Pharmacognostic specification and brazilin content in Caesalpinia sappan heartwoods, macroscopic, microscopic and molecular identification of selected Caesalpinia species in Thailand

Abstract

The genus Caesalpinia L. belongs to Caesalpiniaceae family. This genus was spread throughout Asia and South East Asia. In Thailand, 18 species of Caesalpinia L. genus were reported which were spread in every parts. Due to the similar morphology especially the leaf part and closely vernacular name of eight Caesalpinia species, the identification of these species is complicated. Caesalpinia sappan L. is the well-known species which has many important drug properties. It was used as food, beverage, and cosmetic industries especially the heartwood part that contained brazilin, one of the flavonoid group. This study aimed to establish the pharmacognostic specification, develop the quantitative analysis of brazilin in C. sappan heartwoods by TLC analysis using TLC-densitometry compared to TLC-image analysis. Microscopic leaf characteristics including leaf constant numbers, and phylogenetic relationship by ISSR markers were used to differentiate the selected eight Caesalpinia species in Thailand. The heartwood of C. sappan was hard and rough with orange-red color. The contents of brazilin using TLC-densitometry and TLC-image analysis were 1.259 ± 0.455 and 1.256 ± 0.405 g/100 g crude drug, respectively. Brazilin contents analyses from two methods were not statistically significantly different (P> 0.05). The analytical methods were validated to confirm that the processes were appropriate, reliable, and gain an accurate data in term of specificity, accuracy, precision, limit of detection, limit of quantitation and robustness. The physico-chemical parameters including total ash, acid insoluble ash, ethanol and water soluble extractive values, loss on drying, and water content should not be more than 0.87, 0.44, 8.50, and 8.52 g/100 g crude drug, respectively whereas the content of ethanol and water soluble extractives should not be less than 2.94 and 3.77 g/100 g crude drug. Botanical characteristics and transverse cross section of midrib were illustrated by drawing. Microscopic leaf characteristics including leaf constant numbers (stomatal number, stomatal index, palisade ratio, epidermal cell area, trichome number, and trichome index) were evaluated. The results from microscopic characteristics can be used as the key to classification plants in Caesalpinia species. For ISSR fingerprinting, 15 universal primers were screened and 7 primers were used in this study. This technique produced a total of distinct and reproducible 217 bands. The genetic relationship among Caesalpinia L. genus presented the similarity index between 0.192 and 0.454. The information from this study provided the beneficial data for identification of selected eight Caesalpinia species, provided the pharmacognostic specification and brazilin content in C. sappan heartwoods in Thailand.