12:12 h (day:night), air temperature 24 °С, food and water ad libitum. All procedures were performed in accord with the Directives of the European Communities Council of 24 November 1986 (86/609/EEC), and in line with the standards of the Bioethics Commission of the "Institute of Cytology and Genetics" Federal Research Centre (No. 26 Protocol of 13.03.2015). O. felineus metacercariae were separated from naturally infected Leuciscus idus, inhabiting the Ob river within the boundaries of Novosibirsk. Metacercariae were washed with 0.9 % NaCl sterile solution and contained at 4 °С no longer than 24 h for in vivo experiments and up to 7 days in sterile phosphate buffer with added kanamycin antibiotic (25 µg /ml) for in vitro experiments.
Obtaining of C. cibarius extract. The extracts were obtained from C. cibarius fungi collected in the Novosibirsk region. The extracts were made using the method of T. Kukina with coauthors (2016). Fungi, dried at the temperature not exceeding 40 °С, were powdered on an electric mill, and then the raw material was sieved through 2 mm holes. A 50 g sample was loaded in a Soxhlet extractor for 20 h. Methanol, ethanol and ethanol with methanol reextraction were used as the extraction agents. The obtained substances were evaporated to dryness on a Buchi rotor evaporator (Switzerland) with water bath at 40 °С and reduced pressure (20-30 mm Hg) due to use of a waterjet pump. Methanolic extract was used in in vivo and in vitro experiments according to recommendations of other researchers (Aina et al., 2012). For in vivo studies C. cibarius extract was suspended in 10 % Tween 80, which is used in animal experiments as a solvent (Teufack et al., 2017). For in vitro studies, the extract was dissolved in dimethyl sulfoxide (DMSO).
In vitro studies. Excycstation of juvenile worms was achieved by adding 0.06 % trypsin solution (Sigma, USA) to metacercariae, keeping it for 15 min at 37 °С. Then the worms were washed five times with incubation medium [the composition: RPMI 1640, Lglutamine (Life Technologies, USA), antibiotics (100 µg /ml streptomycin + 100 unit/ ml penicillin, Sigma, USA), an antimycotic agent (25 µg / ml amphotericin В) and 1 % glucose] and put in wells of a standard cultural plate, containing 990 µl of the medium and 10 µl of the studied agent. Dry fungi extract was diluted to the required concentration in 100 % DMSO so that adding 10 µl to the medium made the concentration of DMSO 1 %, and the extract -10, 100 or 1000 µg /ml. There were two wells per concentration, each containing 60-80 juvenile O. felineus specimens. 1 % DMSO was added to the control wells. The plate was put in CO 2 incubator (37 °С, 5 % СО 2 ) for 24 h.
Mobility of juvenile worms was analyzed in 24, 72, 120 and 168 h after adding the agent based on the method of J. Keiser with coauthors (2013). Mobility was assessed visually using a Axiovert 40CFL microscope (Zeiss, Germany) according to a 4score scale (4 -active continuous worm movement, 3 -poorly pronounced, slow movements of the entire body, 2 -very rare movements, mostly limited to a single body part, 1 -complete immobility), and then normalized by the control group (1 % DMSO). A day after adding the agents, the average effective agent concentration, when complete immobility occurred in 50 % of all worms (IC 50 ), was calculated using CompuSyn 1.0 software (ComboSynInc).
In vivo studies. Two schemes of introducing C. cibarius extract to the infected animals were used. In the first series of experiments at 6 pm metacercariae in 0.1 ml saline were injected to half of the animals (100 larvae/mouse), and 0.9 % NaCl to the other half. On the next day at 10 am a single dose of C. cibarius extract solution or a solvent (10 % Tween 80) was introduced. C. cibarius extract was administered to half of the animals in each group, and the solvent to the other half. Each subgroup had 7-14 mice. In the second series of the experiments C. cibarius extract (or solvent) were administered to the infected animals daily during 7 days, 5 weeks after introducing O. felineus metacercariae. Two groups of 14-15 mice were used. The substances were administered to the mice intragastrically via special probes (Braintree Scientific, Inc., USA). A 600 mg/kg dose was chosen based on the literature data (Khalili et al., 2014).
During both experiments the animal body mass was measured every ten days. In 6 weeks after infection the mice were euthanized by decapitaton and the biomaterial for further studies was taken. Liver, spleen and thymus were weighted and the relative organ mass was calculated to gram of body mass. Liver was put in 0.9 % NaCl solution to calculate the number of parasites in biliary ducts and gall bladder and to determine their maturity state. For exposing worms and analyzing the state of biliary ducts, an Axiovert 40CFL light microscope (Zeiss, Germany) with ×4 magnification was used. The blood collected during decapitation was centrifuged at 3000 rpm, 4 °С, 20 min. Serum was separated and stored at -70 °С until performing the biochemical studies.
Studying biochemical composition of C. cibarius extracts. The content of protein, polysaccharides, phenol compounds, flavonoids and carotinoids in C. cibarius extracts were determined applying an earlier described method (Protsenko et al., 2018).
Statistics. Statistical processing of the data was made in Statistica 6.0 (StatSoft) software suit. Twoway analysis of variance (ANOVA) was carried out for the first scheme of administration: the first factor was "infection" (infectednoninfected), the second factor was "agent" (C. cibarius extract, solvent). To analyze the extract effect in the second administration scheme, oneway ANOVA was used. Body mass changes were evaluated by the Wilcoxon test criteria for paired comparison. The data were presented as the mean value ± error of mean. The results were considered statistically significant at p ≤ 0.05 and as a trend at 0.05 < p < 0.1. Parasite survival in the in vitro experiment was estimated with Kaplan-Meier method, the survival curves were compared using χ 2 . The curves differed at p ≤ 0.05.

In vitro study
In the first minutes after introducing the extract or 1 % DMSO no changes in worm appearance or activity were observed. In 24 h worm mobility in wells with 10, 100 and 1000 µg/ml extract concentration was 6.15 %, 7.69 % and 41.15 % below the control mobility respectively. Further on (in 72-120 h) worm mobility was gradually decreasing. In 120 h the group with 10 µg/ml of extract had 64 % less mobility than in the control group; in 100 µg/ml group -71.2 % less than in the control group; and in the 1000 µg/ml group complete termination of activity was observed. In 168 h most worms died. In 24 h after adding C. cibarius extract IC 50 was 1.58 µg/ml.
The appearance of parasites had changed under the agent influence: many worms in wells with 10 and 100 µg/ml extract doses had enlarged excretory bladder in 72 h ( Fig. 1, a, b), maintained further on. No such changes were observed in the control worms during the entire 168hour period (Fig. 1, d) and specimens with 1000 µg/ml extract concentration that had nearly all died by that time (Fig. 1, c).

In vivo study
Singledose C. cibarius extract administered on the first day after infection led to a statistically significant (F 1;20 = 4.36, p = 0.048) decrease of the number of parasites in biliary ducts of liver 6 weeks after infection (18.1 ± 1.5), in comparison with the control group (23.5 ± 2.2). No considerable changes in the appearance of the parasites separated from mice liver with and without introducing C. cibarius extract were found microscopically. The worms were mostly immature, with two properly visualized intestine branches. Both groups had an animal with one mature O. felineus specimen.
No significant effect of the "infection" and "agent" factors on the relative mass of liver and spleen in the analyzed groups was found; there was no interaction between the factors (Table 1) infected animals with administration of C. cibarius extract, in comparison with mice from the control group.
No impact of C. cibarius extract upon animal body weight gain in the 6 weeks of the experiment was discovered: mice in each subgroup were equally gaining weight that increased on average by 2.5-3 g.
In the second series of experiements intriduciton of C. cibarius extract for 7 days in 5 weeks after infecting did not cause any statistically significant change in the number of parasites (26.2 ± 0.89 -C. cibarius, 28.4 ± 1.85 -solvent; F 1;29 = 1.16, р = 0.292). At the same time, the worms separated from biliary ducts after intriducting solvent and C. cibarius extract were different: the latter had considerably enlarged excretory bladder (Fig. 2, a, b). All worms were immature, had properly visualised two intestine branches in all mice, except two in each group that had a single mature O. felineus specimen (Fig. 2, c).

C. cibarius extract composition
Composition of different C. cibarius extracts was analysed. It was found that metanolic extraction is better for separatng phenol compounds, and ethanol extraction -for carotinoids (Table 4). Reextraction by ethanol after methanolic extraction helps increase the number of polysaccharides and phenol compounds, but is accompanied by considerable protein loss. In all three types of extraction the equal and insignificant content of flavonoids was obtained.

Discussion
In the present work, the first data on anthelminthic activity of C. cibarius extracts on a model of O. felineusinduced opisthorchiasis in mice was obtained. Earlier, antiinflammatory effects (Vamanu, Nita, 2014), insecticide  activity (CienieckaRosłonkiewicz et al., 2007), antioxidant and hepatoprotective properties -decreased fibrotic liver changes associated with induced inflammation (Aina et al., 2012;Khalili et al., 2014Khalili et al., , 2015, as well as cytotoxic effects on cancer cells in vitro (Sari et al., 2017) for C. cibarius extracts were demonstrated. In view of the above, one might expect a possible inhibitory action of C. cibarius directly on helminths.
Our in vitro experiments proved that survival and mobility of juvenile O. felineus specimens reduces in accordance with increasing the dose of C. cibarius extract in the incubational medium. In 120 h after starting the experiments, actively moving worms were found only in the control group. Enlarged excretory bladder in parasites also indicate anthelminthic activity of C. cibarius: similar morphological changes were observed as an effect of praziquantel (Pakharukova et al., 2015). It is believed that praziquantel damages parasite's metabolism affecting membrane transport proteins, particularly, proteins of the parasite's excretory system (Greenberg, 2014). It is possible that C. cibarius also affects performance of the excretory system of O. felineus.
IC 50 of C. cibarius extract (1.58 mg/ml) is much higher than IC 50 of other clinically used agents such as praziquantel (IC 50 = 0.33 µg/ml for juvenile specimens and 0.14 µg/ml for adult specimens) (Pakharukova et al., 2015) or tribendimidin which is used to treat clonorchiasis (IC 50 = 0.05 µg/ml for adult specimens) (Keiser et al., 2013). It can be explained by low content of potentially anthelminthic compounds in used C. cibarius extract. Such substances in C. cibarius could be specific betaglucans as immunomodulators with cytotoxic effect on tumor cells (El Enshasy, HattiKaul, 2013;Valverde et al., 2015;Sari et al., 2017), ergosterol -fungal steroid with antioxidant properties, as well as phenol compounds (myricetin and catechine) involved in antioxidant processes Valverde et al., 2015;Muszyńska et al., 2016). Several authors emphasize high activity of flavonoids (Kozarski et al., 2015). The obtained composition of C. cibarius extract shows presence of a considerable number of polysaccharides, with prevalence of betaglucans (Muszyńska et al., 2016). Flavonoids and phenol compounds are present in smaller quantities. Ergosterol was discovered earlier in C. cibarius extracts by other researchers (Muszyńska et al., 2016).
The in vivo model demonstrated a prolonged juvenile stage of parasite development in mice in comparison with hamsters (Avgustinovich et al., 2017a(Avgustinovich et al., , 2018. Almost all parasites were immature: only two intestine branches were visible, and there were no gonades. In natural hosts (humans, cats), parasites mature in a month after infection (Beyer, 2005). It can indicate that mice have special resistance mechanisms to O. felineus infection. Mice groups with solvent and C. cibarius extract under both schemes of administering had only single mature worm specimen. Therefore, it can be assumed that the extract did not influence maturation of O. felineus specimens in case of natural worm development. Enlargement of worm excretory bladder after administering C. cibarius extract to mice, similar to those found in the in vitro study, corroborates the anthelminthic effect of C. cibarius extract on parasites.
It should be emphasized that singledose administration of C. cibarius extract on the first day after infecting caused a statistically significant reduction of the number of worms in the animals' biliary ducts. Therefore, possible preventative anthelminthic effect of C. cibarius extract on the model of O. felineusinduced opisthorchiasis is shown for the first time. There were no statistically significant changes in the number of parasites in the animals' biliary ducts when C. cibarius extract was administered for 7 days, 5 weeks after infection. Probably, some time after O. felineus migrate to biliary ducts, they become resistant to the effects of C. cibarius extract even under longer administration conditions. Thus, C. cibarius extract prevents worm attachment to the walls of biliary ducts and helps wash them out, but is not efficient against already attached parasites. However, we do not exclude that a higher concentration of one of the extract components could have the anihelminth effect at the later stages of infection.
C. cibarius extract did not effect changes in the relative mass of liver, spleen and thymus in intact as well as infected animals under both schemes of introduction of C. cibarius extract. It means that the extract does not have any apparent toxic effect upon host organisms. Since the infected animals that received C. cibarius extracts had increased relative thymus weight in comparison with noninfected animals that were administered the extract, it is possible to assume that C. cibarius extract can stimulate Тcell immunity of host in parasitic infections.
Singledose introduction of C. cibarius extract did not prevent normal body weight gain in animals, which implies absence of any negative effects upon the organism of hosts. The observed decreased mice body weight when C. cibarius extract was administered for 7 days is most likely related to an impact of daily stress from the substance administering procedure, which other authors also pointed out (Charmandari et al., 2005).
O. felineus infection increased activity of ALT, AST, and LDH. Such changes are noted in opisthorchiasis as well as other inflammatory processes in liver (Wonkchalee et al., 2012). Administering of C. cibarius extract did not normalize but also did not worsen these parameters. One can assume that singledose introduction of the extract does not have