Abstract
Safety data of newly developed active pharmaceutical ingredient is of regulatory requirement. The LD50 value is important to estimate the suitable dosage of extract for acute toxicity study and determine its safe dosage. LD50 estimations for aqueous and ethanol extracts of polyherbal Diabecine™ were determined due to its potential to be used as active ingredient in herbal supplement for diabetic patients. Diabecine™ is a proprietary herbal blend of Curcuma xanthorriza, Cinnamomum zeylanicum, Andrographis paniculata, Orthosiphon stamineus and Eugenia polyantha Wight traditionally used as herbal medicine for diabetes. The cytotoxicity towards three cell lines commonly used for in vitro diabetic study namely, 3T3-L1, WRL-68 and 1.1B4, was determined in this study using MTT assay method. The IC50 values were determined and used to estimate LD50 values using ICCVAM’s regression formula: log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024. All the extracts showed non-toxic effects against 3T3-L1, WRL-68 and 1.1B4 cell lines at concentration of 0.5 to 100 µg/mL, but significantly cytotoxic to all the three cell lines at concentration of 1,000 µg/mL and above. The IC50 values for 3T3-L1, 1.1B4 and WRL-68 are in the range of 493.8 to 779.6 µg/mL, 258.4 to 386.7µg/mL and 630.9 to 2,785 µg/mL. The 1.1B4 cell line was shown to have the highest cytotoxic sensitivity to the extracts. The IC50 values for 1.1B4 were used to estimate the LD50 values for ethanol freeze-dried, ethanol spray-dried, aqueous freeze-dried and aqueous spray-dried extracts, which are 956.4 mg/kg, 969.4 mg/kg,834.4 mg/kg and 880.4 mg/kg. The starting dose of each extracts for acute toxicity study could be 3.2 factors lower than the estimated LD50 values, which was suggested to be 298.9 mg/kg, 302.9 mg/kg, 260.8 mg/kg and275.1 mg/kg for ethanol freeze-dried, ethanol spray-dried, aqueous freeze-dried and aqueous spray-dried extracts.
