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Inhibitory effects of (-)-Epigallocatechin-3-gallate from green tea on the growth of Babesia parasites

Published online by Cambridge University Press:  22 December 2009

M. ABOULAILA ,
N. YOKOYAMA  and
I. IGARASHI
M. ABOULAILA
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido 080-8555, Japan
N. YOKOYAMA
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido 080-8555, Japan
I. IGARASHI*
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido 080-8555, Japan
*
*Corresponding author: Tel: +81 155 49 5641. Fax: +81 155 49 5643. E-mail: igarcpmi@obihiro.ac.jp
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Summary

(-)-Epigallocatechin-3-gallate (EGCG) is the major tea catechin and accounts for 50–80% of the total catechin in green tea. (-)-Epigallocatechin-3-gallate has antioxidant, anti-inflammatory, anti-microbial, anti-cancer, and anti-trypanocidal activities. This report describes the inhibitory effect of (-)-Epigallocatechin-3-gallate on the in vitro growth of bovine Babesia parasites and the in vivo growth of the mouse-adapted rodent babesia B. microti. The in vitro growth of the Babesia species was significantly (P<0·05) inhibited in the presence of micromolar concentrations of EGCG (IC50 values=18 and 25 μM for B. bovis, and B. bigemina, respectively). The parasites showed no re-growth at 25 μM for B. bovis and B. bigemina in the subsequent viability test. The drug significantly (P<0·05) inhibited the growth of B. microti at doses of 5 and 10 mg/kg body weight, and the parasites completely cleared on day 14 and 16 post-inoculation in the 5 and 10 mg/kg treated groups, respectively. These findings highlight the potentiality of (-)-Epigallocatechin-3-gallate as a chemotherapeutic drug for the treatment of babesiosis.

Keywords

(-)-Epigallocatechin-3-gallatein vitro growthin vivo growthBabesia microti

Information

Type
Research Article
Information
Parasitology , Volume 137 , Issue 5 , April 2010 , pp. 785 - 791
Copyright
Copyright © Cambridge University Press 2009

INTRODUCTION

Babesia parasites are tick-transmitted protozoa that affect a wide variety of wild and domestic animals and are responsible for enormous economic losses to the livestock industry worldwide (Kuttler, Reference Kuttler and Ristic1988). Moreover, some species are major aetiologic agents of human babesiosis (Homer et al. Reference Homer, Aguilar-Delfin, Telford, Krause and Persing2000; Kjemtrup and Conrad, Reference Kjemtrup and Conrad2000). Babesia microti infects laboratory mice, causes a relatively mild but persistent disease, and has served as a useful experimental model for the analysis of animal and human babesiosis (Cox and Young, Reference Cox and Young1969). The analysis of the disease in this model has enabled a search for anti-babesial drugs (Wittner et al. Reference Wittner, Lederman, Tanowitz, Rosenbaum and Weiss1996; Marley et al. Reference Marley, Eberhard, Steurer, Ellis, McGreevy and Ruebush1997; Bork et al. Reference Bork, Yokoyama, Ikehara, Kumar, Sugimoto and Igarashi2004a). Several babesicidal drugs that have been in use for years have proven to be ineffective owing to problems related to their toxicity and the development of resistant parasites (Bork et al. Reference Bork, Yokoyama and Igarashi2005a; Vial and Gorenflot, Reference Vial and Gorenflot2006). Therefore, there is a need to develop new drugs that have a chemotherapeutic effect against babesiosis with high specificity to the parasites and low toxicity to the hosts.

Tea (Camellia sinensis) is one of the most popular beverages in the world, and its beneficial effects on health have attracted great attention (Yang and Landau, Reference Yang and Landau2000). The most significant groups of tea components are polyphenols, especially the catechin group called flavonols. (–)-Epigallocatechin-3-gallate (EGCG) is the major tea catechin and EGCG accounts for 50–80% of the total catechin (Graham, Reference Graham1992). EGCG have anti-tumorigenic (Chen and Zhang, Reference Chen and Zhang2007), anti-inflammatory (Lin and Lin, Reference Lin and Lin1997), anti-oxidative (Fraga et al. Reference Fraga, Martino, Ferraro, Coussio and Boveris1987), anti-proliferative (Shammas et al. Reference Shammas, Neri, Koley, Batchu, Bertheau, Munshi, Prabhala, Fulciniti, Tai, Treon, Goyal, Anderson and Munshi2006), anti-bacterial (Mabe et al. Reference Mabe, Yamada, Oguni and Takahashi1999), and anti-viral (Fassina et al. Reference Fassina, Buffa, Benelli, Varnier, Noonan and Albini2002; Yamaguchi et al. Reference Yamaguchi, Honda, Ikigai, Hara and Shimamura2002; Williamson et al. Reference Williamson, McCormick, Nance and Shearer2006) effects. EGCG also have anti-trypanocidal activity against Trypanosoma cruzi (Paveto et al. Reference Paveto, Güida, Esteva, Martino, Coussio, Flawiá and Torres2004). In this study we present the inhibitory effect of EGCG on B. bovis and B. bigemina in the in vitro cultures and on the in vivo growth of B. microti.

MATERIALS AND METHODS

Chemical compounds

(-) Epigallocatechin-3-gallate (EGCG) was purchased from Sigma-Aldrich (USA) and used as a test drug. A working stock solution of 1 mm in PBS was prepared and stored at −30°C until use. Diminiazine aceturate (GANASEG) was purchased from (Ciba-Geigy Japan Lit., Tokyo, Japan). A working stock solution of 10 mm in distilled water was prepared and stored at −30°C until use. Tetracycline hydrochloride was purchased from Sigma-Aldrich (USA). A working stock solution of 20 mm in distilled water was prepared and stored at −30°C until use.

In vitro growth inhibition assay

B. bovis Texas strain and B. bigemina Argentina strain were grown in a micro-aerophilous stationary-phase culture system using bovine erythrocytes (RBCs), and sera as described previously (Bork et al. Reference Bork, Yokoyama, Ikehara, Kumar, Sugimoto and Igarashi2004a). Serum-free GIT medium also was used to study the effect of serum-free medium on the drug inhibition of B. bovis (Bork et al. Reference Bork, Okamura, Matsu, Kumar, Yokoyama and Igarashi2005b). The in vitro growth inhibition assay for (-) Epigallocatechin-3-gallate followed a method previously described (Bork et al. Reference Bork, Yokoyama, Matsuo, Claveria, Fujisaki and Igarashi2003a, Reference Bork, Yokoyama, Ikehara, Kumar, Sugimoto and Igarashi2004a) with some modifications. Parasite-infected RBCs were diluted with uninfected RBC to obtain 1% parasitaemia. Twenty μl of RBC with 1% parasitaemia were dispensed to a 96-well microtitre plate together with 200 μl of the culture medium containing the indicated concentration of (-) Epigallocatechin-3-gallate (5, 10, 25, 50, 100 μm), diminazene aceturate (0·5, 1, and 2 μm), and tetracycline hydrochloride (5, 50, and 100 μm) (Matsuu et al. Reference Matsuu, Yamasaki, Xuan, Ikadai and Hikasa2008) and then incubated at 37°C in a humified multi-gas water-jacketed incubator. For the control, cultures without the drug and another containing only PBS or distilled water at a similar concentration to the highest drug concentration were prepared. The experiments were carried out in triplicate for each drug concentration in 3 separate trials for a period of 4 days. The culture medium was replaced daily with 200 μl of fresh medium containing the appropriate concentration of the drug. Parasitaemia was monitored daily by counting the parasitized RBC to approximately 1000 in Giemsia-stained thin blood smears. Values of a 50% inhibitory concentration (IC50) of the tested drugs against all parasites were calculated by interpolation after curve fitting, based on parasitaemia observed at day 3 after drug treatment.

Viability test

After the fourth day of the treatment, 6 μl of each of the control and drug-treated (at the various concentrations) RBC was mixed with 14 μl of parasite-free RBC and suspended in 200 μl of fresh growth medium without drug supplementation. The plates were incubated for the next 10 days. The culture medium was replaced daily, and parasite recrudescence was determined by light microscopy to evaluate the parasite viability (Bork et al. Reference Bork, Yokoyama, Ikehara, Kumar, Sugimoto and Igarashi2004a).

In vivo inhibition assay

The Munich strain of B. microti was maintained by passage in the blood of BALB/c mice as previously described (Yokoyama et al. Reference Yokoyama, Bork, Nishisaka, Hirata, Matsuo, Inoue, Xuan, Suzuki, Sugimoto and Igarashi2003). The in vivo growth inhibitory assay for EGCG was carried out for B. microti twice per parasite according to the method previously described (Bork et al. Reference Bork, Yokoyama, Ikehara, Kumar, Sugimoto and Igarashi2004a; Li et al. Reference Li, Ashok, Li, Yang, Sama and Wang2007) with some modifications. Eighteen 8-week-old female BALB/c mice (CLEA Japan, Tokyo, Japan) were divided into 3 groups. Each group consisted of 6 mice that were inoculated intraperitoneally with 1×106B. microti-infected RBC. EGCG was administered at a dosage of 5 and 10 mg/kg after dissolving in 0·3 ml of phosphate-buffered saline (PBS) for the first and second groups. PBS alone was administered at 0·3 ml to the third group as a placebo control. EGCG-treated groups underwent daily intraperitoneal injections from days 3 to 12 post-infection when the parasitaemia was approximately 1%. The levels of parasitaemia in all mice were monitored daily until day 26 post-infection by examination of stained thin blood smears prepared from venous tail blood. All animal experiments were conducted in accordance with the Standard Relating to the Care and Management of Experimental Animals promulgated by the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.

Statistical analysis

JMP statistical software version 5.1(SAS Institute Inc., USA) was used to compare the means in the in vitro and in vivo experiments by independent Student's t-test and considered to be significantly different when P<0·05.

RESULTS

In vitro growth inhibition assay

The growth of B. bovis (Fig. 1A) and B. bigemina (Fig. 1B) from an initial parasitaemia of 1% was significantly (Student's t-test, P<0·05) inhibited at 10 and 25 μm respectively and significantly suppressed in the presence of 50 μm EGCG. Complete clearance of B. bovis and B. bigemina (Fig. 1A and B) was observed as early as the second day of drug treatment. The concentrations of 50 μm tetracycline hydrochloride and 1 μm diminazene aceturate significantly (Student's t-test, P<0·05) inhibited the growth of B. bovis and B. bigemina. However, higher concentrations of 100 μm tetracycline hydrochloride and 2 μm diminazene aceturate could not completely clear the parasites on the fourth day of culture (Fig. 1A and B). The 50% inhibitory concentration (IC50) values of EGCG on the third day of culture were 18 μm (B. bovis) and 25 μm (B. bigemina) (Table 1). Subsequent cultivation of the parasites without the drug for a 10-day period showed no regrowth of the parasites at 25 μm for B. bovis and B. bigemina (Fig. 1A and B). Parasites exposed to lower drug concentrations started to grow again, as shown by light microscopy. The IC50 values of tetracycline hydrochloride were 48 μm (B. bovis) and 43 μm (B. bigemina), while those of diminazene aceturate were 0·67 μm (B. bovis) and 0·5 μm (B. bigemina) (Table 1). Parasites could not re-grow at the concentrations of 100 μm tetracycline hydrochloride and 1 μm diminazene aceturate in the subsequent viability test (Fig. 1A and B). There was no difference in the growth inhibition of EGCG for B. bovis with GIT medium (data not shown), therefore, serum has no effect on the activity of EGCG. EGCG affected the morphology of the parasites in treated cultures as indicated by light microscopy. Some parasites appeared degenerate, or abnormally multidividing forms were observed (Fig. 2B and D). In addition to these reported changes, treated B. bigemina parasites lost the normal appearance of the acute angle and had an obtuse angle between their pairs (Fig. 2D). Based on light microscopic observations of the changes in the host cell shape, size, and colour, and the appearance of perforations, EGCG was non-toxic to erythrocytes.

Fig. 1.

Inhibitory effect of EGCG on the in vitro growth of Babesia bovis (A) and B. bigemina (B). Diminazene aceturate (Dimin) and tetracycline hydrochloride (Tetra) were used as positive controls. Each value represents the mean±standard deviation of 3 separate experiments carried out in triplicate. Asterisks, indicate significant difference (Student's t-test; * P<0·05) between the 10 μm and 25 μm EGCG-treated and the control cultures of B. bovis and B. bigemina, respectively.

Fig. 2.

EGCG-treated Babesia parasites in an in vitro culture. B. bovis: control (A), treated with 10 μm EGCG (B). B. bigemina: control (C), treated with 10 μm EGCG (D). Drug-treated cultures showed a higher number of degenerated parasites (thin arrows) and abnormally multidividing forms (arrowheads) and parasites with an obtuse angle (thick arrows). Scale bars=10 μm.

Table 1.

IC50 values of EGCG, tetracycline hydrochloride and diminazene aceturate for growth inhibition of Babesia bovis and B. bigemina

a IC50 values expressed as drug concentration are in micromoles of the growth medium and were determined on day 3 of in vitro culture using a curve fitting technique. IC50 values obtained from 3 separate experiments.

In vivo effect of EGCG on the growth of B. microti

In B. microti infected mice, the levels of parasitaemia in the treated groups increased significantly (Student's t-test, P<0·05) more slowly than the control, achieving peak parasitaemia of 10·5 and 9·1% in the presence of 5 and 10 mg/kg on day 11 p.i., respectively. In contrast, in the control group, the peak parasitaemia was 60·8% (PBS) on day 11 p.i. (Fig. 3). The 5 mg/kg treated mice completely cleared the parasites on day 14 p.i. achieving a parasitaemia of zero, while the parasites completely cleared on day 16 p.i. in 10 mg/kg treated mice. There were no signs of toxicity observed in treated mice.

Fig. 3.

Inhibitory effects of EGCG (5 and 10 mg/kg) on the in vivo growth of Babesia microti for observations of 6 mice per experimental group. Each value represents the mean±S.D for 2 experiments. Asterisks, indicate significant difference (Student's t-test; * P<0·05) between drug-treated and PBS groups.

DISCUSSION

In the present study, the inhibitory effect of EGCG on the in vitro growth of bovine Babesia was evident. The presence of a higher concentration of EGCG in the culture completely suppressed the growth of all parasites tested in this study. As the presence of the solvents alone did not affect the growth of the parasites, the growth inhibition observed in this study was due to the effects of EGCG. The IC50 values of EGCG were lower than those of tetracycline hydrochloride, while they were higher than those of diminazene aceturate. The IC50s values of EGCG for Babesia parasites were lower than other drugs used in previous studies: ketoconazole (Bork et al. Reference Bork, Yokoyama, Matsuo, Claveria, Fujisaki and Igarashi2003c), gossypol (Bork et al. Reference Bork, Okamura, Boonchit, Hirata, Yokoyama and Igarashi2004b), heparin (Bork et al. Reference Bork, Yokoyama, Ikehara, Kumar, Sugimoto and Igarashi2004a), EGTA (Okubo et al. Reference Okubo, Wilawan, Bork, Okamura, Yokoyama and Igarashi2006), and chloroquine diphosphate (Matsuu et al. Reference Matsuu, Yamasaki, Xuan, Ikadai and Hikasa2008). They were a similar range to the other drugs tested as babesicidal drugs: triclosan (Bork et al. Reference Bork, Yokoyama, Matsuo, Claveria, Fujisaki and Igarashi2003a), clotrimazole (Bork et al. Reference Bork, Yokoyama, Matsuo, Claveria, Fujisaki and Igarashi2003b, Reference Bork, Yokoyama, Matsuo, Claveria, Fujisaki and Igarashic), tetracyclines (Nott et al. Reference Nott, O'Sullivan, Gero and Bagnara1990; Matsuu et al. Reference Matsuu, Yamasaki, Xuan, Ikadai and Hikasa2008), staurosporine (Bork et al. Reference Bork, Das, Okubo, Yokoyama and Igarashi2006), purvalanol A (Nakamura et al. Reference Nakamura, Yokoyama and Igarashi2007), and ALLN (Okubo et al. Reference Okubo, Yokoyama, Govind, Alhassan and Igarashi2007). The IC50 values of EGCG were higher than other babesicidal drugs: quinuronium sulfate (Brockelman and Tan-ariya, Reference Brockelman and Tan-ariya1991), imidocarb dipropionate (Rodriguez and Trees, Reference Rodriguez and Trees1996; Brasseur et al. Reference Brasseur, Lecoublet, Kapel, Vennec and Ballet1998), Clindamycin phosphate (Brasseur et al. Reference Brasseur, Lecoublet, Kapel, Vennec and Ballet1998), and atovaquone (Pudney and Gray, Reference Pudney and Gray1997; Matsuu et al. Reference Matsuu, Yamasaki, Xuan, Ikadai and Hikasa2008). The IC50s values of EGCG were very high comparable to those reported for T. cruzi (Paveto et al. Reference Paveto, Güida, Esteva, Martino, Coussio, Flawiá and Torres2004) indicating a higher sensitivity of T. cruzi to EGCG than Babesia parasites. The IC50s values of EGCG for Babesia are very low when compared with that reported for the normal mammalian cells of H9 (174·8 μm) and THP-1 (440·3 μm) types (Yamaguchi et al. Reference Yamaguchi, Honda, Ikigai, Hara and Shimamura2002); furthermore, the concentration of 20 μm had no effect on the normal mammalian cells (Shammas et al. Reference Shammas, Neri, Koley, Batchu, Bertheau, Munshi, Prabhala, Fulciniti, Tai, Treon, Goyal, Anderson and Munshi2006). Thus, EGCG is non-toxic to the mammalian cells.

EGCG had a strong inhibitory effect on the growth of the cultured parasites; thus, we were encouraged to investigate its in vivo effect on B. microti infection in mice. The inhibitory effect of EGCG on the growth of B. microti was evident. The difference in growth inhibition between the control and the drug-treated groups was significant on days 4–14 p.i. Interestingly, there was no significant difference in the inhibition of B. microti between the two doses used and this was consistent with the report by Wang et al. (Reference Wang, Mei, Feng and Xu2006) in which doses up to 10 mg/kg did not inhibit Concanavalin A induced alanine aminotransferase (ALT) release from hepatocytes more efficiently than the dose of 5 mg/kg. B. microti-infected mice that were treated with 5 and 10 mg/kg did not show signs of toxicity and were alive during and after the end of the experiment. In the current study, EGCG effectively suppressed B. microti in mice treated for 10 days at 5 and 10 mg/kg with 83·3 and 84% inhibition, respectively, on day 11 p.i., while mice treated with 2·5 mg heparin for 10 days had 86% inhibition on day 8 p.i. (Bork et al. Reference Bork, Yokoyama, Ikehara, Kumar, Sugimoto and Igarashi2004a). In the hamster model, atovaquone at doses of 300, 150, and 80 mg/kg/day for 8 days was effective in the recovery of all animals compared with 50% of those receiving 10 mg/kg/day (Hughes and Oz, Reference Hughes and Oz1995), while recrudescences occurred at the dosage of 100 mg/kg/day (Wittner et al. Reference Wittner, Lederman, Tanowitz, Rosenbaum and Weiss1996). Clindamycin plus quinine was used at 150 mg/kg for 7 days, and the inhibition was 70% on day 7 post-treatment (Marley et al. Reference Marley, Eberhard, Steurer, Ellis, McGreevy and Ruebush1997). Azithromycin, either alone (150 and 300 mg/kg) or in combination (150 mg/kg) with quinine (250 mg/kg), was effective in reducing levels of parasitaemia during the period of drug administration, but parasitaemia rapidly returned to pre-treatment levels when the drug was stopped (Weiss et al. Reference Weiss, Wittner, Wasserman, Oz, Retsema and Tanowitz1993). In the gerbil model, atovaquone at 50 mg/kg for 10 days resulted in rapid reduction of parasitaemia but did not eliminate the infection (Gray and Pudney, Reference Gray and Pudney1999). The direct comparison of EGCG effectiveness with other drugs used for in vivo treatment of Babesia infection requires further study. Our results encourage the potential use of EGCG as a chemotherapeutic tool against animal and human babesiosis.

EGCG was used as anti-oxidative (Fraga et al. Reference Fraga, Martino, Ferraro, Coussio and Boveris1987), anti-inflammatory (Lin and Lin, Reference Lin and Lin1997), anti-proliferative (Ramirez-Mares et al. Reference Ramirez-Mares, Chandra and de Mejia2004; Shammas et al. Reference Shammas, Neri, Koley, Batchu, Bertheau, Munshi, Prabhala, Fulciniti, Tai, Treon, Goyal, Anderson and Munshi2006), anti-cancer (Chen and Zhang, Reference Chen and Zhang2007), anti-bacterial (Mabe et al. Reference Mabe, Yamada, Oguni and Takahashi1999), anti-viral (Fassina et al. Reference Fassina, Buffa, Benelli, Varnier, Noonan and Albini2002; Yamaguchi et al. Reference Yamaguchi, Honda, Ikigai, Hara and Shimamura2002; Williamson et al. Reference Williamson, McCormick, Nance and Shearer2006) and anti-trypanocidal (Paveto et al. Reference Paveto, Güida, Esteva, Martino, Coussio, Flawiá and Torres2004). The mechanism of inhibition of EGCG for Babesia parasites is not known while there are several reported mechanisms of inhibition in the previous research on other microorganisms. EGCG inhibits arginine kinase of T. cruzi (Paveto et al. Reference Paveto, Güida, Esteva, Martino, Coussio, Flawiá and Torres2004), but this enzyme was not found in the genome data-base of either B. bovis or B. bigemina (http://www.sanger.ac.uk) indicating a different mechanism of action for Babesia parasites. EGCG has been shown to be an inhibitor of dihydrofolate reductase (DHFR) of Stenotrophomonas maltophilia (Navarro-Martínez et al. Reference Navarro-Martínez, Navarro-Perán, Cabezas-Herrera, Ruiz-Gómez, García-Cánovas and Rodríguez-López2005); therefore, EGCG may inhibit the homologous enzyme of Babesia parasites, but this requires further investigation. EGCG binds to the haemagglutinin (HA) spike proteins of influenza virus which results in blocking the viral attachment to the receptors of target cells (Nakayama et al. Reference Nakayama, Suzuki, Toda, Okubo, Hara and Shimamura1993); furthermore, EGCG inhibited the binding of HIV1-gp120 to CD4+T cells in a dose-dependent manner (Williamson et al. Reference Williamson, McCormick, Nance and Shearer2006). However, EGCG only affected the morphology of the intracellular parasites, and the percentages of the extracellular parasites in treated cultures were not different from those of the control. Therefore, EGCG had no effect on parasite invasion and further studies are required to identify the potential mechanism of action.

In summary, EGCG inhibited the in vitro growth of 2 bovine Babesia species and the in vivo growth of B. microti. EGCG may have potential for practical use in in vivo therapy of babesiosis; however, further studies will be required to confirm its mechanism of inhibition to Babesia parasites.

ACKNOWLEDGEMENTS

This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Grants from the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), the 21st Century COE Program (A-1), Ministry of Education, Culture, Sports, Science, and Technology, Japan, and Ministry of Higher Education, Egypt.

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Figure 0

Fig. 1. Inhibitory effect of EGCG on the in vitro growth of Babesia bovis (A) and B. bigemina (B). Diminazene aceturate (Dimin) and tetracycline hydrochloride (Tetra) were used as positive controls. Each value represents the mean±standard deviation of 3 separate experiments carried out in triplicate. Asterisks, indicate significant difference (Student's t-test; * P<0·05) between the 10 μm and 25 μm EGCG-treated and the control cultures of B. bovis and B. bigemina, respectively.

Figure 1

Fig. 2. EGCG-treated Babesia parasites in an in vitro culture. B. bovis: control (A), treated with 10 μm EGCG (B). B. bigemina: control (C), treated with 10 μm EGCG (D). Drug-treated cultures showed a higher number of degenerated parasites (thin arrows) and abnormally multidividing forms (arrowheads) and parasites with an obtuse angle (thick arrows). Scale bars=10 μm.

Figure 2

Table 1. IC50 values of EGCG, tetracycline hydrochloride and diminazene aceturate for growth inhibition of Babesia bovis and B. bigemina

Figure 3

Fig. 3. Inhibitory effects of EGCG (5 and 10 mg/kg) on the in vivo growth of Babesia microti for observations of 6 mice per experimental group. Each value represents the mean±S.D for 2 experiments. Asterisks, indicate significant difference (Student's t-test; * P<0·05) between drug-treated and PBS groups.