Abstract
Disseminated candidiasis is a serious problem in public health that results from the invasion of Candida species, in particular Candida albicans. The aim of this study was to compare the efficacies of Zataria multiflora essential oil and itraconazole in clearing C. albicans from the visceral organs of BALB/c mice suffered from disseminated candidiasis. Zataria multiflora essential oil was extracted using Clevenger-type apparatus and analyzed by gas chromatography mass spectrometry (GC-MS). For clearance experiment, mice (20-25 g, N=8 per group) received essential oil at doses of 30, 48 and 64 mg/kg and itraconazole at dose of 200 mg/kg intraperitoneally (IP) 2 days before and after intravenous inoculation of 0.5 × 106 C. albicans blastospores. The treated animals were sacrificed on day 20, and 0.1 g of the tissue homogenates was plated onto specific media. In GC-Mass, the main components of the essential oil were carvacrol (61.29%) and thymol (25.18%). The results demonstrated that IP administration of 64 mg/kg of the essential oil had the highest efficacy in reducing C. albicans and produced 39.5, 21.8, 141.5, 174 and 501-fold reductions in mean CFUs per 0.1 gram in Candida infections of the liver, spleen, lungs, brain and kidneys, respectively, compared to positive control. Itraconazole showed significantly more responsiveness than the essential oil at dose of 30 mg/kg in clearing C. albicans from the kidneys (P<0.02), brain (P<0.02) and spleen (P<0.04), and less responsiveness than that of 64 mg/kg in clearing the organism from the brain (P<0.01), lungs (P<0.0005) and kidneys (P<0.0005), whereas no significant difference was observed between this drug and Z. multiflora at dose of 48 mg/kg. These data explain the increased rate of yeast clearance and reduced dissemination to the viscera of Z. multiflora treated mice.
Keywords: Zataria multiflora essential oil, Candida albicans, Disseminated candidiasis, Carvacrol
INTRODUCTION
With the increasing number of immunocompromised patients and the greater use of broad-spectrum antifungal agents, broad-spectrum antibiotics, abdominal surgery, indwelling central venous lines, parenteral nutrition and cytotoxic chemotherapy, disseminated candidiasis has become a significant cause of mortality and morbidity (19, 21). Despite recent advances in antifungal therapy, the cure rate of disseminated candidiasis remains unsatisfactory because of a lack of nontoxic effective antifungal agents with favorable pharmacokinetic properties (26). The advent of azoles provides new options for the treatment and prevention of invasive candidiasis (24). However, the emergence of resistance to antifungal azoles poses a new challenge to our limited therapeutic strategies (4, 8). New agents with potent antifungal efficacy, improved safety and high levels of tissue penetration are clearly needed.
Plant essential oils have been known to show inhibition of proliferation or killing activity against a wide variety of micro-organisms (11, 17). Zataria multiflora is a plant belonging to the Laminaceae family that geographically grows only in Iran, Pakistan and Afghanistan (1). This plant has the local name of Avishan Shirazi (in Iran) and traditional uses such as antiseptic, anesthetic and antispasmodic (14). The main constituents of the essential oil of this plant are phenolic compounds such as carvacrol, thymol and eugenol (22). The essential oil has excellent in vitro activity against Candida species (7, 9). Up to now, little is known, however, about the in vivo activity of Z. multiflora in the treatment of cutaneous and mucosal fungal infections (15), but there is no study on disseminated candidiasis. The present study was undertaken to evaluate the efficacies of different doses of Z. multiflora essential oil in mice model of disseminated candidiasis and to compare its effect with itraconazole.
MATERIALS AND METHODS
Test organism
The strain of C. albicans (ATCC 10261) was isolated from the blood of a patient with disseminated candidiasis and stored in sterile distilled water at room temperature. To prepare the inoculum, fresh cultures were grown on Sabouraud glucose agar (Merck Co., Darmstadt, Germany) for 48 h at 30°C. The yeast cells were harvested, washed with normal saline and adjusted by hemocytometer approximately 1 × 108 /ml.
Laboratory animals
Forty female BALB/c mice, weighing 20 to 25 g, were used for all experiments. The mice were housed and maintained at the standard conditions. The animals were divided into 5 groups and 8 mice per group were used for each experiment. All experiments were repeated 3 times.
Extraction of the essential oil
Samples of Z. multiflora were collected from Shiraz, Iran on April 2008. Air-dried aerial parts of the plant were subjected to steam distillation for 2 h using Clevenger-type apparatus. The essential oil obtained of the air-dried material was analyzed by gas chromatography (GC) (Thermoquest 2000, UK). The chromatograph was equipped with DB5 capillary column (30 × 0.25 mm ID × 0.25μm film thicknesses) and the data were acquired under the following conditions: initial temperature 50 °C; program rate 2.5 °C; final temperature 265 °C and injector temperature 250 °C. The carrier gas was helium and the split ratio was 120. The essential oil was also analyzed by gas chromatography mass spectrometry (GC-MS) (Termoques Finningan, UK) and the same capillary column and analytical conditions indicated above. The MS was run in the electron ionization mode, using ionization energy of 70 eV.
Itraconazole
Standard antifungal powder of itraconazole (Janssen, Beerse, Belgium) was obtained from their respective manufacturers. Itraconazole stock solution was prepared in polyethylene glycol (Sigma Chemical Co., St. Louis, Mo.) and sterilized through a 0.45μm sterile, disposal and non- pyrogenic syringe filter (Nalgene, USA).
Animal infection
Normal mice were infected by intravenous injection of 0.5 × 106 saline-washed blastospores of C. albicans. Positive control received only IP injection of the organism. Three treatment groups received the essential oil of Z. multiflora (homogenized in distilled water by vortexing in high speed for 2 h) at doses of 30, 48 and 64 mg/kg by IP injection. The first dose was given 2 days before inoculation of C. albicans and the next dose was given 2 days after infection. The positive controls did not receive any drug regimen. For Z. multiflora groups, mice were sacrificed 20 days following the administration of different doses of the essential oils. Mice in the last group received itraconazole (200 mg/kg) via IP injection and sacrificed on day 20. The kidneys, spleen, liver, lungs and brain of each animal were weighed and examined by qualitative and quantitative cultures. The visceral organs from animals were ground with a pestle and mortar and 0.1 gram of tissue was plated on Sabouraud glucose agar. The plates were incubated at 35°C for 48 h, and the number of CFU per 0.1 gram of each tissue was determined.
Statistical analyses
The results were expressed as the mean CFU±standard deviation (SD) from 3 experiments. Mann-whiteny and Kruskal-wallis tests were used to compare the differences among the groups by SPSS software (Version 15). A P value less than 0.05 was statistically considered significant.
RESULTS
Chemical composition of Z. multiflora essential oil
The yield of the essential oil of air-dried aerial parts of the representative sample of Z. multiflora was 1.66% (v/w). Percentages of components of the essential oil (as determined by GC and GC-MS) were summarized in table 1. As shown in table 1, the main components of the essential oil were carvacrol (61.29%) and thymol (25.18%).
Table 1.
Essential oil composition of Z. multiflora identified by gas chromatography and gas chromatography–mass spectrometry
| Component | component (%) |
|---|---|
| Carvacrol | 61.29 |
| Thymol | 25.18 |
| Linalool | 1.96 |
| ρ-Cymene | 1.90 |
| β- Caryophyllene | 1.82 |
| α- Pinene | 0.34 |
| α- Thujene | 0.10 |
| Myrcene | 0.27 |
| α - Terpinene | 0.33 |
| γ- Terpinene | 0.76 |
| Thymul methyl ether | 0.95 |
| Carvacrol methyl ether | 0.95 |
| Camphene | 0.01 |
| 1-Octen-3-ol | 0.02 |
| 3-Octanone | 0.05 |
| β- Penene | 0.09 |
| α – Phellandrene | 0.02 |
| Limonene | 0.21 |
| Linalool oxide trans | 0.07 |
| Linalool oxide cis | 0.06 |
| 4-Terpineol | 0.30 |
| α- Terpineol | 0.54 |
| Bornyl acetate | 0.01 |
| Carvacerol acetate | 0.02 |
| β – Phellandrene | 1.82 |
| Aromadendrene | 0.23 |
| Alloaromadendrene | 0.11 |
| α- Humulene | 0.02 |
| Valencene | 0.14 |
| Spathulenol | 0.37 |
| Widdrol | 0.64 |
| Total | 99.09 |
Clearance of C. albicans from the kidneys: The whole untreated mice died on days 9-10, whereas the different treated groups were sacrificed on day 20. The clearance of C. albicans from untreated mice and mice treated with different doses of Z. multiflora and itraconazole was summarized in table 2. In the untreated mice receiving 0.5 × 106 C. albicans, the number of yeast cells in the kidneys of mice was ranged from 80 to 429 (mean, 350.8) on 10 days postinfection. In the mice receiving the different doses of Z. multiflora, the mean counts were 200 CFUs at dose of 30 mg/kg, 53.8 CFUs at 48 mg/kg and 0.7 CFUs at 64 mg/kg on 20 days postinfection. In mice receiving itraconazole, the mean counts were determined 65.9 CFUs at dose of 200 mg/kg on 20 days postinfection.
Table 2.
Comparative efficacies of Zataria multiflora essential oil and itraconazole on disseminated candidiasis in normal mice (Mean±SD of CFU/0.1 gram)
| Organ | Brain | Lungs | Liver | Spleen | Kidney |
|---|---|---|---|---|---|
| Material | |||||
| Control | 69.7± 0.10 | 28.3± 0.06 | 51.3 ± 0.03 | 8.7±0.01 | 350.8±0.41 |
| Z. multiflora (30mg/kg) | 44.9±0.12 | 13±0.09 | 36.3±0.10 | 6.7 ±0.06 | 200 ±0.44 |
| Z. multiflora (48mg/kg) | 51.8±0.20 | 4.7 ±0.05 | 2.9±0.01 | 1.8 ±0.01 | 3.8±0.21 |
| Z. multiflora (64mg/kg) | 0.4±0.01 | 0.2±0.01 | 1.3±0.09 | 0.4± 0.01 | 0.7±0.03 |
| Itraconazole (200mg/kg) | 27.2±0.10 | 10.7±0.08 | 3.3±0.03 | 1.9±0.01 | 65.9±0.27 |
Clearance of C. albicans from the liver: In the untreated animals, the number of C. albicans from the liver was ranged from 0 to 75.6 (mean, 51.3). In the mice receiving the different doses of Z. multiflora, the organisms decreased at dose of 30 mg/kg from 51.3 to 36.3 CFUs, to 2.9 CFUs at dose of 48 mg/kg and to 1.3 CFUs at dose of 64 mg/kg. Itraconazole had close efficacy with the essential oil at dose of 48 mg/kg, which resulted in decreasing the CFUs from 51.3 to 3.3 CFUs.
Clearance of C. albicans from the spleen: The mean counts of C. albicans from the spleen were 8.7 CFUs in untreated animals, 6.7, 1.8 and 0.4 CFUs at doses of 30, 48 and 64 mg/kg of the essential oil, respectively, and 1.9 CFUs at itraconazole group.
Clearance of C. albicans from the brain: As shown in table 2, there were no significant differences in the number of C. albicans from the brains of untreated and treated animals except for mice receiving 64 mg/kg of Z. multiflora (P<0.05).
Clearance of C. albicans from the lungs: The highest and lowest numbers of C. albicans from the lungs were demonstrated in untreated mice (mean; 28.3) and mice receiving 64 mg/kg (mean; 0.2) of the essential oils, respectively.
In this study, Z. multiflora essential oil demonstrated a significant dose dependent antifungal effect in clearing C. albicans from the organs. In comparison to itraconazole, essential oil had less active at dose of 30 mg/kg in clearing C. albicans from the kidneys (P<0.02), brain (P<0.02) and spleen (P<0.04), similarly clearances at dose of 48 mg/kg from all tissues and higher active at dose of 64 mg/kg in clearing the organism from the brain (P<0.01), lungs (P<0.0005) and kidneys (P<0.0005).
DISCUSSION
Recently new antifungal agents were used for treatment of disseminated candidiasis, including imidazoles and aminoacids (4). In some cases, these drugs showed failure to treatment, side effects and high relapse of disease (5, 12, 20, 25). For these reasons, the widespread efforts were made to identify natural agents to combat these opportunistic infections. Zataria multiflora is a valuable medicinal plant grown extensively in Iran, and the chemical compositions of essential oil have been extensively characterized (18, 19). The results of the clearance experiments using infecting doses of 0.5 × 106 in normal mice demonstrated the reduction of C. albicans from the kidneys, spleen, liver, brain and lungs of mice treated with 48 and 64 mg of Z. multiflora essential oil per kg twice with a 4-day interval. Control mice were infected with the same dose of C. albicans and Candida invasion was appeared in this group on day 9 and the whole animals died on day 10. In treatment groups, mice were sacrificed 20 days after administrating the last dose of the essential oil and itraconazole. In the present study, high-dose IP administration (64 mg/kg) of the essential oil demonstrated the highest efficacy in reducing C. albicans and produced 39.5, 21.8, 141.5, 174 and 501-fold reductions in mean CFUs per 0.1 gram in candidiasis of the liver, spleen, lungs, brain and kidneys in the mice model, respectively. The higher essential oil concentration resulted in higher inhibitory effect. No any major side effects have been shown in using Z. multiflora components in human and animals in this study and other investigations (15, 16). In our previous studies, the essential oil significantly increased innate and cellular immune responses in animal models. It was demonstrated that this essence has multiple biological effects on in vivo condition such as stimulation of immune system and antimicrobial activity (16, 23). Overall, the antifungal activity of the essential oils is related to the respective composition of the plant essential oils, the structural configuration of the constituent components and their functional groups and possible synergistic interactions between components (6). It was demonstrated that the main components with phenolic structures in Z. multiflora, such as carvacrol and thymol, have high activity against tested Candida species (21). In Iran, Fataneh (7) and Mahmoudabadi et al. (17) demonstrated anti-Candida activity of carvacrol and thymol in 2 in-vitro studies. In the present study, these compounds (carvacrol and thymol) were found as 2 main components, which was in agreement with published data (10, 13, 18). Itraconazole is a triazole anti-fungal agent with broad spectrum activity against a wide range of systemic fungal. It has found particular application in the treatment of immuno-compromised patients with fungal infections, especially fungal infections in patients undergoing chemotherapy, or afflicted with AIDS and AIDS-related conditions, or organ transplant recipients (2). Comparing to itraconazole with the essential oil, this drug showed significantly more responsiveness than the essential oil at dose of 30 mg/kg in clearing C. albicans from the kidneys (P<0.02), brain (P<0.02) and spleen (P<0.04), and less responsiveness than that of 64 mg/kg in clearing the organism from the brain (P<0.01), lungs (P<0.0005) and kidneys (P<0.0005), whereas no significant difference was observed between itraconazole and Z. multiflora at dose of 48 mg/kg in all tissues.
In summary, different concentrations of the essential oil exhibited considerable inhibitory effects against disseminated candidiasis. Further studies are needed to evaluate the toxic effect of Z. multiflora essential oil, via injection route, it could be used to treat human with disseminated candidiasis and this treatment would be lower cost when compared with itraconazole.
ACKNOWLEDGEMENTS
This work was supported by the Research Council of University of Tehran.
RESUMO
Eficiência comparada do óleo essencial de Zataria multiflora e itraconazol contra infecçãao disseminada de Candida albicans em camundongos BALB/c
A candidíase disseminada é um problema sério de saúde publica decorrente da invasão por espécies de Candida, e Candida albicans em particular. O objetivo deste estudo foi comparar a eficiência do óleo essencial de Zataria multiflora e itraconazol na remoção de C. albicans das visceras de camundongos BALB/c com candidíase disseminada. O óleo essencial de Zataria multiflora foi extraído empregando um aparelho do tipo Clevenger e analisado por cromatografia a gás e espectrometria de massa (GC-MS). Para os experimentos de remoção, camundongos (20-25g, n=8 por grupo) receberam óleo essencial nas doses de 30, 48 e 64 mg/kg e itraconazol na dose de 200 mg/kg via intraperitoneal (IP) por dois dias antes e após a inoculação intravenosa de 0,5 × 106 blastósporos de C. albicans. Os animais tratados foram sacrificados no vigésimo dia e 0,1g dos tecidos homogeneizados foram semeados em meios específicos. De acordo com o GC-MS, os principais componentes do óleo essencial foram carvacrol (61,29%) e timol (25,28%). Os resultados mostraram que a administração IP de 64 mg/kg de óleo essencial apresentou a eficiência mais alta na redução de C. albicans e resultou na redução de 39,5, 21,8, 141,5, 174 e 501 vezes na contagem média de C. albicans por 0,1g do fígado, baço, pulmões, cérebro e rins, respectivamente, quando comparado ao controle positivo. O itraconazol apresentou redução de C. albicans maior do que o óleo essencial na dose de 30mg/kg nos rins (P<0,02), cérebro (P<0,02) e baço (P<0,04) e menor no cérebro (P<0,01), pulmões (P<0,0005) e rins (P<0,0005) na dose de 64 mg/kg, enquanto não houve diferença entre esse droga e óleo essencial na dose de 48mg/kg. Estes resultados explicam a remoção aumentada de leveduras e a disseminação reduzida para as vísceras de camundongos tratados com Z. multiflora.
Palavras-chave: óleo essencial de Zataria multiflora, Candida albicans, candidíase disseminada, carvacrol
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