Myrrh Oil Case Study

2. Materials and methods
2.1. Chemical reagents
Myrrh oil was purchased from Sigma-Aldrich (Saint Louis, USA). Stock solutions of 100 mg/ml in Ethanol (Eth) were prepared and stored at −30°C until use. Diminazene aceturate (Ganaseg) was purchased from (Ciba-Geigy Japan Ltd., Tokyo, Japan) and used as a positive control drug. A working stock solution of 10 mM dissolved in DDW was prepared and stored at -30◦C until required for use.

2.2. Rodent Babesia and mice
The Munich strain of B. microti was maintained by serial passage in the blood of BALB/c mice (AbouLaila et al. 2010b). Thirty female BALB/c mice (8 weeks old) were purchased from CLEA Japan (Tokyo, Japan) and used for the in vivo studies.

2.3. In vitro cultivation of Babesia parasites

bovis (Texas strain) (Hines et al. 1995), B. bigemina (Argentina strain) (Igarashi et al. 1998), B. caballi (AbouLaila et al. 2010b), and T. equi (U.S. Department of Agriculture) (Melhorn and Schein 1998). Parasites were cultured in bovine or equine red blood cells using a continuous micro-aerophilous stationary phase culture system (Aboulaila et al. 2012; Igarashi et al. 1998). The culture medium, M199, applied to B. bovis, B. bigemina, and T. equi (obtained from Sigma-Aldrich, Tokyo, Japan), was supplemented with 40% bovine or equine serum and 60 U/ml of penicillin G, 60 µg/ml of streptomycin, and 0.15 µg/ml of amphotericin B (Sigma-Aldrich, Tokyo, Japan) (Aboulaila et al. 2010c). Hypoxanthine (ICN Biomedicals, Inc., Aurora, OH) was added to the T. equi culture as a vital supplement at 13.6 mg/ml. For B. caballi, the culture medium RPMI 1640 was supplemented with 40% horse serum, antibiotics, and amphotericin B (Aboulaila et al.

Viability test
After 4 days of treatment, 6 µL of parasite-free bovine or equine packed red blood cells was added to 14 µL of packed red blood cells from the previously drug-treated cultures in 200 µL of a fresh growth medium without the drug. The fresh growth medium was replaced every day for the next 10 days, and parasite recrudescence was determined daily after removal of the drugs by microscopic examination of Giemsa-stained thin erythrocyte smears (Aboulaila et al. 2010c).
2.6. Effect of myrrh oil on host