Anti-microtubular activity of total alkaloids and aqueous extract of Detarium microcarpum a medicinal plant harvested in Mali

Abstract

Detarium microcarpum, is a species confined to drier regions of west and central Africa used to treat various diseases including cancer. Phytochemical screening revealed the presence of secondary metabolites (alkaloids) The aim of this work is to study the effect of total aqueous extracts and alkaloid fractions from D. microcarpum leaves, bark and roots on Nicotiana tabacum L. cv. 'Bright Yellow 2' (BY-2) tobacco cell line GFP-TuA3 expressing a N-terminal fusion of GFP. The plant was harvested in two different regions of Mali with a contrasting climate. The effects of the extracts on the microtubules was followed by spinning disc confocal microscopy. We showed that the anti-microtubular effect of the extracts is dose-dependent, depends of the sampling site and the part of the plant used. Total alkaloids extracted of D. microcarpum bark have more effect on microtubules than leaf and root. The bioactivity-guided fractionation should be used to screen out the biologically active compounds of the total alkaloid extracts of the bark of D. microcarpum.

Keywords: Detarium microcarpum; Alkaloids; Anti‐microtubule activity; Aqueous extract; Mali; Mortality assay; Sanankoroba; Sikasso; Spinning disc microscopy; Traditional African medicine.

Fig. 1

Generation of aqueous extract and alkaloid fractions from Detarium microcarpum leaves, bark and roots

Fig. 2

Representative cells of the tobacco BY-2 cell line GFP-TuA3 expressing a N-terminal fusion of GFP to tobacco tubulin □□ under control of the CaMV 35S promoter to visualise microtubules in vivo. Treatment for 2 h either with culture medium alone (A, B), an aqueous bark extract of Detarium microcarpum (collection site Sanankoroba) at 0.5 mg^.mL.⁻¹ (C, D) or the microtubule eliminating herbicide oryzalin at 10 µM (E, F). Insert in C is zoomed in D to show the partial disassembly of microtubules and the high background of solublte GFP-tagged tubulin. Confocal sections in the cortical region obtained by spinning disc microscopy are shown. Since these are single confocal sections, the illuminated area differs epending on the shape of the cell (round versus flat in diameter)

Fig. 3

a Dose–response relation for microtubule disintegration by an aqueous extract from the bark of D. microcarpum (collection site Sanankoroba) in GFP-TuA3 cells. Treatment time was 2 h, 10 µM of oryzalin was used as positive control (solid line, dotted lines show standard errors). Different letters show significant differences between different genotypes and treatments according to a Duncan test at P < 0.05). b-d representative cells after treatment with either 0.1 mg^.mL⁻¹ (b), 3 mg^.mL⁻¹ (c) or 100 mg^.mL⁻¹ (d), respectively

Fig. 4

Effect of aqueous extracts (0.5 mg. mL⁻¹) from bark of D. microcarpum collected at two different sites in Mali in GFP-TuA3 cells Treatment time was 2 h. a mock control. b extract from bark collected in Sanankoroba. C extract from bark collected in Sikasso. Geometrical projections from confocal z-stacks are shown

Fig. 5

Effect of aqueous extracts (0.5 mg. mL⁻¹) from leaves of D. microcarpum collected at two different sites in Mali in GFP-TuA3 cells. Treatment time was 2 h a mock control b extract from leaves collected in Sanankoroba c extract from leaves collected in Sikasso. Geometrical projections from confocal z-stacks are shown

Fig. 6

Effect of aqueous extracts (0.5 mg. mL⁻¹) from roots of D. microcarpum collected at two different sites in Mali in GFP-TuA3. Treatment time was 2 h a mock control b extract from roots collected in Sanankoroba c extract from roots collected in Sikasso. Geometrical projections from confocal z-stacks are shown

Fig. 7

Quantification of the antimicrotubular effect of aqueous extracts (0.5 mg^.mL⁻¹) from different parts of D. microcarpum collected at the two sites in Mali. A score of 30 (red) corresponds to complete elimination, a score of 60 (blue) corresponds to fully intact microtubules. Treatment time was 2 h, target were tobacco BY-2 cells expressing a N-terminal fusion of GFP to tobacco tubulin α3 under control of the CaMV 35S promoter to visualise microtubules in vivo. Data represent mean values from 13–32 individual cells per experiment

Fig. 8

Phylogenetic position of the two accessions for D. macrocarpum collected in Mali (highlighted in green) compared to other members of the Tribus Detarieae and Cercis canadensis (Tribus Cercidoideae) as outgroup based on the plastidic marker rbcL (a) and trnH-psbA igs (b). The phylogeny was constructed using the Neighbour-Joining algorithm and bootstrap values derived from 1000 burn-ins. Grey: accession Breteler 12528 (WAG) from Gabun declared as D. macrocarpum, but partially clustering with the morphologically similar Sindora klaineana

Fig. 9

Time course for the antimicrotubular effect of aqueous extracts (5 mg. mL⁻¹) from bark of D.microcarpum (collection site Sikasso) in tobacco BY-2 cells expressing a N-terminal fusion of GFP to tobacco tubulin α3 under control of the CaMV 35S promoter to visualise microtubules in vivo. Geometrical projection of z-stacks collected at a step size of 0.5 μm

Fig. 10

Antimicrotubular effect of the alkaloid fraction from different parts of D. microcarpum collected at Sanankoroba. A–C show representative cells after treatment with either 0.05 mg.mL⁻¹ or with 0.50 mg^.mL⁻¹ of alkaloid fractions generated either from leaf (A), bark (B) or root (C). Treatment time was 2 h, target were tobacco BY-2 cells expressing a N-terminal fusion of GFP to tobacco tubulin □3 under control of the CaMV 35S promoter to visualise microtubules in vivo. D Dose–response relation of the antimicrotubular effect for alkaloids extracted from roots of D. microcarpum collected at Sanankoroba. A score of 30 (red) corresponds to complete elimination, a score of 60 (blue) corresponds to fully intact microtubules. Data represent mean values from 10 to 19 individual cells per experiment

Fig. 11

Cytotoxic effect of alkaloid fractions from different parts of D. microcarpum collected at Sikasso and Sanankoroba. A Dose–response relation of mortality in tobacco BY-2 cells expressing a N-terminal fusion of GFP to tobacco tubulin □3 under control of the CaMV 35S scored at day 3 after addition of the alkaloid fraction generated from leaves collected either in Sanankoroba or in Sikasso. Data represent mean and standard error from three independent experimental series. B Relationship between microtubule integrity scored at 2 h after addition of the alkaloid fraction and the mortality scored after day 3 induced by the same fraction pooled over fractions generated from leaves, bark and roots collected at the two sites. The negative correlation of mortality over MT integrity has been inferred by linear regression with a correlation coefficient R.² = 0.73