In vitro anti-parasitic activities of Pulicaria dysenterica and Lycopus europaeus methanolic extracts against Trichomonas gallinae

Introduction Avian trichomoniasis is a parasitic disease caused by the protozoan parasite Trichomonas gallinae. Parasites live mainly in the upper area of the digestive tract anterior to the gizzard (1). Depending on the strain virulence, they can live in the tissue of the head, thorax, or abdomen of various groups of birds, counting Columbiformes, Passeriformes, Psittaciformes and Falconiformes (2-4). Among these groups, the family Columbidae is known as the parasite’s main host, particularly the domestic pigeon (Columba livia domestica) has been considered responsible for the worldwide spread of T. gallinae (1). For more than 40 years, the 5-nitroimidazole drugs family, specifically metronidazole and tinidazole, have been used to treat the infections caused by certain gram-negative bacilli and anaerobic parasitic protozoa, especially trichomoniasis (5,6). Currently, metronidazole is the only drug approved for the treatment of T. gallinae. However, http://www.herbmedpharmacol.com doi: 10.15171/jhp.2018.19


Introduction
Avian trichomoniasis is a parasitic disease caused by the protozoan parasite Trichomonas gallinae.Parasites live mainly in the upper area of the digestive tract anterior to the gizzard (1).Depending on the strain virulence, they can live in the tissue of the head, thorax, or abdomen of various groups of birds, counting Columbiformes, Passeriformes, Psittaciformes and Falconiformes (2)(3)(4).Among these groups, the family Columbidae is known as the parasite's main host, particularly the domestic pigeon (Columba livia domestica) has been considered responsible for the worldwide spread of T. gallinae (1).For more than 40 years, the 5-nitroimidazole drugs family, specifically metronidazole and tinidazole, have been used to treat the infections caused by certain gram-negative bacilli and anaerobic parasitic protozoa, especially trichomoniasis (5,6).Currently, metronidazole is the only drug approved for the treatment of T. gallinae.However, the existence of resistant strains of T. gallinae to these medications has been observed since the 1990s (6)(7)(8).The first therapeutic failures have been described for treatment of avian trichomoniasis.Besides, some metronidazoleresistant strains have been found frequently (5)(6)(7)9).Therefore, alternative curative therapies are necessary to provide a substitute treatment for avian trichomoniasis.Recently, the applications of many plants extract instead of chemicals have been recommended as remedies against parasites (10)(11)(12)(13)(14). Substances of herbal origin may have anti-trichomonas activities and offer new hope in the treatment of trichomoniasis.Two of the promising antiprotozoa herbal sources are Pulicaria dysenterica and Lycopus europaeus.The genus Pulicaria, belonging to Asteraceae (Compositae) family, includes 100 species with a distribution from Europe to North Africa and Asia, particularly around the Mediterranean regions (15).Only five species of the genus grow in Iran, which includes P. dysenterica, P. arabica, P. salvifolia, P. vulgaris, and P. gnaphalodes (16,17).The chemical literature survey shows the presence of flavonoids, sesquiterpenoids, polyacetylenes, polyacetylenes, sesquiterpenoid lactones, diterpenoids, thymol derivatives, and caryophyllene derivatives in the members of the genus Pulicaria (18)(19)(20).Pulicaria dysenterica (Inula dysenterica), commonly called "Flea-bane" and "Meadow false fleabane", is a persistent plant with 10-30 cm high, with gold-yellow flowers.It is hermaphrodite and grows on sandy, stony places in Saudi Arabia, Iran, Afghanistan, and western Tibet (16).New compounds including methoxy-12acetoxycaryophylla, methoxycaryophylla-2, dimethoxy-12-acetoxycaryophyll-2, hydroxycaryophyll-2, and dihydroxycaryophyll-2 have been isolated from this plant (18,19).In Iran, the original source of this plant is Razavi Khorasan province.P. dysenterica commonly is used as a medicinal plant to treat severe heatstroke, diarrhoea, inflammatory, and leishmaniosis diseases (14).Moreover, it has several biological activities such as antioxidant and insecticide properties (21,22).Lycopus europaeus L. (Labiatae) also known as "gypsywort", is a genus of flowering plants of the Lamiaceae family, which is distributed in Europe, Asia, and the United States.Although active components of L. europaeus are not identified completely, some phytochemical studies on Lycopus spp.show that it contains phenolic compounds, cinnamic acid, rosmarinic acid, caffeic acid, gallic acid, luteolin-7-O-glucuronide, flavonoids, tannins, saponins, lithospermic acid, coumarins, saponins, alkaloids, sterols, caryophyllene, α-pinene, terpinene and terpenoids.Some studies on the extract of L. europaeus proved its analgesic and anti-inflammatory activities.L. europaeus is a perennial plant, which is conventionally suggested for treating mild hyperthyroidism.The anti-thyrotropic, antigonadotropic, antioxidant, antimicrobial and cardiotonic effects of this plant are attributed to phenolic compounds, mainly to derivatives of hydroxycinnamic and flavonoids (23)(24)(25)(26).This in vitro research was aimed to study the antitrichomonal activities of methanolic extracts of P. dysenterica and L. europaeus in the treatment of T. gallinae and compare the efficacy of these natural products to metronidazole as a standard anti-trichomonal drug.According to available information, this study is the first research demonstrating the potential anti-trichomonal activities of these plants.

Materials and Methods
In vitro cultivation of parasite Trichomonas gallinae trophozoites were taken by sterile swabs from the oral cavity, oesophagus and the crop of diseased domestic pigeon (C.livia domestica) with clinical features of trichomonosis and the oral cavity inoculated on Hollander's modification of trypticase yeast extract maltose (TYM) complete medium (pH 6.5).The TYM medium contained 10 g of trypticase peptone, 5 g of yeast extract, 2.5 g of maltose, 0.5 g of K 2 HPO 4 , 0.5 g of KH 2 PO 4 , 0.5 g of potassium chloride (KCL), 0.5 g of L-ascorbic acid, 0.5 g of potassium bicarbonate (KHCO 3 ), 0.05 g of ferrous sulfate (FeSO 4 ), and 0.02 g of agar (Merck, Germany) per 300 mL of distilled water.After sterilization of the medium for 15 minutes at 121°C, 30 mL foetal calf serum (Hyclone, USA) and antibiotic/antimycotic solution containing 0.15 μg/mL streptomycin, 0.15 μg/mL penicillin, and 0.15 μg/ mL amphotericin B, (Sigma-Aldrich, Vienna, Austria) were added to the medium (27,28).Following incubation at 38°C, motile trichomonads were monitored daily by light microscopy.Isolates were sub-cultured every 48 hours by transferring 500 µL of cultured medium into a new sterile 15 mL Falcon, containing 5 mL of fresh medium.T. gallinae cells were counted using a haemocytometer (Neubauer Improved, bright line; Germany) and adjusted to 2 × 10 7 /mL in the working suspension.
Preparation of methanolic extract Aerial parts of P. dysenterica and L. europaeus were collected from rural areas of Mazandaran province.The plants were identified by experienced botanists at the faculty of medicinal plants, Amol University of special modern technologies.The collected plants were washed thoroughly with distilled water and crushed into small pieces to facilitate drying.The pieces of plants were dried at room temperature under shade for three weeks.Finally, the dried plants were powdered using an electric blender and a fine powder was obtained.Plant extracts were obtained by the following procedures: Three grams of each plant was suspended in 80 ml of 80% methanol (v/v).Suspensions were homogenized by vortex for 5 minutes, and the mixtures were placed in an ultrasonic bath (Elmasonic S40H, 340 W, 37 kHz) at 30ºC and sonicated for 1 hour.Finally, the extracts were dried using a vacuum rotary evaporator in a water bath at 40°C and dried samples were weighed and transferred into the microtubes and stored at 4°C until the time of the experiments (29,30).

In vitro anti-trichomonas assay
To explore anti-trichomonas effects of P. dysenterica (200, 100, 50, 25, 12.5 and 6.25 mg/mL) and L. europaeus (227, 113.5, 56.75, 28.37.14.1 and 7.09 mg/mL) different extract concentrations were diluted in phosphate buffer saline (PBS) and added to the sterile Eppendorf tubes.Approximately 1×10 7 trophozoites/mL T. gallinae was added to prepare concentrations of each extract.Metronidazole (100 µg/mL) was utilized as a positive control and PBS was utilized as a negative control.The growth of T. gallinae was observed 0, 1, 3, and 6 hours after treatment at 38°C.Each concentration was replicated three times.

Data analysis
In every sample, in each time live cells of T. gallinae were counted using hemocytometer slide.The active parasites and those with moving flagellum were considered as live cells.Percentage of growth inhibition (GI %) was calculated and reported using the Eq. 1 where a and b are a; mean numbers of viable parasites in control tube and mean b number of viable parasites in a test tube, respectively (31).Eq. 1: GI% = a-b/a ×100.

Statistical analysis
The study was conducted as factorial based on a randomized design with three replications.Analysis of variance (ANOVA) procedure followed by Duncan's test using SPSS 16 (SPSS Inc., USA) software was applied to determine the significant differences between treatment means.P values of ≤0.01 were considered significant.

Results
The results showed that both methanolic extracts of P. dysenterica and L. europaeus were able to reduce the viability of T. gallinae trophozoites by causing cell death at each of the six concentrations.The alive trophozoites were decreased remarkably with enhancing the concentrations of extracts and exposure time.In general, there was a little difference between the effectiveness of the higher concentrations of methanolic extract of P. dysenterica (200, 100 and 50 mg/mL) and L. europaeus (227, 113.5 and 56.57mg/mL) with these concentrations at 1 hour after exposure (Figures 1 and 2, respectively).In the highest concentrations of P. dysenterica (200 mg/mL) immediately after adding trophozoites to the dilutions, the motile of trophozoites were decreased and growth inhibitory effect was 10%.Exposure to the methanolic extract of P. dysenterica at 200, 100, and 50 mg/mL concentrations resulted in 100% cell dead within 1 hour after treatment (Figure 1).Also, 100% GI was detected with concentrations of 25, 12.5, and 6.25 mg/ mL after 3 and 6 hours after exposure to the methanolic extract of P. dysenterica (Table 1).Moreover, in a dose-dependent and time-dependent manner, the L. europaeus methanolic extract at all different concentrations had significant anti-trichomonal effects (P ≤ 0.01) even immediately after exposure to trophozoites of T. gallinae (Table 2).According to Table 2, L. europaeus treated culture showed 60% inhibition of the growth of parasite with 227 mg/mL concentration in incubation at 0 hours.The growth inhibitory effect of L. europaeus methanolic extract at 227, 113.5, and 56.57mg/mL concentrations was 100% at 1 hour after treatment.The L. europaeus methanolic extract in lower concentrations

Percentage of Growth Inhibition
Time (h) 200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 6.25mg/ml Metronidazole showed different results.With a concentration of 28.37 mg/mL, 1.66, 5.01 and 35.01%prevention of the parasitic growth was found after 0, 1 and, 3 h, respectively and complete inhibition of growth (100%) 6 hours after exposure.Besides, with both concentrations of 14.1 and 7.09 mg/mL, 1.66% inhibition of the parasitic growth was observed after 0, 1, and 3 hours of treatment.At the concentration of 14.1 mg/mL, deterrence of growth was 30% after 6 hours.Moreover, at the lowest concentration (7.09 mg/mL), 15% inhibition of growth was found after 6 hours (Table 2).
No remarkable reductions in the number of trophozoites were observed in the control samples.Each concentration showed the statistically significant difference (P ≤ 0.01) compared to parasite control and metronidazole groups.

Discussion
Trichomonas gallinae causes severe tissue lesions in the crop and oesophagus of birds.The only drugs recommended for treating trichomoniasis are metronidazole and a related 5-nitroimidazole, tinidazole, which have been utilized over 40 years.Many studies on clinical resistance dysenterica and 60% GI with a 227 mg/mL concentration of L. europaeus was observed instantly following therapy (0 hours).In lower concentrations of methanolic extract of P. dysenterica (25, 12.5 and 6.25 mg/mL) and L. europaeus (28.37, 14.1 mg/mL) longer incubation period (3 and 6 hours) was required to obtain complete loss of viability and motility, while the 7.09 mg/mL concentration of L. europaeus needed 6 hours to completely inhibit the trophozoites growth.The effect of P. dysenterica and L. europaeus on the inhibition growth of T. gallinae showed a statistically significant difference compared to parasite control and metronidazole.The results of all extracts revealed that the inhibition of trophozoites growth and trophozoites motility depended on the concentration and incubation time.These findings are in agreement with the results of Youssefi et al who reported similar inhibitory effect using Artemisia sieberi, and findings of Seddiek et al using garlic against T. gallinae trophozoites in vitro and in vivo assays (12,34).Consequently, the higher the concentration of the extract and the longer the application time, the more significantly lethal effects of the extract.
In conclusion, the present study is the first in vitro investigation evaluating the efficacy of the methanolic extract of P. dysenterica and L. europaeus against T. gallinae trophozoites.Results support our perspective for the possibility of using the methanolic extract of P. dysenterica and L. europaeus as anti-trichomonas agents at several concentrations and can suggest the potential use of these plants for treating metronidazole-resistant isolates of T. gallinae.However, more comprehensive studies are needed to survey antitrichomonal activities of methanolic extracts of P. dysenterica and L. europaeus in vitro and in vivo conditions.

Table 1 .
Effect of Pulicaria dysenterica methanolic extract on the growth of Trichomonas gallinae trophozoites at different incubation periods statistically significant difference in comparison to parasite control in the same time interval.** P<0.01, statistically significant difference in comparison to metronidazole 100 µg/mL in the same time interval.Values having no common superscript are significantly different (P < 0.01).Effect of methanolic extract of Lycopus europaeus on Trichomonas gallinae trophozoites in TYM medium following 0, 1, 3 and 6 hours after treatment.
(32,33)e drugs have been well documented; for example, nitroimidazole resistant of isolates of T. gallinae was found in Belgium, Spain, and the United States(32,33).

Table 2 .
(26)20)of methanolic extract of Lycopus europaeus on the in vitro growth of Trichomonas gallinae trophozoites after different exposure time statistically significant difference in comparison to parasite control in the same time interval.**P<0.01,statisticallysignificantdifference in comparison to metronidazole 100 µg/mL in the same time interval.Values having no common superscript are significantly different (P < 0.01).studies of Pulicaria spp.andL.europaeusindicatedthattherewere various bioactive components in these plants, which might be responsible for their biological activities.Asghari et al and Zahabi et al reported that P. gnaphalodes extract had a good leishmanicidal effects and it seems that the leishmanicidal activity is mostly associated with the existence of components such as alpha-pinene, borneol, 1,8-cineole, p-cymene, geraniol, and thymol in the essential oils, which have already exhibited leishmanicidal activity(14,20).Several studies presented an outline of chemical constituents of different Lycopus species.Peng et al isolated various compounds from L. lucidus Turcz and L. europaeus, including terpenoids, flavonoids, phenolic acids, and steroids(35).Fialova et al demonstrated the anti-staphylococcal activity of L. europaeus leaves water extract on clinical Staphylococcus aureus strains.Data obtained in their study showed that rosmarinic acid and luteolin-7-O-glucuronide were considered involved in biological activities(26).According to other studies, biologically active compounds from plant material are mostly affected by dose and incubation time.Under the empirical conditions, increasing the concentrations of extracts from lowest to the highest dose increased the mortality rate of trophozoites of T. gallinae.On the other hands, inhibition of trophozoites growth and trophozoites motility in lower concentrations increased with a constant incubation time.