Pharmacognostic specification and dioscorine content of dioscorea hispida tubers

Abstract

Dioscorea hispida Dennst. dried tubers have been used as a crude drug in Thai remedy named Thoraneesanthakhat. It has traditionally been used to treat constipation. The aim of this research is to establish the pharmacognostic specification and determine the content of dioscorine in D. hispida tubers. The tubers were collected from 14 different locations throughout Thailand. The drawing of whole plant of D. hispida was illustrated in detail. The crude drug was traditionally prepared by slicing the tuber and sun drying. The macroscopic characters were longitudinal pieces or irregularly shaped, off-white colour with some light brown epidermis. The prominent anatomical and histological characteristics were starch granules and raphide crystal. The total ash, acid insoluble ash, loss on drying and water content should be not more than 3.44, 0.92, 11.50 and 11.55% w/w respectively whereas ethanol-soluble extractive and water-soluble extractive should be not less than 3.00 and 15.07% w/w respectively. The content of dioscorine in D. hispida dried tubers was identified using TLC image analysis compared to TLC-densitometry. The standard dioscorine was prepared from dried tubers by ethanol extraction, picrate crystallization, back extraction and column chromatographic purification. The identification of isolated dioscorine was confirmed by ¹H and ¹³C NMR spectra as well as previously reported spectra prior to be used as dioscorine standard. Dried tuber samples were successively extracted in ethanol by soxhlet apparatus. The extracts were analyzed for dioscorine content by TLC using Aluminium oxide 60 GF₂₅₄ neutral as stationary phase and methanol-chloroform (3 : 97) as mobile phase. The density of dioscorine spot at hRf value of 80 detected under UV254 was analyzed and transformed to peak area by the Scion Image software. Five concentrations of standard and 14 samples were developed on the same TLC plate. Each sample was quantitated in triplicate. For TLC-densitometry, the same protocol was performed using Camag Linomat syringe and Camag TLC scanner with winCATS software instead manual. The dioscorine content of the dried crude drug determined by TLC image analysis and TLC-densitometry were 0.66 and 0.72% w/w respectively. The polynomial regresstion data of both methods for dioscorine showed good linear relationship with a correlation coefficient of 0.999 in the concentration range of 2.50-12.50 μg/spot. The LOD and LOQ were 0.28 and 0.84 μg/spot by TLC image analysis and 0.37 and 1.13 μg/spot by TLC-densitometry respectively. TLC image analysis was valid for quantification of dioscorine in D. hispida tuber. This study provided scientific information for the quality control of D. hispida tuber ingredient in Thai traditional medicine.