Effect of Echinophora platyloba, Stachys lavandulifolia, and Eucalyptus camaldulensis plants on Trichomonas vaginalis growth in vitro.

BACKGROUND: Trichomonas vaginalis is a protozoan parasite which causes vaginitis in women worldwide. Metronidazole with vast side effects is drug of choice for this infection. In search for an alternative drug, in this study the effect of three plants on Trichomonas vaginalis has been investigated in vitro.
MATERIALS AND METHODS: Alcoholic and watery extracts of Echinophora platyloba, Stachys lavandulifolia, and Eucalyptus camaldulensis were prepared. In TYIS culture medium containing alive Trichomonas vaginalis different concentrations of extracts of three plants were added. Following, 24, 48, and 72 h incubation the number of parasite in each test tube was counted.
RESULTS: Eucalyptus camaldulensis showed a strong effect on Trichomonas vaginalis growth. However, no significant effect was observed with Echinophora platyloba or Stachys lavandulifolia extracts.
CONCLUSION: Eucalyptus camaldulensis can be considered as an alternative drug for treatment of infective vaginitis which is caused by bacteria, fungi and parasites.

INTRODUCTION

Trichomonas vaginalis, a protozoan parasite, is a common sexually transmitted infection in women and men.[12] It is usually transmitted by sexual relationship, by sharing contaminated underwear clothes or by nonhygienic vaginal examination.[3] In women, it causes vaginitis and cystitis and in men it causes urethritis and prostatitis.[4] Trichomonas vaginalis is considered as a common cause of vaginitis and as a causative factor for preterm birth and low birth weight.[5] It has also been associated with increased human immunodeficiency virus transmission.[6]

Metronidazole with vast side effects is now considered as a drug of choice for this infection with a cure rate of approximately 95%.[7] Clinical resistance to this drug has been reported since 1962.[8] Therefore, it would be very important to search for an alternative drug.

Echinophora plants have four species in Iran and distributed in the west and western north of Iran.[9] The genus Stachys is widespread throughout the world. In Iran, many species of this genus are present, from which, 13 are endemic. Stachys lavanduifolia has been used as an anxiolytic and sedative in Iranian folk medicine.[10] Analysis of the crude extracts of the Eucalyptus spp. revealed the presence of saponin, saponin glycosides, steroid, cardiac glycoside, tannins, volatile oils, phenols and balsam.[11]

Antibacterial, antifungal or antiparasitic effects of Echinophora platyloba,[12-17] Stachys lavandulifolia,[1819] and Eucalyptus camaldulensis[920] plants have been reported. In some local area of Iran these plants may be used for vaginal infection treatment. In this work, the effect of Echinophora platyloba, Stachys lavandulifolia, and Eucalyptus camaldulensis with antiparasitic activity[15] on Trichomonas vaginalis has been investigated in vitro.

MATERIALS AND METHODS

Echinophora platyloba , Stachys lavandulifolia were collected from Chahar Mahal va Bakhtiari mountains and Eucalyptus camaldulensis was collected form Khostan trees. All plants were collected in spring of 2010 and confirmed by experts in medical plant research center of Shahrekord university o f medical sciences. Leaves of those three plants were washed with water, dried, and then ground finely in a spice small electric mill. The resulting powders were mixed with ethanol or water to make extractions.

In order to prepare alcoholic extracts, 40 g of each dried powder plant was added to 1 L of 96% ethanol and left for 12 days. The mixture clarified by filtration through a sterile filter paper. The product was placed in the vacuum rotary evaporation device to remove solvent alcohol and it was dried in the 40°C oven.

In order to prepare watery extracts, 40 g of each dried powder plant soaked in 1 L distilled water and left for 48 h. The mixture clarified by filtration through a sterile filter paper. Vacuum rotary evaporation device was used to remove the solvent water. Stock solutions of dried extracts were reconstituted in 50% DMSO.

Trichomonas vaginalis parasite was isolated from vaginal discharge of women referred to Hajar hospital clinic in Shahrekord city, Iran and transferred to TYIS culture medium. A pooled of five parasite isolates was used for experiments. In test tubes containing 10 ml of TYIS culture medium different concentrations of dried extracts of three plants were added. In one tube metronidazole (5 μg/ml) was added as positive control and one tube left intact as negative control. Then 50 μl of medium containing about 100 live Trichomonas vaginalis were added to each tube. All tubes incubated at 37°C and the number of parasites in each tube was counted following, 24, 48, and 72 h incubation. For parasite counting, each tube was first shacked and 10 μl of it was observed on a microscope slide. Trichomonas vaginalis parasites with active flagella were considered alive. Each experiment was performed in triplicate.

RESULTS

In tubes contained metronidazole (5 μg /ml), 60 or 90 μg Eucliptus, no parasite was observed after 72 h, while in control test tubes parasite had a normal growth. Details of parasite counts in all test tubes have been presented in Table 1. No significant effect on Trichomonas vaginalis growth was observed when alcoholic or watery extract of aerial parts of Stachys lavandulifolia were used. Details of parasite counts of this experiment presented in Table 2. Also no significant effect was observed when Echinophora platyloba was used.

Table 1

Results of Trichomonas vaginalis counts in test tubes treated with Eucaliptus extract in comparison with appropriate controls

Table 2

Number of Trichomonas vaginalis in 20 μl of TYIS-33 culture medium 72 h after treatment with different concentrations of Stachys lavandulifolia extracts or metronidazole

DISCUSSION

Results of this investigation revealed that Eucalyptus camaldulensis but not Echinophora platyloba or Stachys lavandulifolia had a strong effects on Trichomonas vaginalis growth in vitro.

Antimicrobial, antifungal, and antiparasitic effects of Eucaliptus spp. have been shown in different investigations.[12131516] Adeniyi demonstrated that Eucaliptus camalddlensis had a strong effect on Helicobcter pylori in vitro.[12] Drug of choice for these bacteria is metronidazole, same drug for treatment of Trichomonas vaginalis. In another study Safaei et al. showed that essential oil of Eucaliptus spp. had a strong antimicrobial activities.[13] Nathan et al. also showed that essential oil of Eucaliptus spp. possess anti trypanosomal activity in vitro and this effects was dose dependent.[15] Ramazani et al. demonstrated antifungal activity of Eucaliptus spp.[16] Satorelli et al. investigated antimicrobial and antifungal activities essential oil of Eucaliptus Spp. They demonstrated that this plant presented the highest growth inhibition against Staphylococcus aureus, Esherichia coli, and Candida albicans.[17] Mahdi et al. studied effects of two plants, Myrtus communcs and Eucaliptus comaldensis, on Trichomonas vaginalis growth in vitro. They showed that Eucaliptus extract caused death of parasite at pH 5.3.[14] Results of this investigation are in agreement with our findings.

CONCLUSION

According to results of present investigation Eucalyptus camaldulensis plant could be considered as an alternative drug for Trichomonas vaginalis treatment. So it will be very worthwhile to recognize the efficient components of this plant with anti Trichomonas activities in further investigation. Moreover, antifungal and antiparasitic activities have been reported for Eucaliptus spp.[12-17] Therefore this plant has a potential to be considered as a unique drug for treatment of infective vaginitis which is caused by bacteria, fungi or parasites. Further investigations are recommended to test this hypothesis.