Evaluation of the effect of some medicinal plants on cultured Trichomonas Vaginalis

Introduction: Trichomoniasis is a worldwide sexually transmitted disease caused by Trichomonas vaginalis. It inflicts severe complications to the human genitourinary system. The devastating negative effects and the emergence of resistance to common medication impose the search for safer and effective alternatives. This research aimed to investigate the effect of the Allium sativum, Nigella sativa crude extracts (NsCE) and the combination between their most effective doses with metronidazole. Methodology: Vaginal swabs were obtained from symptomatic patients, and cultured on Diamond's medium. Assessment of various concentrations of these herbs at different follow-up periods was done by counting the number of dead T. vaginalis trophozoites using the hemocytometer and trypan blue staining. Transmission electron microscope study was done. Results: NsCE 9 mg/mL yielded the highest lethal effect on T. vaginalis trophozoites after 72 hours, compared with metronidazole. Combination of NsCE 9 mg/mL and metronidazole 50 μg/mL gave the best result. Additionally, Tomex90 μg/mL, represents a tolerable effect after 72 hours, but metronidazole 100 μg/mL still has higher effect. These results were confirmed by the ultrastructural changes observed in T. vaginalis trophozoites, signifying severe damage of nucleus and cytoplasm with large vacuolization and cell membrane defects. Conclusions: NsCE is a promising anti-Trichomonas especially its combination with metronidazole which showed a high synergistic effect.


Introduction
Trichomoniasisis a common worldwide sexually transmitted disease caused by the flagellated protozoan Trichomonas vaginalis (T. vaginalis) [1]. It can be considered as a re-emerging infectious disease [2]. The prevalence of trichomoniasis estimated between 2012 and 2016 by WHO, is 110.4 million cases [3]. Trichomonas vaginalis affects the urogenital tract of male and female especially at childbearing period [4]. The disease stages vary from an asymptomatic carrier state to obvious vaginitis [5]. T. vaginalis infection is associated with cervicitis, urethritis and serious complications as pelvic inflammatory disease (PID) [1], infertility [6] cervical cancer and HIV transmission. In men, trichomoniasis causes urethritis, complicated with epididymitis, prostatitis and infertility. In addition, T. vaginalis may be associated with cancer prostate [7]. Although, there are different methods for diagnosis of this infection, as staining techniques, immunochromatography and nucleic acid amplification [8], the gold standard method for diagnosis of T. vaginalis is the culture on Diamond's medium [5].
The traditional drug for trichomoniasis is the metronidazole (MTZ) or tinidazole. It is an antibiotic and a member of the 5-nitroimidazole family [9]. Yet, it has many side effects [10] particularly when higher doses are needed in the steadily increased resistant cases. [11]. Metronidazole causes nausea, dizziness, induce hypersensitivity reactions and dermatological symptoms [12], in addition to its teratogenic and carcinogenic effects on fetus [13]. Currently, no alternative safe therapy is approved to overcome the serious side effects of the traditional drugs or treat the refractory cases of trichomoniasis. These facts emphasize the need for other safe effective treatment. Natural products are an attractive resource [2].
Allium sativum (Garlic) is a popular vegetable which is used as a spice and food additive [14] and in modern medicine [15]. Thiosulfinates, sulphurcontaining amino acids, and allicin are responsible for the therapeutic benefit of garlic [16]. These sulphur compounds have antimicrobial, anticancer, antioxidant, anti-inflammatory, cardio protective, antidiabetic and immunomodulatory activities [17]. They also produce glutathione which has antioxidant activity [18]. Garlic strengthens the activity of immune cells through the bioactive properties of allicin [19]. Moreover, it has an antiprotozoal effect [20]. Nigella sativa (N. sativa) is an ancient annual plant. It has therapeutic effects against infections caused by hepatitis C virus, and Helicobacter pylori [21].
Bearing in mind the need for an alternative safe antitrichomonal therapy, this study was planned to evaluate the different concentrations of garlic, the crude alcoholic extract of Nigella sativa and the combination between their highest effective doses and metronidazole as an anti-Trichomonas therapy at 24, 48,72 hours. Assessment of T. vaginalis trophozoites viability was done by trypan blue method. The results were confirmed by studying the ultrastructural changes of T. vaginalis trophozoites using Transmission Electron Microscope (TEM).

Parasite Culture
Vaginal swabs were taken from 90 females with suspected trichomoniasis based on signs and symptoms. Wet mount examination of vaginal swab was done within 2 hours [22] or the samples were preserved in amies transport medium for 24-48 hours [23]. From 90 examined samples, only15 positive samples were found for study. The parasites were axenically grown on modified trypticase-yeast extract-maltose (TYM) Diamond's medium of pH: 6.2 [24] and Broth medium [25] supplemented with fetal bovine serum, antibiotic mixture of penicillin and streptomycin at 37 °C. Maintenance of T. vaginalis stocks was done by subculturing [26]. Examination of these subcultures was done on Diamond's medium ensuring the viability of T. vaginalis trophozoites by an inverted microscope.

Preparation of Drugs and Herbs
MTZ was obtained in the form of tablets 250 mg (Flagyl, Sanofi-Aventis, Egypt). Tablets were crushed and dissolved in distilled water, then diluted in the culture medium to yield the concentrations of 25 µg/mL, 50 µg/mL and 100 µg/mL [27]. Garlic was obtained as 200 mg (Tomex, Sekem) then 30, 60 and 90 µg/mL were prepared [27]. N. sativa crude extract (NsCE) was prepared from air dried seed, the grounded seeds were soaked in aqueous methanol 85% (1/10w/v) then filtered and the plant residue re-extracted with 50% methanol. Removal of methanol by rotatory evaporator below 40% was used [28].

Experimental design
T. vaginalis trophozoites were cultured on Broth and Diamond's media to decide the suitable media for this study. Cultures treated with different concentrations of MTZ were tested and the highest effective dose was used as a positive control. T. vaginalis trophozoites were incubated (5×10 4 cells /tube) with each herb as duplicate for 24, 48 and 72 hours. Both the MTZ treated culture and the control cultures of the parasites (containing the parasite only), were submitted to the same procedure used for the extracts cultures. The combination groups were done by adding half the dose of MTZ and the highest effective dose of the used herbs.

Ethical approval
This experimental random sample study was performed at Medical Parasitology, Gynecology and Obstetrics Departments and Scientific and Medical Research Centre (ZSMRC), Faculty of Medicine, Zagazig University. Written informed consents were taken from all patients before sample collection. The study approval was obtained from the Institutional Review Board (IRB) Unit, Faculty of Medicine, Zagazig University (IRB#:4290/14-1-2018).
Antitrichomonal assessment a) Inverted microscope was used to examine each tube after addition of the drug or herb in 24 hours, 48 hours and 72 hours. b) The haemocytometer and trypan blue staining were used to count the number of dead trophozoites in each tube after 24 hours, 48 hours, and 72 hours [29]. c) Transmission electron microscope (TEM) was used to detect the ultrastructural changes of the parasite in the culture. Trophozoites were fixed with glutaraldehyde and cacodylate buffer. Then the samples were prepared to be examined in a JOEL1200EXII electron microscope [30].

Statistical analysis
The collected data were computerized and statistically analysed using SPSS program (Statistical Package for Social Science) version 25.0 and the ANOVA.

Results
Comparing the modified Diamond's with Broth media, it was better concerning the survival of T. vaginalis trophozoites. Each concentration of MTZ showed very highly significant difference (P < 0.001) at different follow-up periods, whereas, there was an insignificant difference between different concentrations of metronidazole at the follow-up period except at 72 hours, Met 100 µg/mL showed the highest mean number of dead T. vginalis trophozoites, so it was used as the positive control (Table 1).
There was a statistically significant difference between all Tomex concentrations and the positive control after 24and 72 hours as well as between Tomex 90µg/mL and the positive control at 48 hours. Also, there were statistical significant differences between 24, 48 and 72 hours' readings for Tomex 30 µg/mL, highly statistically significant differences for Tomex 60 µg/mL and highly statistically significant difference at Tomex 90 µg/mL, which represent the best result in all Tomex concentrations after 72 hours, yet metronidazole 100 µg/mL still has higher effect. Also, there were a statistically significant difference between the different used concentrations of alcoholic N. sativa crude extract (NsCE): 3 mg/mL, 6 mg/mL and 9 mg/mL, with the positive control at all follow up periods. The NsCE 9 mg/mL gave the best results represented by the highest mean count of dead T. vaginalis trophozoites after 72 hours ( Table 2).
Regarding the combination between the highest effective dose of each herb and MTZ 50 µg/mL, the combination between NSCE 9 mg/mL and Met50 µg/mL gave the highest mean number of dead T. vaginalis trophozoites at different follow up periods (Table 3).
For demonstrating the trophozoite viability, trypan blue was used (Figure 1a and b). In TEM, the untreated cultured T. vaginalis trophozoites at 72 hours, showed a clear nucleus, well organized cytoplasm and well preserved cell membrane (Figure 2a). In contrast, the MTZ treated culture demonstrated an integral nucleus, centripetal displacement of organelles and a large vacuole although, the cell membrane, cytosome and the kinetoplast were intact (Figure 2a and b). The Tomex treated culture showed a homogenized nucleus with condensed chromatin, minimal cytoplasmic degenerative changes and distorted, plugged cell membrane (Figure2c and d).

Discussion
The present study is an in vitro trial to investigate the efficacy of different concentrations of commercially available garlic tablet (Tomex®) and NsCE, the combination between their highest effective doses with metronidazole against T. vaginalis infection at 24, 48,72 hours follow up periods.
Comparison between modified Diamond's and Broth media was done to get the most suitable medium for the growth of T. vaginalis. It was found that the mean number of dead trophozoites on modified Diamond's medium was less than Broth medium with statistical significant difference at all follow up periods. Therefore we settled on using the modified Diamond's medium. Our results agree with Gelbart et al. [31] who proved that modified Diamond's medium is recommended as the medium of choice for T. vaginalis.
Observing the effect of different Tomex concentrations (30 µg/mL, 60 µg/mL and 90 µg/mL) on cultured T. vaginalis. The concentration of 90 µg/mL has the highest mean number of dead trophozoites after 72 hours (47.00 ± 9.00) but MTZ 100 µg/mL still has higher effect (76.67 ± 25.50). Our results are compatible with Ahmed [32] and Ibrahim [27] who established that garlic is as efficient as metronidazole on reduction of trophozoites multiplication and motility. Furthermore, Alyasari et al. [33] stated that the growth rate, viability and motility of T. vaginalis were completely inhibited on using the aqueous garlic extract. The difference in results may be due to the use of commercial Tomex in the present study, but they used the aqueous extract ( Table 2). The effect of Tomex is due to allicin production [34] that leads to disruption of the normal physiological functions of the parasite [35].
Concerning the different concentrations of N.sativa, crude extract, The NsCE 9 mg/mL gave the highest mean count of dead T. vaginalis trophozoites (194.33 ± 98.01) after 72 hours compared with the positive control ( Table 2). These results agree with Tonkal [36] who found that NsCE has a remarkable inhibitory effect on the growth of T. vaginalis trophozoite. Also Mahmoud et al. [28] and Al-Ammash [37] pointed out that NsCE had an in vitro antitrichomanal effect. As the adhesion process plays a crucial role in the pathogenesis of Trichomonas, N. sativa has an anti-adhesion effect for T. vaginalis to human epithelial cells [38].
Comparing between the effect of metronidazole 100 µg/mL alone and its combination with the uppermost effective concentration of each herb (Tomex 90 µg/mL and NsCE 9 mg/mL) on the mean number of dead T. vaginalis trophozoites (Table 3), the combination between metronidazole and NsCE showed the best antitrichomonal effect (760.67 ± 400.020) paving the way towards a new era of treatment. The combination between drug and herb was also, practiced by Mady et al. [39] who emphasized that the combination between pyrimethamine and NSO against murine toxoplasmosis, was better than the herb alone. Also the combination done by Nassef et al. [40] between cisplatin, a cytotoxic drug, and NSO showed an anti-Trypanosome evansi. The explanation for these results could be that the herbs showed a synergistic effect when combined with drugs, as evidenced by Boullata [41] who highlighted that the co-administered herbs altered the drug concentration owing to the modification of intestinal and hepatic metabolizing enzymes.
Concerning the TEM study, it was found that the ultrastructure of the untreated T. vaginalis trophozoites (control group) confirmed an intact cell membrane, one nucleus, hydrogenosomes and few vacuoles. These findings are in accordance with Costamagna and Figueroa [42] who studied the ultra-structure of T. vaginalis in liquid cultures. The T. vaginalis Diamond media: negative control; Met 100µg/mL: positive control; SD: Standard deviation; P^: Repeated measure ANOVA test; P $ : ANOVA test; NS: Non significant (P > 0.05); *: Significant (P < 0.05); **: Highly significant (P < 0.001); Groups with different letters are statistically significant (P < 0.05).
trophozoites treated with metronidazole after 72 hours were swollen loosing much of the material within cytoplasm when compared to the non-treated group. The remaining part of the organelles (nucleus, some vesicles and few hydrogenosomes) seemed as a large vacuole with centripetal displacement (Figure 2b). These results are in agreement with Oxberry et al. [43] who established the loss of the cytoplasmic material, vacuolization and disruption of cytoplasmic membrane after the usage of metronidazole. Tomex treated culture after 72 hours in our research, T. vaginalis trophozoites lost their normal morphology with nuclear condensation, disorganization of internal organelles, abnormal vacuolization and cell membrane distortion (Figure 2c and d). This damage in the morphology may affect the virulence and pathogenesis of the trophozoites. T. vaginalis trophozoites treated with NsCE after 72 hours showed severe cell damage, destroyed nucleus, large vacuolization within cytoplasm and cell membrane defects that may influence the virulence of the parasite (Figure 2e and and f).

Conclusions
NsCE is promising as an anti-Trichomonas particularly its combination with MTZ that showed anactive synergistic effect. The Diamond's medium was superior to Broth medium for T. vaginalis culture.