International Journal of Medical Laboratory

1. Department of Parasitology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Medical Parasitology and Mycology, School of Public Health, Tehran university of Medical Sciences, Tehran, Iran.
4. Department of Laboratory Sciences, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran.

Introduction
Natural products provide a large foundation of medications and are the basis of the drug compounds development. The therapeutic preparations from these plants, being available around, are inexpensive and easily affordable and are used more than chemical remedies in the treatment of different diseases [1]. Today, the number of proper herbal drugs used in the treatment of diseases in comparison to the total number of approved drug in the world is increasing. It is estimated that 250,000 to 500,000 species of plants exist on the Earth. Only 1 to 10% of these are used as foods by both humans and other animals. Therefore, it is conceivable that even more are used for therapeutic purposes [2]. Potential effects of plants as source of new antiprotozoal medications is proven by examples such as emetine, quinine and artemisinin obtained from Cephaelisi pecacuhana, Cinchona species and Artemisia annua, respectively [3]. Satureja hortensis (S. hortensis) and Medicago sativa (M. sativa) have found popularity as important herbal remedies against infectious diseases and for other medical purposes. S. hortensis has been traditionally used as stomachic, stimulant, expectorant, carminative, antidiarrheal, antioxidant, anti-inflammatory, sedative and aphrodisiac for the treatment of different types of infectious diseases [4]. M. sativa which is one of the most reputed medicinal plants is traditionally used to cure kidney pain, cough, antidiabetic, antioxidant, anti-inflammatory, antimicrobial and as well as central nervous system disorders. Phytochemical studies have shown flavonoids, alkaloids, phytoestrogens, coumarins, digestive enzymes, triterpenes, saponins and phytosterols on M. sativa plant [5]. Trichomonas vaginalis (T. vaginalis) is the agent of Trichomoniasis that is related with vaginitis, urethritis, cervicitis, prostatitis, epididymitis, cervical cancer, infertility, pelvic inflammatory, foul-smelling discharge, vaginal orurethral expulsion, pruritus and dysuria. This infection is prevalent in 5 to 74% of women and 5 to 29% of men with the estimate of 250 million patients worldwide. Current available drugs for parasitic diseases are effective in many cases. But they also have
some limitations. Metronidazole and tinidazole are recommended drugs for the treatment of human trichomoniasis. But, many side-effects
such as potential carcinogenic, mutagenicity, embryogenic and drug-resistant of T. vaginalis have been described. Of course, common bad effects comprise headache, glossitis, urticaria, pruritus, vertigo, nausea, dry mouth, bitter metallic taste and vomiting [6]. Given the importance of the disease and the problems of controlling and combating due to sexual transmission, the drug resistance and the absence of appropriate and effective vaccines, seem to be the acceptable, safe, cheap and available medical approaches. Therefore, the present study aimed to examine the anti-trichomonas vaginalis effect of S. hortensis and M. sativa in vitro.
Materials and Methods
Preparation of crude extracts
Leaves of M. sativa and S. hortensis were dried under shade, and were powdered mechanically using a commercial electrical blender. To achieve the ethanolic extract, 50 gr of dry powder was added to 500 ml of ethanol 80% and mixed steadily for 1 hr using amagnetic stirrer. The gained solutions were left at room temperature for 72 hr. After filtration, the solvent was then removed by rotary evaporator. The gained filtrate (5.5 g) was put into a sterile glass and stored at -20°C for further use [7].
Parasites culture
The organisms used in this study were isolated from vaginal discharge of female patients attending to the health care centers of Shahrekord city, Iran. The isolates were cultured in modified Diamonds media and
kept in Parasitology Research Laboratory in Yazd University of Medical Sciences for examination. The protozoa were cultured axenically in modified Diamonds medium (pH 6.2) supplemented with 10% heat-inactivated bovine serum and incubated at 37°C with 5% of CO₂. Log phase culture of T. vaginalis was diluted with modified Diamonds medium for obtaining 10⁵ cells/ml. The parasites were used, showing normal motility and morphology during the logarithmic phase of growth.
In vitro susceptibility assays
To explore anti‑trichomonas effects of S. hortensis and M. sativa, the extracts were diluted with dimethyl sulfoxide with a final concentration of 0.1% in test solution and transferred to microtubes for providing final concentrations of 50, 100, 200, 400, 600, 800 and 1000 µg/ml. T. vaginalis trophozoites were incubated for 24, 48 and 72 hr at 37^◦C in the presence of different concentrations (50- 1000 µg/ml) of the ethanolic extracts and the total numbers of viable protozoa were assessed using a hemocytometer. The eosin stain with the concentration of 0.1% was used to check the viability of the trophozoites. The DMSO and metronidazole were used as negative and positive controls, respectively. The experiments were carried out in triplicate. All microscopic examinations were performed blindly by two investigators. Results of parasite counting have been described as percentage of growth inhibition (%GI). The numbers of parasites were compared with the positive and negative controls and the percentage of %GI was calculated for each experiment with respect to the growth control as follows:



The 50% inhibitory concentration was (IC50) calculated and figure was plotted using SigmaPlot™13 (Systat Software Inc, USA) [7]. This study was approved by the Ethics Committee of Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Statistical analysis
The normal distribution of data was evaluated by Kolmogorov–Smirnov (K-S) test. Afterwards, the interaction between time and concentration was calculated by repeated-measures analysis of variance for each extract. Additionally, two-tailed t test analysis based on different concentrations was used to reveal the statistical difference between each of the extracts and the negative control group at a given hour.
Results and Discussion
The results of Anti-trichomonas activity of various concentrations of M. sativa and S. hortensis were shown in figures 1 and 2, respectively. The IC50 values are shown in table 1 as well. For the positive control, metronidazole killed all the cells after 24 hr
of incubation time. Ethanolic extract of M. sativain in concentration of 600 μg/ml showed 80% GI during 72 hr and the concentrations of 800 μg/ml revealed more than 90% GI after 48 hr. Complete inhibition of growth (100% GI) of the trophozoites was seen in concentrations of 1000 μg/ml after 48 hr. 100% GI was shown in concentrations of 1000 μg/ml after 72 hr using ethanolic extract of S. hortensis. Also, more than 80% GI has been observed in concentration of 600 μg/ml after 72 hr. Regarding the effect of two evaluated extracts on T. vaginalis, there were significant differences between S. hortensis as well as M. sativa and their negative controls in various times (Fig. 2). Furthermore, for M. sativa compared to the negative control at 24, 48, and 72 hr were 0.02, 0.03 and 0.05 respectively. Then for the M. sativa and S. hortensis extracts, these were significant statistically. (p<0.05).

Time	IC50 (ug/ml) with 95% confidence
	Medicago sativain	Satureja hortensis
After 24 hr	474.38	539.96
After 48 hr	361.95	441.34
After 72 hr	197.43	396.99

Metronidazole is the choice treatment for trichomoniasis and most of intestinal protozoan diseases. Many studies indicated the resistance of this drug and its carcinogenic and teratogenic complications (especially in the first trimester). Also safety in pregnancy is unreliable. These defects had the scientists search for alternative drugs with fewer side effects [8]. There is a great need for available and inexpensive therapeutic agents particularly in the underdeveloped and developing countries. Natural products are the talented basis of active molecules that can be widely used for their minimal side effects, better acceptance of patient, as well as availability and non-toxicity profile [9]. This study was designed to survey the anti-Trichomonas vaginalis efficacy of S. hortensis and M. sativa in vitro. According to the results, both extracts tested in the current investigation indicated remarkable anti-Trichomonas activity against trophozoites of T. vaginalis. However, the M. sativa ethanolic extract was more effective than S. hortensis as it demonstrated a lower IC50 values against trophozoite of T. vaginalis with 100% GI at hour 48 and 72. The difference in the effect of two extracts on
the parasite can be explained by the presence of different chemical compounds in the plants. To date, many studies have been conducted around the activity of some medicinal plants against T. vaginalis [10]. Allium hirtifolium, Mikania cordifolia from Asteraceae family, Scutia buxifulia from Asteraceae family and Lobalia neurolarea from Rhamnaceae family, Carcia papaya, Cocos nucifera and Praneem polyherbal tablets are shown to have anti-Trichomonas activity in vivo and in vitro. Calzada et al. in their in vitro study examined the anti-Trichomonas activity of Mexican medicinal plants and showed that thyme
with a minimum inhibitory concentration of 126.4 μg/ml is ineffective for Trichomonas trophozoite [11]. In a similar study, Mirzaei et al. indicated the cytotoxicity of S. hortensis extract and demonstrated that S. hortensis has anti- leishmanial effect with IC50=298/42 ug/ml after 72 hr of exposure time [7]. Diba
et al. revealed that the alcoholic extract of S. hortensis can inhibit the growth of some species of Candida and Aspergillus in vitro and also can kill them in higher concentrations. Species of Satureja (S. khuzestanica, leaf) comprise effective compounds which can serve as an alternative agent in the control of leishmaniasis [12].
Conclusion
Based on the results, it is concluded that ethanolic extracts of S. hortensis and M. sativa are potent inhibitors of the growth of T. vaginalis. The current study provided valuable data as for new active products against T.vaginalis. It may act as a favorable anti-trichomonacidal remedy in the future. Additional investigations are recommended for isolation of active fractions and components.
Conflict of Interest
No conflict of interest is associated with this work.
Acknowledgment
The authors are thankful to the authorities in Shahid Sadoughi University of Medical Sciences, Yazd, Iran for their financial support.
 

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