MACROSCOPIC, MICROSCOPIC AND MOLECULAR AUTHENTICATION OF SELECTED GENERA EURYCOMA, ERYTHROXYLUM AND TINOSPORA ENDEMIC TO THAILAND

Abstract

Genera Eurycoma, Erythroxylum and Tinospora have been widely used as herbal plants for a long time as their pharmacological activities in traditional medicine for antipyretic. Among these three genera, there are some similarity in morphological characteristics which leading to crude drugs substitution. This current study aims to examine the distinguishable characteristics between Eurycoma longifolia Jack and Eurycoma harmandiana Pierre in genus Eurycoma, Erythroxylum novogranatense (Morris) Hieron, Erythroxylum cambodianum Pierre and Erythroxylum cuneatum (Miq.) Kurz in genus Erythroxylum, and Tinospora crispa (L.) Miers ex Hook.f.& Thomson and Tinospora baenzigeri Forman in genus Tinospora using macroscopic examination, microscopic examination and molecular authentication as well as establish the standardization parameter for establish the quantity of Eurycoma longifolia Jack due to its essential medicinal plant. Evaluation of macroscopic characteristics and midrib transverse section of leaf were illustrated by drawing, and described as anatomical and botanical characteristics. Leaf constant values (stomata number, stomata index, epidermal cell number, epidermal cell area, palisade ratio and stomatal type) were evaluated under microscope. AFLP DNA fingerprint analysis was also performed for their genetic assessment. Standardization of Eurycoma longifolia Jack root were evaluated according to WHO guideline. Midrib transverse section of leaf showed the difference in arrangement of fundamental cells in each species. For microscopic examination, genera Eurycoma and Tinospora showed anomocytic type of stomata but genus Erythroxylum showed paracytic type of stomata. The stomata existed only on the abaxial in all species except occurred on both sides of adaxial and abaxial in Tinospora baenzigeri. Microscopically leaf constant numbers any these species were established and capable to be a tool for authentication. AFLP fingerprinting obtained from five primer combinations produced a total of distinct and highly polymorphic fragments (348, 349 and 476 bands), percentage of polymorphic bands (96.26%, 97.42% and 96.01%) and the similarity index (0.331 - 0.957, 0.270 - 0.988 and 0.472 - 0.934) among selected species in genera Eurycoma, Erythroxylum and Tinospora respectively. The UPGMA dendrogram was clearly separated individual species into each cluster. Pharmacognostic specification of Eurycoma longifolia root were founded unique of arrangement and type of cell components. The standardization parameters of Eurycoma longifolia root including total ash, acid insoluble ash, loss on drying and water contents should be not more than 1.97, 0.67, 7.52 and 13.35 while water soluble extractive and ethanol soluble extractive values should be not less than 4.89 and 1.73 of dry weight respectively. Thin layer chromatographic patterns of methanolic extract were also demonstrated. In conclusion, the data obtained from these results provided highly useful information and could be applied for the authentication of selected genera Eurycoma, Erythroxylum and Tinospora, and established the standardization of Eurycoma longifolia.