- Email: [email protected]
Anti-Toxoplasma gondii RH strain activity of herbal extracts used in traditional medicine
International Journal of Antimicrobial Agents 32 (2008) 360–362
Short communication
Anti-Toxoplasma gondii RH strain activity of herbal extracts used in traditional medicine Kyung-Min Choi a , Jingu Gang b , Jisoo Yun c,∗ a Ecodigm Co., Ltd., 305DTV Post BI, 707 Tamnip-dong, Yuseong-gu, Daejeon 305-510, South Korea Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea Research Center for Resistant Cells, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, South Korea b
c
Received 22 March 2008; accepted 14 April 2008
Abstract Methanolic extracts of 15 traditional medicines used to treat Toxoplasma gondii were tested in vitro for their anti-T. gondii activity and cytotoxicity. The median effective concentration (EC50 ) values for the herbal extracts ranged from 0.11 mg/mL to 2.28 mg/mL. Significant antiT. gondii RH strain activity was observed with Zingiber officinale extracts (EC50 = 0.18 mg/mL), which displayed a highly selective toxicity (selectivity = 10.1). Sophora flavescens Aiton extracts also showed high anti-T. gondii activity (EC50 = 0.20 mg/mL) and a high selective toxicity (4.6). This indicates that Z. officinale and S. flavescens Aiton extracts may be sources of new anti-T. gondii compounds. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Toxoplasma gondii; Anti-Toxoplasma gondii RH strain activity; Traditional medicine
1. Introduction Toxoplasma gondii, an obligate intracellular coccidian, has the ability to infect and propagate in virtually all nucleated host cells [1,2]. T. gondii infection causes toxoplasmosis, an infectious disease existing worldwide. This infection is normally innocuous in individuals with an intact immune response [3] who can promote a well-balanced interaction between the host and the parasite [2]. The RH strain of T. gondii is a virulent and non-cyst-forming strain that grows considerably faster than avirulent strains [4,5]. To control toxoplasmosis, a combination of antifolates, such as pyrimethamine and sulfadiazine, has been used with success and it is the first choice in most clinical settings [6,7]. On the other hand, a large percentage of undesirable side effects such as haematological toxicity (caused by pyrimethamine), cutaneous rash, leukopenia and thrombocytopenia (caused by sulphonamides) are often reported [7–9]. ∗
Corresponding author. Tel.: +82 62 233 6054; fax: +82 62 233 6052. E-mail address: [email protected] (J. Yun).
In an effort to improve the therapies used against T. gondii RH strain, we have examined numerous natural products for potential sources of new compounds with high activity and low toxicity. In this study, we describe the preliminary screening of 15 methanolic extracts used in Korean traditional medicine for anti-T. gondii RH strain activity and low cytotoxicity.
2. Materials and methods 2.1. RH strains of T. gondii RH strains of T. gondii were maintained by routine intraperitoneal passage with RPMI 1640 medium collected at 4 days post infection [5,10]. ICR mice weighing ca. 25 g were infected with 1 × 106 parasites at each passage. The tachyzoite concentration was determined using a haemocytometer at 200× magnification. The parasites were re-suspended at a density of 4 × 107 parasites/mL in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS).
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.04.012
K.-M. Choi et al. / International Journal of Antimicrobial Agents 32 (2008) 360–362
2.2. Cell culture HeLa cells were maintained in RPMI 1640 medium supplemented with 10% FBS. Cells were incubated at 37 ◦ C with humidified air and 5% CO2 . Cells were harvested by treatment with trypsin and washed twice in phosphate-buffered saline solution (pH 7.4). Viable cells were counted using a trypan blue exclusion test [11,12].
2.3. Preparation of traditional medicine samples All herbal drugs screened in this study were purchased from the University Oriental Drugstore, Iksan, South Korea. They were authenticated by matching against herbarium specimens at the College of Pharmacy, Wonkwang University, South Korea. Methanolic extracts were obtained by sonication of plant material (100 g dry weight) for 3 h in methanol (1 L). After filtration, the solvent was removed by evaporation in vacuo. The dried extract was dissolved in dimethyl sulphoxide (DMSO) before use. The anti-T. gondii RH strain drugs pyrimethamine, sulfadiazine and spiramycin were purchased from Sigma (St Louis, MO).
361
Table 1 In vitro anti-Toxoplasma gondii RH strain activity and cytotoxicity of traditional medicines Plant name
Arecae pericarpium Sophora flavescens Aiton Meliae cortex Rheum undulatum L. Pulsatilla radix Acorus gramineus Soland Dryopteris crassirhizoma Quisqualis indica L. Fraxinus rhynchophylla Hance Glycyrrhiza glabra L. Portulaca oleracea L. Schizandra chinensis Baillon Zingiber officinale Angelica gigas Artemisia scoparia Sulfadiazine Pyrimethamine Spiramycin
EC50 (mg/mL) HeLa cells
T. gondii RH
1.35 0.92 0.28 0.21 1.16 0.21 0.37 1.32 1.35 0.22 1.34 1.34 1.81 1.28 0.11 0.06 0.21 0.77
1.30 0.20 0.77 2.28 1.52 0.11 0.15 1.78 1.57 0.13 1.53 1.57 0.18 1.40 1.53 0.07 0.10 0.31
Selectivitya
– 4.6 – – – 1.9 2.5 – – 1.7 – – 10.1 – – – 2.1 2.5
EC50 , median effective concentration. a Ratio of the EC value for HeLa cells to the EC value for T. gondii 50 50 RH strain.
3. Results and discussion 2.4. In vitro infection and effectors HeLa cells were harvested during exponential growth (Day 2) and cultured in 96-well plates (ca. 6 × 104 cells/mL). Then, 3 × 105 parasites/mL were added to each well (parasite:cell ratio = 5:1, final volume 200 L) [13]. Six hours after inoculation, the cultures were washed twice with RPMI 1640 medium without FBS to remove any non-adherent parasites. After 18 h incubation, RPMI 1640 medium supplemented with 2% FBS was added to each well along with different concentrations of the 15 methanolic extracts. After 24 h of treatment, anti-T. gondii RH activity and cytotoxicity of the traditional medicines were examined using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (Promega, Madison, WI) assay [14]. The assay was conducted in 96-well plates and assayed using a microplate reader (Spectra Max Gemini XG, parameters chosen in SOFT max pro 4.0; Molecular Devices, Sunnyvale, CA) using a wavelength of 490 nm. All of the test compounds were assayed in triplicate at each concentration. All data points represent the mean of three independent experiments. The median effective concentration (EC50 ) value refers to the concentration of the compound necessary to inhibit at 50% of the control values. Selectivity refers to the mean of the EC50 value for HeLa cells relative to the mean of the EC50 value of the T. gondii RH strain [15].
Methanolic extracts of Korean herbal medicines used for T. gondii therapy were assessed for their activity against anti-T. gondii RH strain. The results of the in vitro anti-T. gondii and cytotoxicity assays against both the T. gondii RH strain and HeLa cells are summarised in Table 1. Extracts of Glycyrrhiza glabra L., Acorus gramineus Soland and Dryopteris crassirhizoma showed high anti-T. gondii RH strain activity (EC50 = 0.11–0.15 mg/mL) but had no selective toxicity (selectivity = 1.7–2.5). Another extract, Sophora flavescens Aiton, showed high anti-T. gondii RH strain activity (EC50 = 0.20 mg/mL) and selective toxicity (selectivity = 4.6). In addition, Zingiber officinale had high anti-T. gondii activity (EC50 = 0.18 mg/mL) as well as cytotoxicity against HeLa cells (at 1.81 mg/mL) and strong anti-T. gondii RH strain activity (selectivity = 10.1) relative to pyrimethamine (selectivity = 2.1) and spiramycin (selectivity = 2.5). The investigation of anti-T. gondii RH strain activity of Korean traditional medicines led to the finding that methanolic extracts of S. flavescens Aiton and Z. officinale showed potent anti-T. gondii RH strain activity. These results allowed us to suggest that these two traditional medicines are likely sources of new compounds that could be used to treat T. gondii infections. Further studies will be necessary to identify and isolate these active compounds. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required.
362
K.-M. Choi et al. / International Journal of Antimicrobial Agents 32 (2008) 360–362
References [1] Werk R. How does Toxoplasma gondii enter host cells? Rev Infect Dis 1985;7:449–57. [2] 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;24:271–9. [3] Yap GS, Sher A. Cell-mediated immunity to Toxoplasma gondii: initiation, regulation and effector function. Immunobiology 1999;201:240–7. [4] Appleford PJ, Smith JE. Toxoplasma gondii: the growth characteristics of three virulent strains. Acta Trop 1997;65:97–104. [5] Zhu S, Lai DH, Li SO, Lun ZR. Stimulative effects of insulin on Toxoplasma gondii replication in 3T3-L1 cells. Cell Biol Int 2006;30:149–53. [6] Harris C, Salgo MP, Tanowitz HB, Wittner M. In vitro assessment of antimicrobial agents against Toxoplasma gondii. J Infect Dis 1988;1570:14–22. [7] Dantas-Leitz L, Urbina JA, de Souza W, Vommaro RC. Antiproliferative synergism of azasterols and antifolates against Toxoplasma gondii. Int J Antimicrob Agents 2005;25:130–5. [8] Alder J, Hutch T, Meulbroek JA, Clement JC. Treatment of experimental Toxoplasma gondii infection by clarithromycin-based combination
[9] [10]
[11]
[12]
[13]
[14]
[15]
therapy with minocycline or pyrimethamine. J Acquir Immune Defic Syndr 1994;7:1141–8. Derouin F, Chastang C. In vitro effects of folate inhibitors on Toxoplasma gondii. Antimicrob Agents Chemother 1989;33:1753–9. Murray HW, Cohn ZA. Macrophage oxygen-dependent antimicrobial activity. I. Susceptibility of Toxoplasma gondii. J Exp Med 1979;150:938–49. Choi KM, Park JK, Hwang SY. Pathogenetic impact of vacuolar degeneration by accelerated transport of Helicobacter pylori VacA. J Microbiol Biotechnol 2003;13:666–72. Döskaya M, Degirmenci A, C ¸ ic¸ek C, Ak M, Korkamz M, Guruz Y, et al. Behavior of Toxoplasma gondii RH Ankara strain tachyzoites during continuous production in various cell lines. Parasitology 2006;132:315–9. 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;89:207–13. Williams C, Espinosa OA, Montenegro H, et al. Hydrosoluble formazan XTT: its application to natural products drug discovery for Leishmania. J Microbiol Methods 2003;55:813–6. Park H, Kim MS, Jeon BH, Kim TK, Kim YM, Ahnn J, et al. Antimalarial activity of herbal extracts used in traditional medicine in Korea. Biol Pharm Bull 2003;26:1623–4.
Short communication
Anti-Toxoplasma gondii RH strain activity of herbal extracts used in traditional medicine Kyung-Min Choi a , Jingu Gang b , Jisoo Yun c,∗ a Ecodigm Co., Ltd., 305DTV Post BI, 707 Tamnip-dong, Yuseong-gu, Daejeon 305-510, South Korea Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea Research Center for Resistant Cells, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, South Korea b
c
Received 22 March 2008; accepted 14 April 2008
Abstract Methanolic extracts of 15 traditional medicines used to treat Toxoplasma gondii were tested in vitro for their anti-T. gondii activity and cytotoxicity. The median effective concentration (EC50 ) values for the herbal extracts ranged from 0.11 mg/mL to 2.28 mg/mL. Significant antiT. gondii RH strain activity was observed with Zingiber officinale extracts (EC50 = 0.18 mg/mL), which displayed a highly selective toxicity (selectivity = 10.1). Sophora flavescens Aiton extracts also showed high anti-T. gondii activity (EC50 = 0.20 mg/mL) and a high selective toxicity (4.6). This indicates that Z. officinale and S. flavescens Aiton extracts may be sources of new anti-T. gondii compounds. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Toxoplasma gondii; Anti-Toxoplasma gondii RH strain activity; Traditional medicine
1. Introduction Toxoplasma gondii, an obligate intracellular coccidian, has the ability to infect and propagate in virtually all nucleated host cells [1,2]. T. gondii infection causes toxoplasmosis, an infectious disease existing worldwide. This infection is normally innocuous in individuals with an intact immune response [3] who can promote a well-balanced interaction between the host and the parasite [2]. The RH strain of T. gondii is a virulent and non-cyst-forming strain that grows considerably faster than avirulent strains [4,5]. To control toxoplasmosis, a combination of antifolates, such as pyrimethamine and sulfadiazine, has been used with success and it is the first choice in most clinical settings [6,7]. On the other hand, a large percentage of undesirable side effects such as haematological toxicity (caused by pyrimethamine), cutaneous rash, leukopenia and thrombocytopenia (caused by sulphonamides) are often reported [7–9]. ∗
Corresponding author. Tel.: +82 62 233 6054; fax: +82 62 233 6052. E-mail address: [email protected] (J. Yun).
In an effort to improve the therapies used against T. gondii RH strain, we have examined numerous natural products for potential sources of new compounds with high activity and low toxicity. In this study, we describe the preliminary screening of 15 methanolic extracts used in Korean traditional medicine for anti-T. gondii RH strain activity and low cytotoxicity.
2. Materials and methods 2.1. RH strains of T. gondii RH strains of T. gondii were maintained by routine intraperitoneal passage with RPMI 1640 medium collected at 4 days post infection [5,10]. ICR mice weighing ca. 25 g were infected with 1 × 106 parasites at each passage. The tachyzoite concentration was determined using a haemocytometer at 200× magnification. The parasites were re-suspended at a density of 4 × 107 parasites/mL in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS).
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.04.012
K.-M. Choi et al. / International Journal of Antimicrobial Agents 32 (2008) 360–362
2.2. Cell culture HeLa cells were maintained in RPMI 1640 medium supplemented with 10% FBS. Cells were incubated at 37 ◦ C with humidified air and 5% CO2 . Cells were harvested by treatment with trypsin and washed twice in phosphate-buffered saline solution (pH 7.4). Viable cells were counted using a trypan blue exclusion test [11,12].
2.3. Preparation of traditional medicine samples All herbal drugs screened in this study were purchased from the University Oriental Drugstore, Iksan, South Korea. They were authenticated by matching against herbarium specimens at the College of Pharmacy, Wonkwang University, South Korea. Methanolic extracts were obtained by sonication of plant material (100 g dry weight) for 3 h in methanol (1 L). After filtration, the solvent was removed by evaporation in vacuo. The dried extract was dissolved in dimethyl sulphoxide (DMSO) before use. The anti-T. gondii RH strain drugs pyrimethamine, sulfadiazine and spiramycin were purchased from Sigma (St Louis, MO).
361
Table 1 In vitro anti-Toxoplasma gondii RH strain activity and cytotoxicity of traditional medicines Plant name
Arecae pericarpium Sophora flavescens Aiton Meliae cortex Rheum undulatum L. Pulsatilla radix Acorus gramineus Soland Dryopteris crassirhizoma Quisqualis indica L. Fraxinus rhynchophylla Hance Glycyrrhiza glabra L. Portulaca oleracea L. Schizandra chinensis Baillon Zingiber officinale Angelica gigas Artemisia scoparia Sulfadiazine Pyrimethamine Spiramycin
EC50 (mg/mL) HeLa cells
T. gondii RH
1.35 0.92 0.28 0.21 1.16 0.21 0.37 1.32 1.35 0.22 1.34 1.34 1.81 1.28 0.11 0.06 0.21 0.77
1.30 0.20 0.77 2.28 1.52 0.11 0.15 1.78 1.57 0.13 1.53 1.57 0.18 1.40 1.53 0.07 0.10 0.31
Selectivitya
– 4.6 – – – 1.9 2.5 – – 1.7 – – 10.1 – – – 2.1 2.5
EC50 , median effective concentration. a Ratio of the EC value for HeLa cells to the EC value for T. gondii 50 50 RH strain.
3. Results and discussion 2.4. In vitro infection and effectors HeLa cells were harvested during exponential growth (Day 2) and cultured in 96-well plates (ca. 6 × 104 cells/mL). Then, 3 × 105 parasites/mL were added to each well (parasite:cell ratio = 5:1, final volume 200 L) [13]. Six hours after inoculation, the cultures were washed twice with RPMI 1640 medium without FBS to remove any non-adherent parasites. After 18 h incubation, RPMI 1640 medium supplemented with 2% FBS was added to each well along with different concentrations of the 15 methanolic extracts. After 24 h of treatment, anti-T. gondii RH activity and cytotoxicity of the traditional medicines were examined using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (Promega, Madison, WI) assay [14]. The assay was conducted in 96-well plates and assayed using a microplate reader (Spectra Max Gemini XG, parameters chosen in SOFT max pro 4.0; Molecular Devices, Sunnyvale, CA) using a wavelength of 490 nm. All of the test compounds were assayed in triplicate at each concentration. All data points represent the mean of three independent experiments. The median effective concentration (EC50 ) value refers to the concentration of the compound necessary to inhibit at 50% of the control values. Selectivity refers to the mean of the EC50 value for HeLa cells relative to the mean of the EC50 value of the T. gondii RH strain [15].
Methanolic extracts of Korean herbal medicines used for T. gondii therapy were assessed for their activity against anti-T. gondii RH strain. The results of the in vitro anti-T. gondii and cytotoxicity assays against both the T. gondii RH strain and HeLa cells are summarised in Table 1. Extracts of Glycyrrhiza glabra L., Acorus gramineus Soland and Dryopteris crassirhizoma showed high anti-T. gondii RH strain activity (EC50 = 0.11–0.15 mg/mL) but had no selective toxicity (selectivity = 1.7–2.5). Another extract, Sophora flavescens Aiton, showed high anti-T. gondii RH strain activity (EC50 = 0.20 mg/mL) and selective toxicity (selectivity = 4.6). In addition, Zingiber officinale had high anti-T. gondii activity (EC50 = 0.18 mg/mL) as well as cytotoxicity against HeLa cells (at 1.81 mg/mL) and strong anti-T. gondii RH strain activity (selectivity = 10.1) relative to pyrimethamine (selectivity = 2.1) and spiramycin (selectivity = 2.5). The investigation of anti-T. gondii RH strain activity of Korean traditional medicines led to the finding that methanolic extracts of S. flavescens Aiton and Z. officinale showed potent anti-T. gondii RH strain activity. These results allowed us to suggest that these two traditional medicines are likely sources of new compounds that could be used to treat T. gondii infections. Further studies will be necessary to identify and isolate these active compounds. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required.
362
K.-M. Choi et al. / International Journal of Antimicrobial Agents 32 (2008) 360–362
References [1] Werk R. How does Toxoplasma gondii enter host cells? Rev Infect Dis 1985;7:449–57. [2] 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;24:271–9. [3] Yap GS, Sher A. Cell-mediated immunity to Toxoplasma gondii: initiation, regulation and effector function. Immunobiology 1999;201:240–7. [4] Appleford PJ, Smith JE. Toxoplasma gondii: the growth characteristics of three virulent strains. Acta Trop 1997;65:97–104. [5] Zhu S, Lai DH, Li SO, Lun ZR. Stimulative effects of insulin on Toxoplasma gondii replication in 3T3-L1 cells. Cell Biol Int 2006;30:149–53. [6] Harris C, Salgo MP, Tanowitz HB, Wittner M. In vitro assessment of antimicrobial agents against Toxoplasma gondii. J Infect Dis 1988;1570:14–22. [7] Dantas-Leitz L, Urbina JA, de Souza W, Vommaro RC. Antiproliferative synergism of azasterols and antifolates against Toxoplasma gondii. Int J Antimicrob Agents 2005;25:130–5. [8] Alder J, Hutch T, Meulbroek JA, Clement JC. Treatment of experimental Toxoplasma gondii infection by clarithromycin-based combination
[9] [10]
[11]
[12]
[13]
[14]
[15]
therapy with minocycline or pyrimethamine. J Acquir Immune Defic Syndr 1994;7:1141–8. Derouin F, Chastang C. In vitro effects of folate inhibitors on Toxoplasma gondii. Antimicrob Agents Chemother 1989;33:1753–9. Murray HW, Cohn ZA. Macrophage oxygen-dependent antimicrobial activity. I. Susceptibility of Toxoplasma gondii. J Exp Med 1979;150:938–49. Choi KM, Park JK, Hwang SY. Pathogenetic impact of vacuolar degeneration by accelerated transport of Helicobacter pylori VacA. J Microbiol Biotechnol 2003;13:666–72. Döskaya M, Degirmenci A, C ¸ ic¸ek C, Ak M, Korkamz M, Guruz Y, et al. Behavior of Toxoplasma gondii RH Ankara strain tachyzoites during continuous production in various cell lines. Parasitology 2006;132:315–9. 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;89:207–13. Williams C, Espinosa OA, Montenegro H, et al. Hydrosoluble formazan XTT: its application to natural products drug discovery for Leishmania. J Microbiol Methods 2003;55:813–6. Park H, Kim MS, Jeon BH, Kim TK, Kim YM, Ahnn J, et al. Antimalarial activity of herbal extracts used in traditional medicine in Korea. Biol Pharm Bull 2003;26:1623–4.