Pharmacognostic specification, mangiferin content, biological activities of aquilaria Crassna leaves and DNA barcodes of selected plants in genus aquilaria

Abstract

Aquilaria crassna Pierre ex Lecomte (Thymelaeaceae) has been used as a medicinal plant in many aspects. Previous research has revealed that A. crassna leaves contain mangiferin as an active compound. Although the active component has been investigated, the pharmacognostic specification and quantification of mangiferin from A. crassna leaves have never been established. There still have little findings about biological activities of A. crassna leaves extract and its metabolite, mangiferin as well as the molecular evaluation of plant in genus Aquilaria. The study aimed to conduct and develop a pharmacognostic standard according to WHO guidance as well as the validated method for quantifying mangiferin content, and also investigated some biological activities such as alpha-glucosidase inhibitory activity, antioxidation activity, cytotoxic activity, and cytoprotective property; in addition, it also investigated the efficient DNA barcoding loci and suggested the most suitable one for Aquilaria species identification. The dried A. crassna leaves from 15 separated locations throughout Thailand were investigated for pharmacognostic specification and their mangiferin contents were quantitatively analysed by TLC densitometry with winCATS software and TLC image analysis. Macroscopic-, microscopic- characteristics, TLC fingerprinting, and physicochemical parameters were reported in this study. The loss on drying, moisture content, and total ash content as well as acid-insoluble ash content were determined to be 8.62±0.13, 8.16±0.14, 6.82±0.09 and 1.49±0.03%, respectively. Ethanol- and water-extractive values were found to be 9.05 ± 0.39 and 16.94 ± 0.22 %, respectively. In addition, the validation method for quantifying the mangiferin content was developed. The contents of mangiferin in A. crassna leaf extract determined by TLC-densitometry and TLC-image analysis were found to be 1.2992± 0.5980 and 1.3036±0.5874 % by dried weight, respectively. The results between these two analytical methods were shown to have an insignificant difference. The yeast alpha-glucosidase inhibitory assay was performed, and the IC50 of A. crassna leaf extract and mangiferin were found to be 0.1840±0.0032, 0.5714±0.0044 mg/ml, respectively. In addition, these samples were analyzed in term of the in vitro antioxidant activities using standard antioxidant assays. The results showed that A. crassna leaf extract and mangiferin possessed antioxidant properties. Moreover, the cytotoxicity of A. crassna leaf extract and mangiferin was also evaluated against three human cancer cell lines using MTT assay. A. crassna leaf extract could inhibit cell proliferation of MDA-MB-231; breast cancer cells (IC50 = 33.89 ± 0.50 µg/ml) greater than HT-29; colorectal cancer cells (IC50 = 51.74 ± 1.42 µg/ml) and HepG2; hepatic cancer cells (IC50 = 53.63 ± 1.54 µg/ml). Mangiferin showed the toxicity against these cancer cell lines but the inhibition was around 33-38% at the highest concentration (100 µg/ml). In addition, the cytoprotective properties of EA.hy926 cell was determined via cell viability testing by MTT assay and intracellular reactive oxygen species (ROS) investigation by DCFH-DA assay. Only mangiferin performed cellular protective attribute from the reduction of H2O2-induced ROS generation, and no cytotoxic effect on EA.hy926 cells at the concentration not more than 200 µg/ml. Western blotting analysis revealed that the mangiferin incubation before exposure to 0.25 mM of H2O2 for 6 h could increase the SOD-1 expression, whereas the HO-1 expression was down-regulated. For determination of apoptosis proteins, mangiferin treatment prior exposure to 0.25 mM H2O2 for 6 h resulted in augmentation of the expression level of anti-apoptotic factor (Bcl-2), decline the level of proapoptotic factor (Bax) compared to H2O2-induced injury control. For DNA barcoding study, three Aquilaria species; A. crassna, A. malaccensis Lam., and A. subintegra Ding Hou, and Enkleia siamensis (Kurz) Nervling were investigated. The DNA barcoding sequences from six candidate of barcoding loci (ITS, matK, rbcL, rpoC1, psbA-trnH intergenic spacer, and ycf1) were established. The phylogenetic tree of each locus was reconstructed and the genetic distances were also determined using a maximum likelihood method. According to ML phylogenetic tree reconstruction and the optimum length of genetic distance, only ITS was suitable marker for Aquilaria species identification. All of these results provide highly useful information for the authentication of A. crassna leaves, and also the contribution to the effectiveness and safety of A. crassna uses.