Effects of different extracts of Eucalyptus camaldulensis on Trichomonas vaginalis parasite in culture medium.

BACKGROUND: Trichomonas vaginalis is considered one of the main causes of vulvovaginitis in women. Metronidazole with vast side effects is now the drug of choice for treatment of this infection. In an attempt to find an alternative drug, the effect of Eucalyptus camaldulensis on this parasite was shown in previous studies. In this investigation, the effect of different extracts of this plant on T. vaginalis in culture medium has been investigated.
MATERIALS AND METHODS: Five different extracts including total extract, diethyl ether, chloroform, ethyl acetate, and water fractions were prepared. The extracts were dried using vacuum rotary evaporator and then they were used for in vitro anti-trichomonas experiments.
RESULTS: Crude extract of E. camaldulensis showed 80% growth inhibition (GI) in a concentration of 12.5 mg/ml during 24 h. Diethyl ether extract in a concentration of 25 mg/ml showed 100% GI during 24 h. With ethyl acetate extract, 100% GI was detected with the minimum concentration of 12.5 mg/ml in the first 24 h. Finally, water extract in a concentration of 50 mg/ml showed 80% and 100% GI after 48 and 72 h, respectively.
CONCLUSION: Ethyl acetate fraction is the extract which showed the highest percentage of GI (100%) with the least concentration (12.5 mg/ml) after 24 and 48 h.

INTRODUCTION

Reported as a common medical problem in women, vulvovaginitis can lead to considerable discomfort and repeated medical visits. Infection, allergy, irritation, and systemic diseases can happen after vulvovaginitis. The most widespread causes of vaginal discharge before menopause are bacterial vaginitis, vulvovaginal candidiasis, and Trichomonas vaginalis.[1]

Trichomoniasis, the most prevalent nonviral sexual infection, with roughly 170 million new infected people worldwide per year, has been related to various discomforts such as pre-term delivery, high infant mortality, or low birth weight, and makes patients more susceptible to HIV infection.[2345]

The mainstay medication for trichomoniasis is metronidazole, but some resistant strains to this treatment have been detected;[67] thus, efforts need to be taken for finding new alternative drugs in order to control trichomoniasis.

For a long time, there is widespread acceptance of using medical herbs in both developing and non-developing societies because of their significant benefits like fewer side effects, better patient tolerance, lesser cost, and treating the main cause of disease.[8]

Eucalyptus, a large genus of Myrtaceae, represented by 900 species and subspecies, can be found the world over. Its leaves were used by Australians for its benefits like wound healing and for treating fungal infection.[910] In addition, the leaf extract of Eucalyptus has been confirmed to be used as a food additive. Also, in some cosmetics’ formulation, Eucalyptus extracts can be found. Based on researches, various biological effects of Eucalyptus, including antimicrobial, antihyperglycemic, and antioxidant activities, have been shown,[11] and it seems that the essential oils of Eucalyptus, containing different compounds such as terpenoid and phenolic ones, play an important role in its biological activities.[12] Moreover, it has been reported that the extracts of Eucalyptus camaldulensis have anti-trichomonas activity.[1314]

The aim of this study was to prepare phenolic and non-phenolic extracts of E. camaldulensis leaves and evaluate which extract fractions are more effective against T. vaginalis.

MATERIALS AND METHODS

Preparation of extract

The crude extracts were prepared by maceration of 500 mg of dried powder of leaves of E. camaldulensis, which were collected from Shooushtar area, a city in Khouzestan, Iran, in July 2011, in 50% methanol and 1% HCl 6 N for 3 days continuously at room temperature.

The solvents of extract were evaporated using rotary evaporator. Then, the crude extract was partitioned into distilled water and diethyl ether phases. The pH of aqueous phase was adjusted to 8-9 with 20% NaOH, which converted phenolic compound to its sodium salt. Then chloroform was used for extraction in order to remove the impurities. Then, the pH of residual aqueous phase was decreased to 3-4 using HCl 6 N and extracted with ethyl acetate to provide phenolic fraction. Five extracts were obtained including total extract, diethyl ether, chloroform, ethyl acetate, and water fractions. The extracts were dried using vacuum rotary evaporator. The dried extracts were used for in vitro anti-trichomonas experiments.[15]

Preparation of test microorganism

Test microorganism was obtained from parasitological lab of Isfahan University of Medical Science, which was isolated from vaginal discharge of female patients attending Obstetric and Gynecology Clinic in Shahrekord, Iran. In all experiments, a pool of four isolates was used. The parasite, T. vaginalis, was cultured in vitro at 37°C in TYIS33. Log phase culture of T. vaginalis was diluted with TYIS33 medium for obtaining 2 × 105 cells/ml.

In vitro anti-trichomonas assay

To explore anti-trichomonas effects of E. camaldulensis, the extracts were diluted with dimethyl sulfoxide (DMSO) or phosphate buffer (depending on their solvents) and transferred to Eppendorf tubes for providing final concentration of 50, 25, 12.5, 6.25, 3.125 mg/ml. The DMSO and phosphate buffer were used as negative control accordingly, and metronidazole at a concentration of 100 μg/ml was used as positive control. All tubes were incubated at 37°C. After 24, 48, and 72 h, the samples were taken from each tube and viable parasites were counted with hemocytometer. Complete active and flagella active parasites were considered as viable ones.

Results of parasite counting have been reported as percentage of growth inhibition (GI %) using the following equation in which “a” stands for mean number of viable parasites in negative control tube and “b” stands for mean number of viable parasites in test tube.[1617] For comparing GI in case and control tubes, statistical values and tests such as mean, standard deviation, and analysis of variance (ANOVA) test were used.

RESULTS

Crude extract of E. camaldulensis showed 80% GI in a concentration of 12.5 mg/ml during 24 h. Most of the growth inhibition was detected in a concentration of 50 mg/ml after 72 h. This is shown in detail in Figure 1.

Figure 1

Effect of crude extract of Eucalyptus camaldulensis on Trichomonas vaginalis in culture medium following 24, 48, or 72 h. Results are presented as mean ± SD, and ANOVA followed by post hoc test was used for statistical analysis

Diethyl ether extract of E. camaldulensis in a concentration of 25 mg/ml showed 100% growth inhibition during 24 h, and this extract in concentrations of 6.25 and 12.5 mg/ml resulted in more than 80% GI after 48 h. Following 24 h, the minimum concentration which showed 100% GI was 6.25 mg/ml. This is shown in detail in Figure 2.

Figure 2

Effect of diethyl ether extract of Eucalyptus camaldulensis on Trichomonas vaginalis in culture medium following 24, 48, or 72 h. Results are presented as mean ± SD, and ANOVA followed by post hoc test was used for statistical analysis

When ethyl acetate extract of E. camaldulensis was used, 100% GI was shown with the minimum concentration of 12.5 mg/ml, at the first 24 h. Also, more than 80% GI was detected with a concentration of 6.25 mg/ml after 72 h. This is shown in detail in Figure 3.

Figure 3

Effect of ethyl acetate extract of Eucalyptus camaldulensis on Trichomonas vaginalis in culture medium following 24, 48, or 72 h. Results are presented as mean ± SD, and ANOVA followed by post hoc test was used for statistical analysis

Water extract of E. camaldulensis in a concentration of 50 mg/ml showed 80% and 100% GI after 48 and 72 h, respectively, while at concentrations of 6.25 and 12.5 mg/ml it improved the survival of the parasite. This is shown in detail in Figure 4.

Figure 4

Effect of water extract of Eucalyptus camaldulensis on Trichomonas vaginalis in culture medium following 24, 48, or 72 h. Results are presented as mean ± SD, and ANOVA followed by post hoc test was used for statistical analysis

DISCUSSION

Considering the percentage of GI results showed in Figures 1 and 4, at concentrations of 50 and 25 mg/ml, diethyl ether and ethyl acetate extracts showed 100% of GI after 24 h. In these concentrations, the least GI % was shown by water fraction. When the concentration of 12.5 mg/ml was used, 100% of GI was observed in ethyl acetate fraction after 24 h. However, the same results were recorded for diethyl ether extract after 48 h. The interesting point to note is that water fraction improved the growth of T. vaginalis. Diethyl ether fraction, at a concentration of 6.25 mg/ml, was the only extract that caused 100% of GI after 72 h. Diethyl ether fraction in a concentration of 3.12 mg/ml revealed 50% and 68% of GI after 48 and 72 h, respectively.

It can be concluded that ethyl acetate fraction showed the highest percentage of GI (100%) at the least concentration (12.5 mg/ml) after 24 and 48 h (P > 0.05 in comparison to positive control. It seems the difference between the activities of the extracts can be due to the presence of different compounds related to the solvent polarity used to form the extract.

Ethyl acetate with a polarity of 4.4 extracted the polar compounds of E. camaldulensis including phenolic materials. It seems polar materials are more effective than semi-polar and non-polar compounds in E. camaldulensis. Also, other polar compounds like allicine and ajoene, which exist in Allium hirtifolium, could exhibit anti-trichomonas activity in comparison to metronidazole.[18] It has been reported in another study that berberin isolated from Berberis arisata had in vitro activity compared with metronidazole, on T. vaginalis.[19] Moreover, alcoholic extracts of Calendula officinalis and Echinacea angustifolia had in vitro efficacy against T. vaginalis.[20] Emodine present in the rhizome and root of Rheum palmatum manifested an inhibitory effect on T. vaginalis.[21]

Also, it is reported that ethyl acetate extract of Arbutus anedo leaves showed 100% GI at a concentration of 500 μg/ml.[22]

CONCLUSION

Growth stimulation was shown with lower concentration of water fraction in our study. It may be because of the presence of sugar and amino acids in this extract, which are water soluble, culture medium of the parasite contains also both sugar and amino acid. While 100% GI was reported after 24 h of T. vaginalis exposure to Nigella sativa aqueous extract in a concentration of 10 mg/ml,[17] the N. sativa water extract was prepared directly from the plant, but the water fraction used in our study was a fraction which was prepared after consequent extraction of the organic solvent.

In general, anti-trichomonas activity seems to depend on the solvent and plant used for extraction.