- Email: [email protected]
Anti-Parasitic Activity of Myristica Fragrans Houtt. Essential Oil Against Toxoplasma Gondii Parasite
Available online at www.sciencedirect.com
APCBEE Procedia 2 (2012) 92 – 96
ICBFS 2012: April 7-8, 2012, Bangkok, Thailand
Anti-parasitic Activity of Myristica fragrans Houtt. Essential Oil against Toxoplasma Gondii Parasite Suthagar Pillai∗, Roziahanim Mahmud, Wei Cai Lee, Shanmugapriya Perumal School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia
Abstract Nutmeg (Myristica fragrans Houtt.) is the seed kernel of inside the fruit. It has been studied to having number of ethnopharmacological properties. Essential oils are secondary metabolites which originated from different parts of plants and made up of volatile compounds. In the present study, the essential oil extracted from nutmeg was investigated for its in vitro cytotoxicity on Vero cell line (normal cell line) and anti-parasitic activity against Toxoplasma gondii parasite. Nutmeg essential oil showed very low cytotoxic activity against Vero cells with IC50 value of 24.83μg/mL. In the in vitro anti-T.gondii assay, the oil extract showed significant inhibiting activity with EC50 value of 24.45μg/mL. This result was comparable to the standard drug clindamycin (EC50=16.57μg/mL.). Based on the promising results, nutmeg essential oil may be further studied for its mechanism of action against T.gondii and to isolate the bioactive compound(s) to treat toxoplasmosis.
© 2012 2012Published PublishedbybyElsevier ElsevierB.V. B.V. Selection and/or review under responsibility of Asia-Pacific © Selection and/or peerpeer review under responsibility of Asia-Pacifi c Chemical,Biological Biological&&Environmental Environmental Engineering Society Chemical, Engineering Society Keywords: Myristica fragrans, Toxoplasma gondii,Vero cell line, essential oil
1. Introduction Since long time back, aromatic herbs and spices have been added to various foods as ingredients to improve the taste, flavor and organoleptic properties. Essential oils are secondary metabolites which derived from plants, herbs and spices are made up of volatile compounds comprising volatile constituents like lipids, terpenoids, ketones, phenols and oxygenated derivatives. The aromatic plants and spices are commonly used
∗
Corresponding author. Tel: +604-653 4701 E-mail address: [email protected]
2212-6708 © 2012 Published by Elsevier B.V. Selection and/or peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society doi:10.1016/j.apcbee.2012.06.017
Suthagar Pillai et al. / APCBEE Procedia 2 (2012) 92 – 96
in phytotherapy and mostly related to various activities of their essential oils, such as antimicrobial, antioxidant, antifungal spasmolytic, carminative, hepatoprotective, antiviral and anticarcinogenic activities [1]. Nutmeg (Myristica fragrans Houttuyn) is the seed kernel of inside the fruit and mace is the lacy covering (aril) on the kernel (Nadkarni, 1988). It has been studied to having anthelmintic, hepatoprotective antiinflammatory, aphrodisiac, insecticide properties and a treatment for rheumatism, diarrhea, asthma, atherosclerosis and flatulence [2-4]. Toxoplasma gondii is an obligate intracellular apicomplexan parasite that has the ability to infect and propagate in virtually all nucleated host cells [5,6]. Infection of T.gondii causes toxoplasmosis, an infectious disease that occurs worldwide. Infections are often asymptomatic in immunocompetent hosts, but severe toxoplasmosis can result from infection of immunocompromised hosts and fetuses [7,8]. It is important to point out that the standard therapies for toxoplasmosis - the combination of pyrimethamine and sulfadiazine, clindamycin, azithromycin, or atavaquone [9-11] -do not eliminate the intracellular parasite and are often associated with severe side effects [7]. Thus, alternative therapeutic compounds for treating toxoplasmosis are urgently needed. The aim of this study is to to evaluate the efficacy of essential oil of Myristica fragrans against T. gondii and in vitro cytotoxicity on normal cell line. 2. Materials and methods 2.1. Plant material The fresh fruits of nutmeg (Myristica fragrans) were obtained from Balik Pulau, Penang, Malaysia. Voucher specimen (11253) Myristica fragrans was authenticated by botanist, Mr. V. Shunmugam and deposited at the Herbarium Unit of the School of Biological Sciences, Universiti Sains Malaysia, Penang. 2.2. Extraction of essential oil The fresh fruits of the Myristica fragrans were submitted to water distillation for 3 h using a modified Clevenger’s apparatus. The ground samples (200 g) were boiled with water (200 ml) for 3 h in a 1 liter round bottom flask fitted with a condenser. The extracted essential oils were dried over anhydrous sodium sulphate and after filtration, stored in amber glass vial at -20°C until tested and analyzed. 2.3. Cytotoxicity Assay The cytotoxicity of the nutmeg essential oil was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay [12] with some modifications. On the day of the test, Vero cells were harvested and counted and the number of cell was adjusted to 12x104cells/mL. 100 μL of the cell suspension were seeded into each well of a 96-well plate and incubated at 37ºC in 5 % CO2 incubator for 24 hr. The final concentrations ranged from 100μg/mL to the lowest 3.125 μg/mL. All tests were performed in triplicates. The control was cell suspension without sample. The culture was incubated for 24 hr. The medium (200 μL) was aspirated out from the well and the 100 μL of MTT- Phosphate Buffered Saline (PBS) (5mg/mL) solution in culture medium in ratio of 1: 9 were added to each well. The plate was covered with aluminium foil and further incubated for 4 hr in the incubator. The medium was then removed and replaced with 100 μL of dimethyl sulfoxide (DMSO) to solubilise the purple MTT formazan. The plates were shaken for 5 min before measuring the absorbance at 540 nm with a microplate reader. The percentage of growth inhibition (GI) was calculated using formula below:
93
94
Suthagar Pillai et al. / APCBEE Procedia 2 (2012) 92 – 96
GI =
A treatment − A control x 100%, where A control
A treatment = Absorbance of treatment A control = Absorbance of control The mean 50 % growth inhibition concentration (IC50) was estimated from a dose-response curve. 2.4. In vitro anti-parasitic assay Vero cells were harvested during exponential growth, cultured in 96-well plates (density= 12×104 cells/mL) and incubated at 37ºC in 5 % CO2 incubator for 24 hr. Then, 6×105 parasites/mL of T.gondii parasite were added to each well (parasite: cell ratio = 5:1, final volume 200 μL) [3]. Six hours after inoculation, the cultures were washed twice with Dulbecco’s Modified Eagle Medium (DMEM) without Fetal bovine serum (FBS) to remove any non-adherent parasites. After 18 hr incubation, DMEM medium was added to each well along with serial concentrations of essential oil. After 24 hr of treatment, anti-T.gondii activity of nutmeg essential oil was examined using the MTT assay [12] with some modifications. Clindamycin served as the positive control. The assay was conducted in 96-well plates and assayed using a microplate reader using a wavelength of 540 nm. All of the test compounds were assayed in triplicate at each concentration. All data points represent the mean of three independent experiments. The percentage of growth inhibition (GI) was calculated manually using the formula: GI =
A treatment − A control x 100%, where A control
A treatment = Absorbance of treatment A control = Absorbance of control The median effective concentration (EC50) value refers to the concentration of the compound necessary to inhibit at 50% of the control values. A dose-response curve was plotted to calculate EC50. 3. Results and discussion The results of cytotoxicity evaluation of the essential oil are shown in Table 1. The results showed that nutmeg essential oil possessed very low cytotoxicity against Vero cell line with IC50 at 24.83μg/ml. Table 1. The in vitro anti-parasitic activity of nutmeg essential oil and clindamycin on Toxoplasma gondii and cytoxicity activity on Vero cells.
Sample Nutmeg essential oil Clindamycin
Cytotoxicity. IC50 (μg/mL) 24.83
In vitro antiT.gondii assay, EC50 (μg/mL) 24.45
-
16.57
The in vitro cytotoxicity study on Vero cell line is important since the cells will be used as the host cell to infect T.gondii parasites. Since the nutmeg oil showed very low cytotoxic effect, it may not affect the host cell
95
Suthagar Pillai et al. / APCBEE Procedia 2 (2012) 92 – 96
performance in the anti-parasitic assay and the anti- T.gondii activity results of the oil obtained would not be due to the toxic effects. Furthermore, previous study [13] reported that the propagation of T.gondii parasites in Vero cells produced high yield and viability of tachyzoites, with minimal host cell contamination. Hence, selection of Vero cells as the host cells for propagation of parasites will be appropriate to use in the investigation of anti- T.gondii activity. The EC50 value of the essential oil from the in vitro anti-T.gondii assay was determined and shown in Table 1. Nutmeg oil showed significant difference of inhibition against the T.gondii (p<0.01) in a dosedependent manner with IC50 of 24.45μg/mL. The EC50 value of clindamycin (positive control) was determined at 16.57μg/mL. The promising result suggested that nutmeg oil may contain some potential active compounds against T.gondii parasite such as as myristicin, limonene, eugenol and terpinen-4-ol.(14-17). 4. Conclusion Summarizing these results, it can be concluded that nutmeg essential oil exhibited strong anti- T.gondii activity with low cytotoxic effects against normal cell line. Thus, the essential oil can be further studied on its action of mechanism and fractionation will be done to determine the strength of its anti- T. Gondii and the potential bioactive compound(s) will be determined. Acknowledgements This project was funded (1001/PFARMASI/813021).
by
Universiti
Sains Malaysia
Research University
(RU) Grant
References [1] Bakkali F., Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils – a review. Food Chem. Toxicol., 2008;46: 446–475. [2] Burkill IH. A dictionary of the economic products of Malay Peninsular. Kuala Lumpur: Ministry of Agriculture, Malaysia. 1996;pp. 1547-1556. [3] Ozaki Y, Soedigdo S, Wattimena YR, Suganda AG. Anti-inflammatory effects of mace, aril of Myristica fragrans Houtt., and its active principles. Jpn. J. Pharmacol., 1989;49: 155-163. [4] Mila J, Olivera P, Mladen M. Chemical Composition and Antioxidant Effect of Free Volatile Aglycones from Nutmeg (Myristica fragrans Houtt.) Compared to Its Essential Oil. Croatica Chemica Acta.,2006;79 (2): 209-214 [5] Derouich-Guergour D, Aldebert D, Vigan I, Jouvin-Marche E, Marche PN, Aubert D et al. Toxoplasma gondii infection can regulate the expression of tumour necrosis factor-alpha receptors on human cells in vitro. Parasite Immunol 2002;24: 271–279. [6] Montoya JG and Liesenfeld O. Toxoplasmosis. Lancet 2004;363: 1965-1976. [7] Werk R. How does Toxoplasma gondii enter host cells? Rev Infect Dis 1958;7: 449–457. [8] Vogel N, Kirisits M, Michael E et al. Congenital toxoplasmosis transmitted from an immunologically competent mother infected before conception. Clin Infect Dis 1996; 23:1055–1060. [9] Chirgwin K, Hafner R, Leport C, Remington J, Andersen J, Bosler EM et al.. Randomized phase II trial of atovaquone with pyrimethamine or sulfadiazine for treatment of toxoplasmic encephalitis in patients with acquired immunodeficiency syndrome: ACTG 237/ANRS 039 Study. AIDS Clinical Trials Group 237/Agence Nationale de Recherche sur le SIDA, Essai 039. Clin Infect Dis 2002;34: 1243-1250.
96
Suthagar Pillai et al. / APCBEE Procedia 2 (2012) 92 – 96
[10] Djurkovic-Djakovic O, Nikolic T, Robert-Gangneux F, Bobic B and Nikolic A. Synergistic effect of clindamycin and atovaquone in acute murine toxoplasmosis. Antimicrob Agents Chemother 1999;43: 2240-2244. [11] Belloni A, Villena I, Gomez JE, Pelloux H, Bonhomme A, Guenounou M, et al. Regulation of tumor necrosis factor alpha and its specific receptors during Toxoplasma gondii infection in human monocytic cells. Parasitol Res 2003;89: 207–213. [12] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J. Immunol. Methods1983;65:55-63 [13] Saadatnia G, Haj Ghani H, Khoo BY, Maimunah A, Rahmah N. Optimization of Toxoplasma gondii cultivation in Vero cell line. Trop. Biomed 2010;27(1):125-130. [14] Lee BK, Kim JH, Jung JW, Choi JW, Han ES, Lee SH, Ko KH, Ryu JH, Myristicin-induced neurotoxicity in human neuroblastoma SK-N-SH cells. Toxicology Letters 2005;157(1): 49–56. [15] Parija T, Das B, Involvement of YY1 and its correlation with c-myc in NDEA induced hepatocarcinogenesis, its prevention by d-limonene. Plant Molecular Biology Reporter 2003; 30: 41–46. [16] Calcabrini A, Stringaro A, Toccacieli L, Meschini S, Marra M, Colone M, Salvatore G, Mondello F, Arancia G, Molinari A. Terpinen-4-ol, The Main Component of Melaleuca Alternifolia (Tea Tree) Oil Inhibits the In Vitro Growth of Human Melanoma Cells, J Invest Dermatol. 2004;122:349-360 [17] Hussain A, Sasidharan S, Ahmed T, Ahmed M, Sharma C, Clove (Syzygium aromaticum) Extract Potentiates Gemcitabine Cytotoxic Effect on Human Cervical Cancer Cell Line. International Journal of Cancer Research2009;5: 95-104
APCBEE Procedia 2 (2012) 92 – 96
ICBFS 2012: April 7-8, 2012, Bangkok, Thailand
Anti-parasitic Activity of Myristica fragrans Houtt. Essential Oil against Toxoplasma Gondii Parasite Suthagar Pillai∗, Roziahanim Mahmud, Wei Cai Lee, Shanmugapriya Perumal School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia
Abstract Nutmeg (Myristica fragrans Houtt.) is the seed kernel of inside the fruit. It has been studied to having number of ethnopharmacological properties. Essential oils are secondary metabolites which originated from different parts of plants and made up of volatile compounds. In the present study, the essential oil extracted from nutmeg was investigated for its in vitro cytotoxicity on Vero cell line (normal cell line) and anti-parasitic activity against Toxoplasma gondii parasite. Nutmeg essential oil showed very low cytotoxic activity against Vero cells with IC50 value of 24.83μg/mL. In the in vitro anti-T.gondii assay, the oil extract showed significant inhibiting activity with EC50 value of 24.45μg/mL. This result was comparable to the standard drug clindamycin (EC50=16.57μg/mL.). Based on the promising results, nutmeg essential oil may be further studied for its mechanism of action against T.gondii and to isolate the bioactive compound(s) to treat toxoplasmosis.
© 2012 2012Published PublishedbybyElsevier ElsevierB.V. B.V. Selection and/or review under responsibility of Asia-Pacific © Selection and/or peerpeer review under responsibility of Asia-Pacifi c Chemical,Biological Biological&&Environmental Environmental Engineering Society Chemical, Engineering Society Keywords: Myristica fragrans, Toxoplasma gondii,Vero cell line, essential oil
1. Introduction Since long time back, aromatic herbs and spices have been added to various foods as ingredients to improve the taste, flavor and organoleptic properties. Essential oils are secondary metabolites which derived from plants, herbs and spices are made up of volatile compounds comprising volatile constituents like lipids, terpenoids, ketones, phenols and oxygenated derivatives. The aromatic plants and spices are commonly used
∗
Corresponding author. Tel: +604-653 4701 E-mail address: [email protected]
2212-6708 © 2012 Published by Elsevier B.V. Selection and/or peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society doi:10.1016/j.apcbee.2012.06.017
Suthagar Pillai et al. / APCBEE Procedia 2 (2012) 92 – 96
in phytotherapy and mostly related to various activities of their essential oils, such as antimicrobial, antioxidant, antifungal spasmolytic, carminative, hepatoprotective, antiviral and anticarcinogenic activities [1]. Nutmeg (Myristica fragrans Houttuyn) is the seed kernel of inside the fruit and mace is the lacy covering (aril) on the kernel (Nadkarni, 1988). It has been studied to having anthelmintic, hepatoprotective antiinflammatory, aphrodisiac, insecticide properties and a treatment for rheumatism, diarrhea, asthma, atherosclerosis and flatulence [2-4]. Toxoplasma gondii is an obligate intracellular apicomplexan parasite that has the ability to infect and propagate in virtually all nucleated host cells [5,6]. Infection of T.gondii causes toxoplasmosis, an infectious disease that occurs worldwide. Infections are often asymptomatic in immunocompetent hosts, but severe toxoplasmosis can result from infection of immunocompromised hosts and fetuses [7,8]. It is important to point out that the standard therapies for toxoplasmosis - the combination of pyrimethamine and sulfadiazine, clindamycin, azithromycin, or atavaquone [9-11] -do not eliminate the intracellular parasite and are often associated with severe side effects [7]. Thus, alternative therapeutic compounds for treating toxoplasmosis are urgently needed. The aim of this study is to to evaluate the efficacy of essential oil of Myristica fragrans against T. gondii and in vitro cytotoxicity on normal cell line. 2. Materials and methods 2.1. Plant material The fresh fruits of nutmeg (Myristica fragrans) were obtained from Balik Pulau, Penang, Malaysia. Voucher specimen (11253) Myristica fragrans was authenticated by botanist, Mr. V. Shunmugam and deposited at the Herbarium Unit of the School of Biological Sciences, Universiti Sains Malaysia, Penang. 2.2. Extraction of essential oil The fresh fruits of the Myristica fragrans were submitted to water distillation for 3 h using a modified Clevenger’s apparatus. The ground samples (200 g) were boiled with water (200 ml) for 3 h in a 1 liter round bottom flask fitted with a condenser. The extracted essential oils were dried over anhydrous sodium sulphate and after filtration, stored in amber glass vial at -20°C until tested and analyzed. 2.3. Cytotoxicity Assay The cytotoxicity of the nutmeg essential oil was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay [12] with some modifications. On the day of the test, Vero cells were harvested and counted and the number of cell was adjusted to 12x104cells/mL. 100 μL of the cell suspension were seeded into each well of a 96-well plate and incubated at 37ºC in 5 % CO2 incubator for 24 hr. The final concentrations ranged from 100μg/mL to the lowest 3.125 μg/mL. All tests were performed in triplicates. The control was cell suspension without sample. The culture was incubated for 24 hr. The medium (200 μL) was aspirated out from the well and the 100 μL of MTT- Phosphate Buffered Saline (PBS) (5mg/mL) solution in culture medium in ratio of 1: 9 were added to each well. The plate was covered with aluminium foil and further incubated for 4 hr in the incubator. The medium was then removed and replaced with 100 μL of dimethyl sulfoxide (DMSO) to solubilise the purple MTT formazan. The plates were shaken for 5 min before measuring the absorbance at 540 nm with a microplate reader. The percentage of growth inhibition (GI) was calculated using formula below:
93
94
Suthagar Pillai et al. / APCBEE Procedia 2 (2012) 92 – 96
GI =
A treatment − A control x 100%, where A control
A treatment = Absorbance of treatment A control = Absorbance of control The mean 50 % growth inhibition concentration (IC50) was estimated from a dose-response curve. 2.4. In vitro anti-parasitic assay Vero cells were harvested during exponential growth, cultured in 96-well plates (density= 12×104 cells/mL) and incubated at 37ºC in 5 % CO2 incubator for 24 hr. Then, 6×105 parasites/mL of T.gondii parasite were added to each well (parasite: cell ratio = 5:1, final volume 200 μL) [3]. Six hours after inoculation, the cultures were washed twice with Dulbecco’s Modified Eagle Medium (DMEM) without Fetal bovine serum (FBS) to remove any non-adherent parasites. After 18 hr incubation, DMEM medium was added to each well along with serial concentrations of essential oil. After 24 hr of treatment, anti-T.gondii activity of nutmeg essential oil was examined using the MTT assay [12] with some modifications. Clindamycin served as the positive control. The assay was conducted in 96-well plates and assayed using a microplate reader using a wavelength of 540 nm. All of the test compounds were assayed in triplicate at each concentration. All data points represent the mean of three independent experiments. The percentage of growth inhibition (GI) was calculated manually using the formula: GI =
A treatment − A control x 100%, where A control
A treatment = Absorbance of treatment A control = Absorbance of control The median effective concentration (EC50) value refers to the concentration of the compound necessary to inhibit at 50% of the control values. A dose-response curve was plotted to calculate EC50. 3. Results and discussion The results of cytotoxicity evaluation of the essential oil are shown in Table 1. The results showed that nutmeg essential oil possessed very low cytotoxicity against Vero cell line with IC50 at 24.83μg/ml. Table 1. The in vitro anti-parasitic activity of nutmeg essential oil and clindamycin on Toxoplasma gondii and cytoxicity activity on Vero cells.
Sample Nutmeg essential oil Clindamycin
Cytotoxicity. IC50 (μg/mL) 24.83
In vitro antiT.gondii assay, EC50 (μg/mL) 24.45
-
16.57
The in vitro cytotoxicity study on Vero cell line is important since the cells will be used as the host cell to infect T.gondii parasites. Since the nutmeg oil showed very low cytotoxic effect, it may not affect the host cell
95
Suthagar Pillai et al. / APCBEE Procedia 2 (2012) 92 – 96
performance in the anti-parasitic assay and the anti- T.gondii activity results of the oil obtained would not be due to the toxic effects. Furthermore, previous study [13] reported that the propagation of T.gondii parasites in Vero cells produced high yield and viability of tachyzoites, with minimal host cell contamination. Hence, selection of Vero cells as the host cells for propagation of parasites will be appropriate to use in the investigation of anti- T.gondii activity. The EC50 value of the essential oil from the in vitro anti-T.gondii assay was determined and shown in Table 1. Nutmeg oil showed significant difference of inhibition against the T.gondii (p<0.01) in a dosedependent manner with IC50 of 24.45μg/mL. The EC50 value of clindamycin (positive control) was determined at 16.57μg/mL. The promising result suggested that nutmeg oil may contain some potential active compounds against T.gondii parasite such as as myristicin, limonene, eugenol and terpinen-4-ol.(14-17). 4. Conclusion Summarizing these results, it can be concluded that nutmeg essential oil exhibited strong anti- T.gondii activity with low cytotoxic effects against normal cell line. Thus, the essential oil can be further studied on its action of mechanism and fractionation will be done to determine the strength of its anti- T. Gondii and the potential bioactive compound(s) will be determined. Acknowledgements This project was funded (1001/PFARMASI/813021).
by
Universiti
Sains Malaysia
Research University
(RU) Grant
References [1] Bakkali F., Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils – a review. Food Chem. Toxicol., 2008;46: 446–475. [2] Burkill IH. A dictionary of the economic products of Malay Peninsular. Kuala Lumpur: Ministry of Agriculture, Malaysia. 1996;pp. 1547-1556. [3] Ozaki Y, Soedigdo S, Wattimena YR, Suganda AG. Anti-inflammatory effects of mace, aril of Myristica fragrans Houtt., and its active principles. Jpn. J. Pharmacol., 1989;49: 155-163. [4] Mila J, Olivera P, Mladen M. Chemical Composition and Antioxidant Effect of Free Volatile Aglycones from Nutmeg (Myristica fragrans Houtt.) Compared to Its Essential Oil. Croatica Chemica Acta.,2006;79 (2): 209-214 [5] Derouich-Guergour D, Aldebert D, Vigan I, Jouvin-Marche E, Marche PN, Aubert D et al. Toxoplasma gondii infection can regulate the expression of tumour necrosis factor-alpha receptors on human cells in vitro. Parasite Immunol 2002;24: 271–279. [6] Montoya JG and Liesenfeld O. Toxoplasmosis. Lancet 2004;363: 1965-1976. [7] Werk R. How does Toxoplasma gondii enter host cells? Rev Infect Dis 1958;7: 449–457. [8] Vogel N, Kirisits M, Michael E et al. Congenital toxoplasmosis transmitted from an immunologically competent mother infected before conception. Clin Infect Dis 1996; 23:1055–1060. [9] Chirgwin K, Hafner R, Leport C, Remington J, Andersen J, Bosler EM et al.. Randomized phase II trial of atovaquone with pyrimethamine or sulfadiazine for treatment of toxoplasmic encephalitis in patients with acquired immunodeficiency syndrome: ACTG 237/ANRS 039 Study. AIDS Clinical Trials Group 237/Agence Nationale de Recherche sur le SIDA, Essai 039. Clin Infect Dis 2002;34: 1243-1250.
96
Suthagar Pillai et al. / APCBEE Procedia 2 (2012) 92 – 96
[10] Djurkovic-Djakovic O, Nikolic T, Robert-Gangneux F, Bobic B and Nikolic A. Synergistic effect of clindamycin and atovaquone in acute murine toxoplasmosis. Antimicrob Agents Chemother 1999;43: 2240-2244. [11] Belloni A, Villena I, Gomez JE, Pelloux H, Bonhomme A, Guenounou M, et al. Regulation of tumor necrosis factor alpha and its specific receptors during Toxoplasma gondii infection in human monocytic cells. Parasitol Res 2003;89: 207–213. [12] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J. Immunol. Methods1983;65:55-63 [13] Saadatnia G, Haj Ghani H, Khoo BY, Maimunah A, Rahmah N. Optimization of Toxoplasma gondii cultivation in Vero cell line. Trop. Biomed 2010;27(1):125-130. [14] Lee BK, Kim JH, Jung JW, Choi JW, Han ES, Lee SH, Ko KH, Ryu JH, Myristicin-induced neurotoxicity in human neuroblastoma SK-N-SH cells. Toxicology Letters 2005;157(1): 49–56. [15] Parija T, Das B, Involvement of YY1 and its correlation with c-myc in NDEA induced hepatocarcinogenesis, its prevention by d-limonene. Plant Molecular Biology Reporter 2003; 30: 41–46. [16] Calcabrini A, Stringaro A, Toccacieli L, Meschini S, Marra M, Colone M, Salvatore G, Mondello F, Arancia G, Molinari A. Terpinen-4-ol, The Main Component of Melaleuca Alternifolia (Tea Tree) Oil Inhibits the In Vitro Growth of Human Melanoma Cells, J Invest Dermatol. 2004;122:349-360 [17] Hussain A, Sasidharan S, Ahmed T, Ahmed M, Sharma C, Clove (Syzygium aromaticum) Extract Potentiates Gemcitabine Cytotoxic Effect on Human Cervical Cancer Cell Line. International Journal of Cancer Research2009;5: 95-104