Microscopic and molecular authentication of selected cassia species endemic to Thailand and evaluation of aloe-emodin contents in cassia garrettiana and cassia grandis leaves

Abstract

The genus Cassia L, belongs to the Caesalpiniaceae family, has been widely used as herbs, foods and ornamental plants for a long time. Due to the similar morphology and vernacular name, the identification of these species is perplexed. This study aimed to distinguish 16 Cassia spp. existing in Thailand using macroscopic examination, microscopic examination and AFLP fingerprinting as well as the quantitative analysis of aloe-emodin contents in C. garrettiana and C. grandis leaves collected from 15 different locations in Thailand was also developed and validated using thin-layer chromatography densitometry and thin-layer chromatography image analysis with ImageJ software. The macroscopic characteristics and transverse section of leaf through midrib were illustrated by drawing. Transverse section of leaf through midrib observed under the light microscope showed the anatomical characteristics of epidermis, palisade cells, spongy cells, vascular bundle, parenchyma and collenchyma. Leaf trichome characteristic and number observed under the light microscope and scanning electron microscopy showed the uniseriate, uni- or multicellular non-glandular and multicellular glandular trichome. However, some Cassia spp. had absent of trichome. Cassia javanica L. had the highest trichome number in both dorsal (78.94±2.86) and ventral (127.39±2.46) surfaces of the leaf whereas Cassia surattensis Burm. f. had the lowest trichome number found only on ventral (3.46±0.80) surface. The AFLP fingerprint among 16 selected Cassia spp. indicated that eleven primer combinations produced a total of distinct and reproducible 849 bands with an average 77.18 bands per primer combinations. The genetic relationship based on amplified AFLP bands showed the similarity index (SI) ranged from 0.25 to 0.78. The quantitative analysis of aloe-emodin contents from 15 sources of Cassia garrettiana Craib and Cassia grandis L. f. using TLC-densitometry with winCATS software and TLC-image analysis with ImageJ software found that C. garrettiana dried crude drug had 0.035±0.007 and 0.035±0.006 g% of aloe-emodin contents whereas in C. grandis dried crude drug had 0.412±0.067 and 0.413±0.075 g% of aloe-emodin contents, respectively. Both methods were developed and validated in term of specificity, linearity, accuracy, precision, limit of detection, limit of quantitation and robustness. The data obtained from this study provided useful information for identification of selected Thai Cassia spp. and provided the aloe-emodin contents in C. garrettiana and C. grandis leaves.