Spatial Distribution and Stability of Cholinesterase Inhibitory Protoberberine Alkaloids from Papaver setiferum

Abstract

During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehydroprotoberberine alkaloids (1 and 2) and nine known compounds (3-11) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range (1-4, 6, 7), offering new lead compounds for the development of cholinesterase inhibitory drugs.

Figure 1

New 7,8-didehydroprotoberberine alkaloids isolated from P. setiferum: 7,8-didehydromecambridine TFA salt (1) and 7,8-didehydroorientalidine TFA salt (2).

Figure 2

Key HMBC (solid arrows), ROESY (dashed arrows), and COSY (bold bonds) correlations in 1.

Figure 3

Comparison of experimental (solid black line) with TDDFT-calculated ECD spectra (dashed lines) of 1 at the B3LYP/6-311++G(d,p) level of theory.

Figure 4

Known compounds (3–11) isolated from P. setiferum.

Figure 5

DESI-IMS data of the distribution of compounds 2/3 (m/z 396) and 7 (m/z 326) in P. setiferum capsules and flower petals. (A) Capsule with simple anatomy marked. (B) Horizontal cross-section of the capsule taken from the lower half with alkaloids shown in the pod walls and vascular bundles (marked with arrowheads and magnified in B1). (C) Vertical cross sections through two different capsules (1, 2). (D) P. setiferum flower. (E) DESI-IMS data of a flower petal fragment. Vascular tissues are marked with arrowheads. The folds occurring during preparation of the petals for imaging caused the appearance of some imaging artifacts (marked with asterisk). Bar = 3 mm (B); 3.5 mm (C); 4.5 mm (E).

Figure 6

Conversion of 13,14-dihydrocoptisine to coptisine in C. majus after tissue injury

Figure 7

Concentration of 3 and 5 across several flowering stages of P. setiferum. Compound 5 was below the limit of detection (LOD) at the first two time points.

Figure 8

Concentration of 1–5 in P. setiferum capsules stored in an oven (2 weeks, 40 °C) vs those stored in a freezer (−20 °C). Compound 5 remained below the limit of detection (LOD) in all samples.