@2024 Afarand., IRAN

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ISSN: 2252-0805    The Horizon of Medical Sciences 2018;24(1):35-39

Background
Giardia lambria, a factor of giardiasis, or fatty diarrhea (Steadtorrhea) is one of the most important pathogens in humans, that its cyst is transmitted by fecal-oral route. This protozoan, due to its feature in the active phase (trophozoite) proliferates and nourishes in the small intestine of humans and its cyst which is contaminating is excreted through feces [1. 2].
Previous Studies
… [2, 4]. Iran is reported as an endemic region for this species, and its prevalence has been reported in different regions [5-7]. Given the anticipation of parasite resistance to drugs reported from different parts of world and from Iran, preventing and treating of this disease, especially in children, is very important [8-10]. … [9, 10]. Currently, for the treatment of Giardia, various compounds such as nitroimidazole, benzimidazole, acridine, and nitro furan such as metronidazole and furazolidone are commonly used that these drugs all have adverse effects, especially in children and women [4, 9, 10]. On the other hand, there are reports of resistance of these parasites to these drugs especially metronidazole, and the carcinogenic and mutagenic effects of these drugs have been reported in animal models [3, 4. 10]. … [11-14].Therefore, to eliminate cysts and trophozoites, different methods are recommended that one of the methods suggested by WHO, is the use of natural plans and foods in the elimination of the parasite cyst [15].
Aim(s)
Considering the very beneficial effects of saffron that is one of the Iranian native plants, its availability, its abundant cultivation and harvest in Khorasan Razavi, especially Torbat-e-Heidarieh, and its domestic and industrial use, the importance of the issue, and no previous studies in this domain in Iran, we decided to investigate the lethal effect of this drug in vitro. Therefore, the aim of this study was to investigate the lethal effect of hydroalcoholic extract of saffron on Giardia lamblia cysts under vitro condition.
Research type
This study was experimental.
Research society, place and time
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Sampling method and number
To collect and isolate Giardia cyst, fresh stools with different consistency (firm, soft and watery) and contaminated (12 samples with high levels of contamination) of the referred people (asymptomatic and symptomatic) to the laboratories of 9th Dey Hospital and laboratory of Health Center of Torbat-e-Heydariyeh University of Medical Sciences were collected.
Used devices&materials
The samples were condensed with 0.85 molar sucrose method. For the separation of Giardia cyst, 10 to 15 gr of stool samples were mixed with 10 times water and blotted using a double layer gas. After blotting the samples, the obtained solution was centrifuged at 400 rpm for 5 minutes. The sediment was re-mixed with 10 times the volume of water and centrifuged at the same speed and time and 5 milliliter water was added to the sediment. To the above solution, 3 ml of pre-prepared o.85 molar sucrose solution was added slowly. The samples, then, centrifuged at 600 rpm for 10 minutes. From the three formed layers after centrifugation, the cysts were carefully collected from the first and second layers by Pasteur pipette and then prepared after washing twice with dilute water for testing. To determine the life of cysts, vital staining of cysts with 0.1% eosin was used. The number of live and dead cysts (stained) is determined by random counting of 100 Giardia cysts. In order to be more precise, each experiment was repeated five times. The percentage of death of the cysts was calculated from the following formula [16]: K%=[(MCC-MCT)/MCC] ×100 In the above formula, K%=the percentage of the death of cysts, MCC= mean number of live cysts in the control group, and MCT=mean number of live cysts in the test group. For cysts counting, Neubauer chamber was used. In addition, the samples can be stored at 4 ° C for performing the next step tests [17]. To extract saffron, this plant was collected from rural area of the city of Torbat-e-Heydariyeh and recorded by Dr. Ahmad Ahmadian at Torbat-e-Heydarieh Saffron Research Center. After confirmation, the amount of 500 gram of the plant was transferred to the laboratory after collection. The plant was dried under the room condition, away from the sun, and was turned into powder with electric grinder. In the next step, 300 g of the powder was dissolved in a solution of water and alcohol with equal ratio (50% water and 50% alcohol) and placed at room temperature for 24 hours. Since alcohol, in contrast to other solvents, separates more substances (both polar and non-polar compounds), absolute ethanol alcohol was used in this study. After smoothing the solution, the solvent (water and alcohol) was evaporated by rotary evaporator, and then the remaining material was dried to become powder, in which the amount of 1970 mg powder was obtained. At the final stage, the obtained powder was diluted in different concentrations of water and alcohol (1, 10, 50, and 100 mg/ml). For conducting the study, the results of effect of the plant were compared with the control group physiological serum) and the concentrations were determined according to the previous studies. In order to investigate the effect of plant extract on cysts, concentrations of 1, 10, 50, and 100 mg/ml of saffron were prepared. Then, 2 milliliter of each concentration was prepared in a separate test tube and to each test tube, 10000 Giardia lambia cysts were added. Finally, the effect of different concentrations of the plant on the cyst life at different times (one minute, 2 minutes, 30 minutes, one hour, 3 hours, and 24 hours) were evaluated. To determine the percentage of cysts life, the vital staining of cysts with 0.1 % eosin was used. Data were analyzed using SPSS 20 software and two-way ANOVA, multiple scales, and paired t-test.
Findings by Text
By increasing the concentration of hydroalcoholic extract of saffron and the contact time of the extract with Giardia lamblia, the mean lethal percentage was increased compared to the control group. This is, while, in the control group, the mean survival rate of Giardia cyst at different times was 0.5% to 3%. In the control group, with increase in time and concentration, a small percentage of cysts were lost, which was not statistically significant (p>0.05). The mean effect of hydroalcoholic extract of saffron on Giardia cysts, especially at concentrations of 50, and 100 mg/ml, after 3 and 24 hours i.e. with the time increase, increased and caused the loss of cysts. Although the mean percentage of effect of plant extract on cysts was different at each contact time, the difference was different at different concentrations at each time (p<0.05; Table 1).
Main comparison to the similar studies
… [8-10, 15, 18]. In the study by Shahabi et al., the lethal effect of hydroalcoholic extract and essential oil of Ajwain plant on Giardia lambia cysts was evaluated in vitro that after 60 minutes of contract between extract of essential oil with Giardia lamblia cysts, the minimum inhibitory concentrations of hydroalcoholic extract and essential oil were respectively 100 and 8 mg/ml after 60 minutes of contact, respectively 75 and 6 mg/ml after 120 minutes of contact, and respectively 75 and 4 mg/ml after 180 minutes of contact [13]. … [19]. Also, the results of research conducted by Farsangi at Boushehr University of Medical Sciences indicate that time has a very important effect on the killing of Giardia cysts by different herbal extracts [18]. In a study done by Esboei et al., hydroalcoholic extract of Gandana plant at 3 and 24 hours had the most lethal effect on Giardia cyst that is consistent with the results of the present study [20]. In a study by Rezaiemanesh et al., also, the lethal effect of aquatic and alcoholic extracts of Turkish Artemisia plant increased significantly with increasing concentration, time and temperature, which is consistent with the results of the present study [21].
Suggestions
It is suggested that the effect of hydroalcoholic extract of saffron be investigated in higher temperatures (37 ° C) and more concentrations of this study in order to obtain a more favorable result and more lethal effect of this extract.
Limitations
One the limitations of this study was the lack of possibility to test saffron extract on laboratory animals.
Conclusions
Hydroalcoholic extract of saffron is effective in eliminating Giardia lamblia cysts in vitro at concentrations of 50 and 100 mg/ml and at 3 and 24 hours.
Acknowledgments
Researchers need to thank the Vice-Chancellor of Research at Torbat-e-Heydarieh University of Medical Sciences and the staff of the Laboratory of Health Center and Saffron Research Institute in Torbat-e-Heydarieh.
Conflict of interest
Non-declared
Ethical Permissions
The protocol of this study was approved by the Ethics Committee in Biomedical Research at Torbat-e-Heydarieh University of Medical Sciences with ethics code IR.THUMS.REC.1394.10.
Funding Sources
This research has been sponsored by the vice-chancellor of Research in Torbat-e-Heydarieh University of Medical Sciences.

TABLES and CHARTS

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