Pharmacognostic specification of usnea siamensis and quantitative analysis of usnic acid by thin layer chromatography

Abstract

Usnea siamensis Wainio was Thai herbal drug that used to treat diseases in folk medicine. Because there was lacked of quality control in U. siamensis crude drug, thus this research aimed to report the pharmacognostic specification and analysis usnic acid content in U. siamensis by TLC-densitometry and TLC image analysis. Dried U. siamensis crude drugs were purchased from 15 various traditional drug stores throughout Thailand. The whole plant of U. siamensis was illustrated in detail. The morphological character of U. siamensis was fruticose lichen, up to 5 m long, grayish-green strands hanging from the branches of trees and showed mycelium on the surface of thallus and many densely distributed holes in central axis by scanning electron microscopy. The foreign matter, total ash, acid insoluble ash, loss on drying and water content should be not more than 6.24, 0.96, 0.16, 10.80 and 13.08% w/w respectively whereas ethanol soluble extractive and water soluble extractive values should be not less than 5.59 and 3.47% w/w respectively. Thin layer chromatographic fingerprint of U. siamensis's ethanol extract used toluene, ethyl acetate and formic acid (139:83:8) as mobile phase, observed under 254 and 365 nm wavelength ultraviolet light and sprayed with 10% sulfuric acid in methanol showed hRf value at 74. The quantitative analysis by thin layer chromatography used chloroform and methanol (9:1) as mobile phase. The usnic acid content was analyzed by TLC-densitometry performed with winCATS software and TLC image analysis performed with ImageJ software. The regression lines of both methods were polynomial in the range of 0.2-1.0 mg/spot and correlation coefficients were 0.9981 and 0.9994 respectively. The precisions calculated by the %RSD of repeatability and intermediate precision, were between 10.25-19.39 and 8.49-11.50 %RSD respectively. The average recoveries were between 83.77-100.45 and 99.17-120.49 %recoveries respectively. LOD and LOQ were 0.06, 0.18 mg and 0.11, 0.34 mg respectively. The robustness was 0.803 and 1.094 %RSD of peak area respectively. The usnic acid contents in U. siamensis were 2.32 and 2.26 g/100g of dried crude drug respectively. The comparison of usnic acid contents between both methods was statistically analysed by paired t-test. It was found that usnic acid contents by two methods were not significantly different (t = 1.183, P = 0.256). TLC-bioautographic method showed the inhibition zone at Rf of usnic acid against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and Candida albicans. This study provided scientific information for the quality control of U. siamensis including usnic acid in Thailand