Bryonolic acid: a large-scale isolation and evaluation of heme oxygenase 1 expression in activated macrophages

Abstract

Bryonolic acid (BA) is a triterpenoid found in the Cucurbitaceae family of plants. Our interests in the immunomodulatory effects of this class of natural products led us to discover that BA induces a marked increase in the expression of a phase 2 response enzyme, heme oxygenase 1 (HO-1), in a dose-dependent manner. This phenotype has translational implications in malarial disease progression, and consequently we developed a large-scale isolation method for BA that will enable future in vitro and in vivo analyses. We have determined ideal growth conditions and time scale for maximizing BA content in the roots of Cucurbita pepo and analyzed BA production by HPLC. Large-scale extraction yielded 1.34% BA based on dry weight, allowing for the isolation of BA on a multigram scale.

Figure 1

Structure and numbering for bryonolic acid.

Figure 2

Western blot analysis and quantification of LPS induced RAW 264.7 cells treated with increasing concentrations of bryonolic acid for 24 h.

Figure 3

HPLC traces for extracts from the fine hairy root, stemroot, and dicotyledon leaf body of 14 day germinations. Bryonolic acid is detected only in the root portion of Cucurbita pepo L.

Figure 4

Bryonolic acid production in Cucurbita pepo L. roots under two growth conditions, peat-based media (a) and moist blotting paper (b). Bryonolic acid content is observed to increase from day 2 to day 16 in peat-based media and from day 2 to day 24 in moist blotting paper, as observed by growing HPLC peak areas (c and d). At its maximum, BA content in roots from moist blotting paper is roughly tenfold greater than that detected in roots from peat-based media, as is apparent in the respective scales.

Figure 5

Comparison of maximum and total BA production in roots from peat-based media versus roots from moist blotting paper (a). Comparison of HPLC peak area for maximum BA production under both conditions (b).