Abstract:

An Alaskan ethnobotanical, Oplopanax horridus (Devil's Club) was evaluated for its anti-TB active principles with focuses on the quantitative evaluation of the activity.

Stepwise counter-current chromatographic fractionation, isobole method based recombination, and bioassay experiments of crude extract and fractions confirmed the presence of synergy among active fractions with two-fold enhancement maximum. In vivo experiments of concentrated active fractions showed no toxicity at 1,000 mg/kg dose level.

The phytochemical investigation led to the isolation of five compounds including two new sesquiterpenes, 3,10-epoxy-11-hydroxynerolid-6-ene and 3 S ,6R ,7S ,10R-cis -6,11-dihydroxy-7,10-epoxynerolidol, and two active compounds, oplopandiol and falcarindiol. A newly developed gradient-array CCC method was proven to be an effective primary separation method. Full spin analyses of the isolated compounds reproduced unique and highly complex 1 H NMR spectra, which facilitate future dereplication.

The results of both the biological and phytochemical experiments suggest that O. horridus is an ethnobotanical in which active constituents concertedly work together to exhibit the overall anti-TB activity. Hence, it is necessary to evaluate the activity of O. horridus at the level of a complex mixture. Accordingly, a new model, named biochemometrics, which allows evaluation of botanical preparations in a quantitative manner without the need for isolation or elucidation of active constituents was developed. The biochemometric analysis of O. horridus led to the assignment of one hundred active principles in its crude extract. The model allowed successful identification of active compounds that were confirmed by isolation, while excluding the inactive compounds from the positive matrix. Furthermore, the MS-based dereplication of anti-TB active representing principles, distinctive chemical entities identified via biochemometrics, revealed that the method is capable of identifying the skeletal structures among active principles. The capabilities of biochemometrics in handling a large number of active principles as well as identifying the structural scaffold of the active constituents opens a new perspective for both natural product drug discovery and ethnobotanical standardization.

The present study confirms the recent trend to recognize the interactions among multiple active principles in herbal preparations by proving the presence of both active principles and synergy, as well as by developing biochemometrics as a new tool for their integrated phytochemical and biological analysis.