Effect of Viscum album L. ssp. austriacum (WIESP.) Vollman on metronidazole resistant and sensitive strains of Trichomonas vaginalis

Effect of Viscum album L. ssp. austriacum (W

İESP.) Vollman on

metronidazole resistant and sensitive strains of Trichomonas vaginalis

H. Ozpinar

, N. Ozpinar

⁎

, N. Eruygur

a

Department of Pharmaceutical Botany, Cumhuriyet University Faculty of Pharmacy, Sivas 58140, Turkey

b_{Cumhuriyet University Animal Hospital, Sivas 58140, Turkey} c

Department of Pharmacognosy, Selcuk University Faculty of Pharmacy, Konya 42130, Turkey

a b s t r a c t

a r t i c l e i n f o

Article history: Received 1 March 2019

Received in revised form 6 May 2019 Accepted 2 July 2019

Available online 17 July 2019 Edited by V Steenkamp

Trichomonas vaginalis (T. vaginalis) is a parasitic protozoan that causes trichomoniasis. Metronidazole is the stan-dard treatment for trichomoniasis; however, metronidazole-resistant strains are implicated in an increasing number of refractory cases. Therefore, the discovery of an antiprotozoal agent that is effective on metronidazole-resistant T. vaginalis will prevent major health problems. In this study, we investigated the antiprotozoal effects of leaf, fruit and body extracts of Viscum album L. ssp. austriacum (VA) on metronidazole-resistant and -sensitive T. vaginalis protozoa. The VA used in our study was collected from 20 different pine trees in September and October. The leaves, fruits and bodies of the VA plants collected were separated and dry extracts were obtained with hexane, chloroform, n-butanol, ethyl acetate and water. The minimum lethal dose (MLD) of the metronidazole-sensitive strain, T. vaginalis ATCC50148 and the metronidazole-resistant strain, T. vaginalis ATCC50143 against metronidazole was tested in comparison with the plant extracts. In addition, GC– MS analysis was performed on the plant extracts that has antiprotozoal effects. Some of the substances identified from GC–MS analysis were purchased commercially and their antiprotozoal effects were further investigated. The VA leaf and fruit ethyl acetate extracts and body butanol extract had the most effect on T. vaginalis. GC–MS qualitative analysis detected 2,4-heptadienal, 3-methylsilane, and methylfuran. Of these substances, only 2-methylfuran was found to be effective on T. vaginalis strains. Ourfindings suggest that the 2-methylfuran found in Viscum album L. ssp. austriacum extracts may be an alternative chemotherapeutic agent to metronidazole.

© 2019 SAAB. Published by Elsevier B.V. All rights reserved. Keywords:

Viscum album Trichomonas vaginalis Metronidazole

1. Introduction

Trichomonas vaginalis (T. vaginalis) is a parasitic protozoan that causes trichomoniasis. Trichomoniasis is usually transmitted from per-son to perper-son through sexual contact. Therefore, the incidence of the disease is high in women who are sexually active. The parasite can live asymptomatically in the vagina for a long time in some infected people. Immunosuppression, HIV, cervical cancer, prostate cancer, use of contraceptives, and polygamy are predisposing factors for this disease (Lehker and Alderete, 2000).

Trichomonal infections have a cosmopolitan distribution and have been identified in all racial groups and socioeconomic statuses. Tricho-monas vaginalis is the most common non-viral sexually transmitted dis-ease (STD) in the world, with roughly 170 million cases annually (W.H.O., 2012).

Metronidazole is the standard treatment for trichomoniasis; how-ever, metronidazole-resistant strains have been detected in an increas-ing number of refractory cases (Kirkcaldy et al., 2012; Ozcelik et al., 2018; Schwebke and Barrientes, 2006; Snipes et al., 2000). Therefore, the discovery of an effective antiprotozoal agent on metronidazole re-sistant T. vaginalis could prevent major health problems.

The use of herbal drugs has been increasing in recent year. Currently, many plants are used for treatments for various diseases and can be as effective a treatment as synthetic drugs. Mistletoe, Viscum album L, (VA) is a semi-parasitic plant that is able to grow on several host trees; the Viscum spp. most frequently used for therapeutic purposes are the ones grown onfir, apple, pine, poplar, and oak trees (Bonamin et al., 2017). VA is a plant with various medical effects and has com-monly been used in traditional medicine for centuries to treat various neurological diseases, including epilepsy, hysteria, nervousness, hyster-ical psychosis, dizziness, and headaches (Szurpnicka et al., 2019). In vivo studies have shown the cytotoxic and growth-inhibiting effects of VA on a variety of adult tumour cell lines such as breast cancer (Marvibaigi et al., 2014), pancreatic cancer (Tröger et al., 2013), and leukaemia (Park et al., 2012) as well as paediatric tumour cell lines such as ⁎ Corresponding author at: Cumhuriyet University Animal Hospital, Sivas 58140;

Turkey.

E-mail address:necatiozpinar@gmail.com(N. Ozpinar).

https://doi.org/10.1016/j.sajb.2019.07.008

0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.

Contents lists available atScienceDirect

South African Journal of Botany

medulloblastoma (Zuzak et al., 2006), osteosarcoma (Kleinsimon et al., 2017), and Ewing sarcoma (Twardziok et al., 2016). Studies have shown that VA also has antibacterial and antifungal effects (Ertürk et al., 2004; Hussain et al., 2011) However, we found no studies on antitrichomonal activity in the literature.

In our study, the antiprotozoal effects were investigated for leaf, fruit, and body extracts of VA on metronidazole-resistant and -sensitive T. vaginalis protozoa. In addition, GC–MS analysis was per-formed on the plant extracts that had antiprotozoal effects. Some of the substances identified from GC–MS analysis were purchased com-mercially and their antiprotozoal effects were further investigated. 2. Material and method

2.1. Plant materials

The VAs used in our study was collected from 20 different pine trees in September and October. The collection location was the Camlibel re-gion along the Sivas–Tokat highway in Turkey near the coordinates of 39° 59′ 4″ N; 36° 30′ 28″ E meters altitude. The leaves, fruits and bodies from the collected VAs were separated, and dry extracts were obtained with hexane, chloroform, n-butanol, ethyl acetate and water.

2.2. Preparation of the plant extract

The dried plant materials were powdered using a grinder. The ex-traction was done at room temperature. 100 g of dried and grounded herbs were extracted with 80% ethanol (250 mL × 4) for 24 h with inter-mittent shaking. Then mixture was filtered through filter paper (Whatman, No.1). Thefiltrates were combined together and concen-trated under vacuum on a rotary evaporator (Buchi R-100 equipped with Vacuum Pump V-300 and Control unit I-300) at 40 °C until dry and then dissolved in distilled water. The aqueous extract was fractioned by successive solvent extractions with hexane, chloroform, n-butanol, ethyl acetate and water (Fig. 1). All of the fractions obtained through solvent extractions were then evaporated to until dry and stored at−20 °C until use.

Plant Materials (100 g)

Extraction with Ethanol (3 Times)

Ethanol extract

Water suspension fractionation with n-hexane

Fractionation with n-butanol

Hexane fraction Residual water suspension

Chloroform fractionation

Chloroform fraction _{Residual water suspension}

Residual water suspension Ethyl acetate fraction

Ethyl acetate fractionation

n-butanol fraction Water fraction Fig. 1. Preparation of the plant extract.

Table 1

T. vaginalis viability rates after exposure to Viscum album extracts for 12 h. Plant section Extraction solvent Study doses (mg/mL) 10 5 2.5 1.25 Control T. vaginalis ATCC 50143 Leaf Ethyl acetate 0⁎ 10.103 _17.103 _18.103 _18.103 Butanol 0⁎ 10.103 16.103 18.103 Hexane 8.103 12.103 15.103 18.103 Chloroform 8.103 10.103 14.103 18.103 Water 6.103 9.103 14.103 18.103 Body Ethyl acetate 0⁎ 9.103 16.103 18.103 Butanol 0⁎ 8.103 _11.103 _18.103 Hexane 6.103 9.103 13.103 18.103 Chloroform 8.103 10.103 15.103 18.103 Water 8.103 10.103 15.103 18.103 Fruit Ethyl acetate 0⁎ 4.103 9.103 18.103 Butanol 10.103 _14.103 _16.103 _18.103 Hexane 9.103 _12.103 _15.103 _18.103 Chloroform 10.103 13.103 16.103 18.103 Water 0⁎ 9.103 11.103 18.103 T. vaginalis ATCC 50148 Leaf Ethyl acetate 0⁎ 0⁎ 6.103 18.103 Butanol 0⁎ 0⁎ 6.103 18.103 Hexane 10.103 _12.103 _12.103 _18.103 Chloroform 10.103 _13.103 _13.103 _18.103 Water 8.103 10.103 11.103 18.103 Body Ethyl acetate 0⁎ 0⁎ 5.103 18.103 Butanol 0⁎ 0⁎ 5.103 18.103 Hexane 8.103 10.103 11.103 18.103 Chloroform 7.103 _9.103 _12.103 _18.103 Water 6.103 8.103 10.103 18.103 Fruit Ethyl acetate 0⁎ 0⁎ 6.103 18.103 Butanol 4.103 6.103 8.103 18.103 Hexane 4.103 7.103 9.103 18.103 Chloroform 6.103 7.103 9.103 18.103 Water 0⁎ 0⁎ 6.103 _18.103

2.3. Trichomonas vaginalis culture and determination of antiprotozoal effect

The metronidazole-resistant T. vaginalis ATCC 50143 and the metronidazole-sensitive T. vaginalis ATCC 50148 strains (obtained from American Type Culture Collection (ATCC)) were used in this study. 2.4. Culture of Trichomonas vaginalis

Trichomonas Broth (TB, Liofilchem, 610061) medium was purchased commercially and was prepared according to the manufacturer's instruc-tions. After preparation of the TB, it was distributed among the experi-mental tubes and placed in the autoclave at 121 °C for 15 min, then cooled to 37 °C, and 10% inactive horse serum (Sigma, 1234598765) was added to the medium. The T. vaginalis strains were added to the TB medium and incubated for 3 d at 37 °C under anaerobic conditions. 2.4.1. Anti-trichomonas assay

The metronidazole minimum lethal dose (MLD) for the metronidazole-sensitive strain, T. vaginalis ATCC 50148, and the metronidazole-resistant strain, T. vaginalis ATCC50143 was tested in comparison with that of the plant extract. For this purpose, 96-well plates were used. The T. vaginalis strains produced from seeding in the TB medium at 37 °C were incubated in metronidazole (Sigma, 1711544348111) concentrations of 400, 200, 100, 50, 25, 12.5, 0.6, and 0.3μM as well as plant extract concentrations of 10, 5, 2.5, and 1.125 mg/mL. After 12 and 24 h, the incubated live protozoa were checked on a Thoma slide for movement offlagella and undulating

membranes and they were also were counted in a 1% eosin solution. A dose where no live parasites were found (determined microscopically) was accepted as the MLD.

2.4.2. GC–MS analysis

For GC–MS determinations, a system combining a GC 7890A with a quadrupole MS 5975C (Agilent Technologies, Waldbronn, Germany) was used. The injection volume was 1μL (splitless injection; injection port temperature 250 °C). A (5%-phenyl) methylpolysiloxane column, 30 m × 0.25 mm i.d., 0.25μm film thickness (DB-5 ms from Agilent) was used, and the carrier gas was helium. The temperature of the column started at 50 °C for 2 min, was raised to 120 °C at a rate of 50 °C/min, then was raised to 260 C at a rate of 3 °C/min, then was held for 2 min, then was raised to 300 °C at a rate of 15 °C/min, andfinally held for 4 min. The sub-stances in VA extracts were detected with electronic ionisation (70 eV) in the scan mode (mass range 20_{–600 GeV). The components were} identi-fied by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 11 mass spectral database.

GC_{–MS analysis was performed on the extracts from the plant parts} for which the anti-trichomonas effect was observed. Some of the sub-stances detected by GC–MS analysis were purchased commercially (2,4-heptadienal (Sigma 180548), 3-methylsilane (Sigma 197734), 2-methylfuran (Sigma W417920)). The antiprotozoal tests were also done with these substances. The GC–MS analysis was performed by the Giresun University Central Research Laboratory Application and Re-search Centre, Giresun, Turkey.

2.5. Statistical analysis

The data obtained in the study were evaluated using SPSS v 22.0 software. The 2 × 2 sets were evaluated with the Fisher exact test and a value of p_{b .05 was accepted as statistically significant.}

3. Results

3.1. Effects of VA extracts on metronidazole-resistant and -sensitive T. vaginalis strains

After 12 h of exposure of the metronidazole-resistant T. vaginalis strain to leaf and body ethyl acetate and butanol extracts, the minimum lethal dose (MLD) value was determined to be 10 mg/mL. In addition, the MLD value of the fruit ethyl acetate and water extracts was also 10 mg/mL. When these data were compared with the control group, the difference was found to be statistically significant (p b .05, Table 2

T. vaginalis viability rates after exposure to Viscum album extracts for 24 h. Plant section Extraction solvent Study doses (mg/mL) 10 5 2.5 1.25 0 T. vaginalis ATCC 50143 Leaf Ethyl acetate 0⁎ 0⁎ 18.103 _19.103 _20.103 Butanol 0⁎ 10.103 18.103 20.103 Hexane 8.103 13.103 15.103 20.103 Chloroform 8.103 11.103 16.103 20.103 Water 6.103 9.103 15.103 20.103 Body Ethyl acetate 0⁎ 2.103 16.103 20.103 Butanol 0⁎ 0⁎ 10.103 20.103 Hexane 5.103 10.103 10.103 20.103 Chloroform 6.103 11.103 14.103 20.103 Water 5.103 8.103 18.103 20.103 Fruit Ethyl acetate 0⁎ 0⁎ 6.103 19.103 Butanol 8.103 _11.103 _16.103 _20.103 Hexane 6.103 12.103 15.103 20.103 Chloroform 9.103 12.103 16.103 20.103 Water 0⁎ 3.103 14.103 20.103 T. vaginalis ATCC 50148 Leaf Ethyl acetate 0⁎ 0⁎ 3.103 19.103 Butanol 0⁎ 0⁎ 4.103 _18.103 Hexane 6.103 _9.103 _12.103 _20.103 Chloroform 8.103 12.103 11.103 20.103 Water 8.103 9.103 15.103 20.103 Body Ethyl acetate 0⁎ 0⁎ 1.103 18.103 Butanol 0⁎ 0⁎ 2.103 19.103 Hexzane 5.103 _9.103 _15.103 _19.103 Chloroform 6.103 _8.103 _14.103 _20.103 Water 5.103 6.103 10.103 19.103 Fruit Ethyl acetate 0⁎ 0⁎ 4.103 20.103 Butanol 0⁎ 5.103 8.103 19.103 Hexzane 0⁎ 7.103 10.103 18.103 Chloroform 4.103 _6.103 _8.103 _20.103 Water 0⁎ 0⁎ 2.103 _20.103

⁎ The difference is significant compared to the control group (p b .05).

Table 3

Selected compounds detected with GC–MS in Viscum album extracts. Active substance

name

Plant section

Solvent Retention time (min.)

Peak area % 2,4-heptadienal Body Ethyl acetate 15.166 0.09

Body Hexane 15.475 0.11

3-methylsilane Body Butanol 4.941 0.01 Body Ethyl acetate 5.444 4.27

Body Hexane 4.884 4.98

Fruit Butanol 10.194 0.04

Fruit Ethyl acetate 5.468 2.12 Fruit Chloroform 11.985 0.02

Fruit Water 12.316 0.43

Leaf Butanol 11.195 0.03

Leaf Ethyl acetate 6.091 0.06

Leaf Hexane 6.039 5.98

Leaf Water 38.889 0.50

2-methylfuran Leaf Butanol 15.132 0.98 Leaf Ethyl acetate 10.943 1.63

Body Butanol 4.941 1.34

Body Ethyl acetate 14.119 1.04 Fruit Ethyl acetate 12.403 2.25

Table 1). From the evaluation on the metronidazole-sensitive T. vaginalis strain, the MLD value of the ethyl acetate and butanol extracts of leaves and bodies was determined to be 5 mg/mL. In addition, MLD value was 5 mg/mL for ethyl acetate and water fruit extracts. When these data were compared with the control group, the difference was found to be statistically signi_{ficant (p b .05,}Table 1).

The MLD values of the metronidazole-resistant T. vaginalis strain after 24-h exposure were 5 mg/mL for the ethyl acetate leaf extract, 10 mg/mL for the butanol leaf extract, 5 mg/mL for the ethyl acetate fruit extract, 5 mg/mL butanol body extract and 10 mg/mL water fruit extract. When these data were compared with the control group, the differences were found to be statistically significant (p b .05,Table 2). From the evaluation on the metronidazole-sensitive T. vaginalis strain, the MLD values of the ethyl acetate and butanol extracts of leaves and bodies were all 5 mg/mL. In addition, the MLD value was 5 mg/mL in ethyl acetate and water fruit extracts and 10 mg/mL for fruit butanol and hexane extracts. When these data were compared with the control group, the differences were found to be statistically significant (p b .05, Table 2).

The viability rates of T. vaginalis strains exposed to different concen-trations of metronidazole for 12 and 24 h are shown inTable 7. Accord-ingly, the MLD value of metronidazole-resistant T. vaginalis strain was N400 μM at 12 and 24 h, whereas the MLD value of metronidazole-sensitive T. vaginalis strain was found as 0.6μM.

3.2. GC–MS analysis

According to the GC–MS analysis of prepared plant extracts, 2,4-heptadienal was only found in body ethyl acetate and hexane extracts. In addition, 3-methylsilane was found in butanol, ethyl acetate, and hexane extracts of the bodies; the butanol, ethyl acetate, chloroform, and water extracts of the fruits; and the butanol, ethyl acetate, hexane, and water extracts of the leaves. Additionally, 2-methyl furan was found in the butanol and ethyl acetate extracts of the leaves and bodiess, as well as in the ethyl acetate and water extracts of the fruit (Table 3). 3.3. Effects of 2,4-heptadienal, 3-methylsilane, and 2-methylfuran on met-ronidazole-resistant and -sensitive T. vaginalis strains

The substances identified from the GS-MS analysis were purchased commercially, and the effects on metronidazole resistant and suscepti-ble T. vaginalis strains at concentrations of 1.6, 0.8, 0.4 and 0.2 mM were tested. At 12 and 24 h after the treatment, the MLD value of 2-methylfuran for the metronidazole-resistant T. vaginalis strain was 1.6 mM, and the MLD value for the metronidazole-sensitive T. vaginalis strain was 0.8 mM. When these data were compared with the control group, the differences were found to be statistically significant (p b .05,Tables 4 and 5). However, 2,4-heptadienal and 3-methylsilane had no effect on T. vaginalis strains after both 12- and 24-h treatment pe-riods (Tables 4 and 5).

According to the results of qualitative GC–MS, only 2-methylfuran was found to be effective on T. vaginalis strains. For this reason, the amount of plant material and extracts determined by GC–MS analysis

was determined. The ethyl acetate fruit extract had the highest 2-methylfuran content at 1.9 ppm, which was determined based on the GC_{–MS data (}Table 6).

4. Discussion

The use of plant-derived drugs as an alternative to synthetic drugs is quite common in developed countries and especially in developing countries. Turkey is one of the most important and rich centres of the world in terms of plant resources due to geography and topography cre-ating different climatic zones varying from cold temperate to subtropi-cal, which in turn contributes to a rich plantflora. VA is a semi-parasitic plant that is able to grow on several host trees; the mistletoe types most frequently used for therapeutic purposes are the ones grown onfir, apple, pine, poplar and oak trees. A broad range of biologically active substances have been identi_{fied in VA and include viscotoxins,} flavo-noids, triterpene acids, and mistletoe lectins (Twardziok et al., 2016). Significant evidence has been found that VA lectins have anticancer properties that enable activation of the apoptotic process (Kim et al., 2000).

There is not enough information about the possible anti-parasitic ef-fects of this plant in the literature. In a study byMoncho et al. (2012) with Viscum verrocosum, the anti-helminthic properties of the dry ex-tracts of this plant on Tswana goats were tested, and a significant de-crease was observed, especially in the number of faecal eggs. It has been suggested that the anti-helminthic effect may be due to the tan-nins of this plant species. Another study was conducted in Konya, Cen-tral Anatolia Region, Turkey. In the study, the bodiess and leaves of the Viscum album L. ssp. austriacum subspecies were used to kill stom-ach and intestinal parasites in ruminants (Yasar et al., 2015).

Metronidazole is the standard treatment for trichomoniasis; how-ever, metronidazole-resistant strains have been cultured from an in-creasing number of refractory cases. Recently, metronidazole was found to be unsuccessful in some patients. According to studies in the United States, metronidazole resistant T. vaginalis has been reported at rates varying between 4.3% and 9.6%, as determined by conventional methods (Kirkcaldy et al., 2012).Kirkcaldy et al. (2012)evaluated 538 patients in six different cities in the United States between 2009 and 2010. All the patients were recorded as HIV-negative and 3.0% were pregnant. Using conventional methods, metronidazole resistance was determined to be low (23 cases identified, 4.3% of the total sample) (Schwebke and Barrientes, 2006). In another study in Finland, 10 clini-cal isolates tested positive for metronidazole resistance using Table 6

Amounts of 2-methylfuran in Viscum album extracts.

Substance Plant section Solvent Amount (ppm)

2-methylfuran Leaf Butanol 0.85

Leaf Ethyl acetate 0.92

Body Butanol 1.1

Body Ethyl acetate 1.3

Fruit Ethyl acetate 1.9

Fruit Water 1.8

Table 5

T. vaginalis viability rates after exposure to 2,4-heptadienal, 3-methylsilane and 2-methylfuran for 24 h.

Substance Study doses (mM)

1.6 0.8 0.4 0.2 Control T. vaginalis ATCC 50143 2,4-heptadienal 19.103 _19.103 _20.103 _20.103 _20.103 3-methylsilane 19.103 20.103 20.103 20.103 2-methylfuran 0⁎ 3.103_⁎ 5.103_⁎ 19.103 T. vaginalis ATCC 50148 2,4-heptadienal 20.103 20.103 20.103 20.103 3-methylsilane 19.103 19.103 20.103 20.103 2-methylfuran 0⁎ 0⁎ 4.103⁎ 18.103

⁎ The difference is significant compared to the control group (p b .05). Table 4

T. vaginalis viability rates exposed to 2,4-heptadienal, 3-methylsilane and 2-methylfuran for 12 h.

Substance Study doses (mM)

1.6 0.8 0.4 0.2 Control T. vaginalis ATCC 50143 2,4-heptadienal 17.103 _18.103 _18.103 _18.103 _18.103 3-methylsilane 17.103 17.103 18.103 18.103 2-methylfuran 0⁎ 5.103 5.103 18.103 T. vaginalis ATCC 50148 2,4-heptadienal 17.103 18.103 18.103 18.103 3-methylsilane 17.103 17.103 18.103 18.103 2-methylfuran 0⁎ 0⁎ 4.103 18.103

conventional methods under aerobic conditions; of these, 3 (30%) resis-tant strains were identified (Meri et al., 2000). In a study in Iran, T. vaginalis was observed in 15 (2.2%) of 683 patients. Using metronida-zole assays and conventional methods,five of the isolates (33.3%) were determined to be metronidazole-resistant (Rabiee et al., 2012). In a 2018 study in Turkey, metronidazole resistance was tested using conventional and molecular methods and resistant isolates were deter-mined to occur at a rate of 33.3% (Ozcelik et al., 2018). We did notfind any study on the antiprotozoal effect of VA on T. vaginalis in literature. In our study, we investigated the antiprotozoal effects of leaf, fruit and body extracts of VA on metronidazole-resistant and -susceptible T. vaginalis protozoa. In recent years, cases of metronidazole-resistant trichomoniasis have increased. However, there is no alternative chemo-therapeutic to metronidazole in the treatment of trichomoniasis. The development of alternative chemotherapeutics for the drugs used could be very important for preventing the development of resistance. There are studies on the biological activity of furan and its derivatives in the literature. These studies are related to the antibacterial (Rahmathullah et al., 1999; Tripathi et al., 2010), antifungal (Pour et al., 2001), antiprotozoal (Das and Boykin, 1977) and antitumoral (Dong et al., 2009) effects of the furans. Ourfindings suggest that 2-methylfuran found in VA extracts may be a chemotherapeutic agent al-ternative for metronidazole.

Financial Interest

The authors listed above have nofinancial interest with any com-pany or organisation in the subject matter or materials discussed in this manuscript.

Edited by V Steenkamp. Acknowledgements

This study was funded by the Cumhuriyet University Scientific Re-search Projects Unit (ECZ-33).

References

Bonamin, L.V., de Carvalho, A.C., Waisse, S., 2017.Viscum album (L.) in experimental ani-mal tumors: a meta-analysis. Exp. Ther. Med. 13, 2723–2740.

Das, B.P., Boykin, D.W., 1977.Synthesis and antiprotozoal activity of 2, 5-bis (4-guanylphenyl) furans. J. Med. Chem. 20, 531–536.

Dong, Y., Shi, Q., Liu, Y.-N., Wang, X., Bastow, K.F., Lee, K.-H., 2009.Antitumor agents. 266. Design, synthesis, and biological evaluation of novel 2-(furan-2-yl) naphthalen-1-ol derivatives as potent and selective antibreast cancer agents. J. Med. Chem. 52, 3586–3590.

Ertürk, Ö., Kati, H., Yayli, N., Demirbağ, Z., 2004.Antimicrobial activity of Viscum album L. subsp. abietis (Wiesb). Turk. J. Biol. 27, 255–258.

Hussain, M.A., Khan, M.Q., Hussain, N., Habib, T., 2011.Antibacterial and antifungal poten-tial of leaves and twigs of Viscum album L. J. Med. Plants Res. 5, 5545–5549.

Kim, M.-S., So, H.-S., Lee, K.-M., Park, J.-S., Lee, J.-H., Moon, S.-K., Ryu, D.-G., Chung, S.-Y., Jung, B.-H., Kim, Y.-K., 2000.Activation of caspase cascades in Korean mistletoe

(Viscum album var. coloratum) lectin-II-induced apoptosis of human myeloleukemic U937 cells. Gen. Pharmacol. Vasc. S. 34, 349–355.

Kirkcaldy, R.D., Augostini, P., Asbel, L.E., Bernstein, K.T., Kerani, R.P., Mettenbrink, C.J., Pathela, P., Schwebke, J.R., Secor, W.E., Workowski, K.A., 2012.Trichomonas vaginalis antimicrobial drug resistance in 6 US cities, STD Surveillance Network, 2009–2010. Emerg. Infect. Dis. 18, 939.

Kleinsimon, S., Kauczor, G., Jaeger, S., Eggert, A., Seifert, G., Delebinski, C., 2017.ViscumTT induces apoptosis and alters IAP expression in osteosarcoma in vitro and has syner-gistic action when combined with different chemotherapeutic drugs. BMC Comple-ment. Altern. Med. 17, 26.

Lehker, M.W., Alderete, J.F., 2000.Biology of trichomonosis. Curr. Opin. Infect. Dis. 13, 37–45.

Marvibaigi, M., Supriyanto, E., Amini, N., Majid, A., Adibah, F., Jaganathan, S.K., 2014. Pre-clinical and Pre-clinical effects of mistletoe against breast cancer. Biomed. Res. Int. 2014.

Meri, T., Jokiranta, T.S., Suhonen, L., Meri, S., 2000.Resistance of Trichomonas vaginalis to metronidazole: report of thefirst three cases from Finland and optimization of in vitro susceptibility testing under various oxygen concentrations. J. Clin. Microbiol. 38, 763–767.

Moncho, T., Madibela, O., Machete, J., Bonang, V., Motlhanka, D., Books, R., OER, R., SCARDA, R., 2012.Effect of crude extracts of Viscum verrocosum treatment on nema-tode parasite faecal egg count in female Tswana goats. Third RUFORUM Biennial Meeting, pp. 24–28.

Ozcelik, S., Ozpinar, N., Karakus, S., Akyildiz, F., Karakaya, O., 2018.Metronidazole resis-tance in Trichomonas vaginalis determined by molecular and conventional methods. Trop. Biomed. 35, 188–194.

Park, Y.-K., Do, Y.R., Jang, B.-C., 2012.Apoptosis of K562 leukemia cells by Abnobaviscum F®, a European mistletoe extract. Oncol. Rep. 28, 2227–2232.

Pour, M.,Špulák, M., Buchta, V., Kubanová, P., Vopršalová, M., Wsól, V., Fáková, H., Koudelka, P., Pourová, H., Schiller, R., 2001.3-phenyl-5-acyloxymethyl-2 H, 5H-furan-2-ones: synthesis and biological activity of a novel group of potential antifungal drugs. J. Med. Chem. 44, 2701–2706.

Rabiee, S., Bazmani, A., Matini, M., Fallah, M., 2012.Comparison of resistant and suscepti-ble strains of Trichomons vaginalis to metronidazole using PCR method. Iran. J. Parasitol. 7, 24.

Rahmathullah, S.M., Hall, J.E., Bender, B.C., McCurdy, D.R., Tidwell, R.R., Boykin, D.W., 1999.

Prodrugs for amidines: synthesis and anti-pneumocystis carinii activity of carba-mates of 2, 5-bis (4-amidinophenyl) furan. J. Med. Chem. 42, 3994–4000.

Schwebke, J.R., Barrientes, F.J., 2006.Prevalence of Trichomonas vaginalis isolates with re-sistance to metronidazole and tinidazole. Antimicrob. Agents Chemother. 50, 4209–4210.

Snipes, L.J., Gamard, P.M., Narcisi, E.M., Beard, C.B., Lehmann, T., Secor, W.E., 2000. Molec-ular epidemiology of metronidazole resistance in a population of Trichomonas vaginalis clinical isolates. J. Clin. Microbiol. 38, 3004–3009.

Szurpnicka, A., Zjawiony, J.K., Szterk, A., 2019.Therapeutic potential of mistletoe in CNS-related neurological disorders and the chemical composition of Viscum species. J. Ethnopharmacol. 231, 241–252.

Tripathi, R.P., Yadav, A.K., Ajay, A., Bisht, S.S., Chaturvedi, V., Sinha, S.K., 2010.Application of Huisgen (3+ 2) cycloaddition reaction: synthesis of 1-(2, 3-dihydrobenzofuran)-2-yl-methyl [1, 2, 3]-triazoles and their antitubercular evaluations. Eur. J. Med. Chem. 45, 142–148.

Tröger, W., Galun, D., Reif, M., Schumann, A., Stanković, N., Milićević, M., 2013.Viscum album [L.] extract therapy in patients with locally advanced or metastatic pancreatic cancer: a randomised clinical trial on overall survival. Eur. J. Cancer 49, 3788–3797.

Twardziok, M., Kleinsimon, S., Rolff, J., Jäger, S., Eggert, A., Seifert, G., Delebinski, C.I., 2016.

Multiple active compounds from Viscum album L. synergistically converge to promote apoptosis in Ewing sarcoma. PLoS One 11, e0159749.

W.H.O, 2012.Global Incidence and Prevalence of Selected Curable Sexually Transmitted Infections-2008. World Health Organization.

Yasar, A., Sinmez, C.C., Aslim, G., 2015.Ruminant parasitic diseases and treatment methods at folklore of Konya area in Central Anatolia Region. Kafkas Univ. Vet. Fak. Derg 21, 1–7.

Zuzak, T.J., Rist, L., Eggenschwiler, J., Grotzer, M., Viviani, A., 2006.Paediatric medulloblas-toma cells are susceptible to Viscum album (mistletoe) preparations. Anticancer Res 26, 3485–3492.

Table 7

T. vaginalis viability rates after exposure to metronidazole for 12 and 24 h.

Time Strains Metronidazole concentrations (μM)

400 200 100 50 25 12.5 0.6 0.3 0 12 h T. vaginalis ATCC 50143 14.103 _16.103 _16.103 _17.103 _17.103 _18.103 _18.103 _18.103 _18.103 T. vaginalis ATCC 50148 0 0 0 0 0 0 0 6.103 24 h T. vaginalis ATCC 50143 16.103 18.103 19.103 19.103 19.103 20.103 20.103 20.103 20.103 T. vaginalis ATCC 50148 0 0 0 0 0 0 0 2.103