A.M. Zobel, G. Zobel, S.E. Schellenberger
SURFACE COMPOUNDS EXTRUDED BY FOOD AND FEED PLANTS AFFECT MITOSES
Department of Chemistry, Trent University
Surface compounds from Ruta graveolens, Brassica oleracea, Medicago sa-tiva, Uncaria tomentosa, and a mixture of surface compounds from Taxus wal-lichiana with coumarin inhibited mitoses. They thus could act as antimicrobial or even anticancer agents used in food and feed. The compounds removed from the interior of the same plants either stimulate mitoses, as in B. oleracea and M. sativa or inhibit mitoses, although much less than do surface deposits. The sampled leaves or twigs were treated fi rst for 1-2 seconds by dipping them into almost-boiling water (Zobel and Brown, 1988) to remove melted waxes and na-tural products deposited in and on the wax. A standard method was then used to remove fl avonoids and other phenolics from the rest of the tissue, with prolonga-tion of the extracprolonga-tion to overnight. In Ruta, Brassica and Medicago, coumarins were found in the highest concentrations. In Uncaria alkaloids and tannins were found. In Taxus taxoids were found, but because these are mutagenic the couma-rin was added to avoid the mutagenic effect; thus even at 10-15 ppm of taxoids 50
ppm of coumarin inhibited mitosis.
Key words: Ruta, Brassica, Medicago, Uncaria, Allium, mitoses.
Allium sativum bulbs were grown in tap water until the roots reached 1.0-1.5 cm
in length. Root segments 1 cm long were removed from the bulbs and trans-ferred to 1 mL of the following paclitaxel solutions: saturated, and 10-3, 10-6, 10-9, 10-12 and 10-5 ppm. Roots were also transferred to two solutions of taxoids from either the sur-face or interior of T. wallichiana combined with coumarin at varying concentrations and volume ratios:
Solution 1 500 μL of 10-15 ppm taxoids: 500 μL of 50 ppm coumarin Solution 2 100 μL of 10-15 ppm taxoids: 900 μL of 50 ppm coumarin
Roots were placed in 1 mL of tap water as a control. Three root segments were collected after 1, 3 and 24 h of incubation and then fi xed in glacial acetic acid: abso-lute ethanol (1:3 v/v). After 1 and 3 h of incubation the changes observed within the fi rst mitotic phases were evaluated and, after 24 h of incubation, those observed over the whole cell cycle were evaluated. The specifi c numbers of mole-cules of paclitaxel alone and paclitaxel combined with coumarin in 1 mL of each solu-tion are given in Table 1.
The average mitotic activity of root tip meristem was evaluated in three indivi-dual squash preparations of 1 mm root tips stained with acetoorceine (Levan, 1938). BROMAT. CHEM. TOKSYKOL. – XLII, 2009, 3, str. 1063 – 1066
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The mitotic activity of the meristem was characterized by the number of divisions per thousand cells for every squash preparation, the particular phases of mitosis being distinguished according to the method of Lopez-Saez and Fernandez-Gomez (1965). The degree of mitodepression was evaluated by comparison of the number of mitoses in the treated material with those of the control. The frequency of each of the mitotic phases was analyzed as the percentage of cells in a particular phase in 200 dividing cells according to Lopez-Saez and Fernandez-Gomez (1965). The ratios of metaphase to anaphase plus telephase (M/A+T) and metaphase to prophase (M/P) were calculated to ascertain whether any blockage of a particular phase had occurred (Podbielkowska et al., 1994). Light microscopy was employed to assess morphological changes to the cell population of promeristematic cells.
RESULTS AND DISCUSSION
In Table 1 the percentages of mitoses compared to the control are given after tre-atment of A. cepa root promeristem with surface and interior compounds of R.
gra-veolens, B. oleracea, M. sativa, U. tomentosa, and a mixture of T. wallichiana with
coumarin. Treatment continued for 24 h and postincubation for 24 and 48 h. The in-terior compounds of cabbage and alfalfa were stimulating mitoses in 24 h and those of the rest were inhibitory. The most inhibition was in the case of T. wallichiana with added coumarin, in the presence of both surface and interior extracts with 50 ppm added coumarin. The concentrations of taxoids were very low (10-15 ppm), and that of coumarin was high (50 ppm); thus coumarin possibly acted mostly by a synergi-stic reaction with taxoids (Zobel and Schellenberger, 2000; Zobel et al., 2007). Ta b l e 1. Percentage of mitoses compared to control, under treatment with surface and interior compounds of
Ruta graveolens, Brassica oleracea, Medicago sativa, Uncaria tomentosa, and a mixture of Taxus wallichiana with
coumarin
Source 24 h Treatment 24 h Treatment /
24 h Postincubation 24 h Treatment / 48 h Postincubation R. graveolens surface –38 –42 –9 R. graveolens interior –18 –16 –32 B. oleracea surface –37 –46 –46 B. oleracea interior +120 +60 +18 M. sativa surface –30 –42 –40 M. sativa interior +122 +61 +18 U. tomentosa surface –63 –61 –60 U. tomentosa interior –23 –25 –30
T. wallichiana surface plus coumarin –92 –90 –90
T. wallichiana interior plus coumarin –80 –80 –90
Surface compounds of a medicinal plant from Peru, U. tomentosa, retarded mito-ses over twice as much as the interior extracts. Both extracts of R. graveolens, which
Nr 3 Surface compounds extruded by food and feed plants affect mitoses 1065 cause photophytodermatitis (Zobel and Brown, 1990), retarded mitoses, but the sur-face ones were twice as active. In continuous postincubation 24 h/48 h interior extra-cts of R. graveolens increased mitoses but they were only 32% of the control.
CONCLUSION
The surface natural products were more active in lowering the frequency of cell division, and there were no chromosomal aberrations when prophase, metaphase, anaphase and telophase were examined under a light microscope, suggesting that the dose employed is safe for human and animal consumption.
Paclitaxol, Taxol®, and Taxoter® could cause mutations in genes (Tinwell and
Ashby, 1994; Schiff et al., 1979; Jordan et al., 1993; Jagetia and Adiga, 1995), but
coumarins have been found not to cause chromosomal aberrations (Keightley et al., 1996).
A.M. Z o b e l, G. Z o b e l, S.E. S c h e l l e n b e r g e r
POWIERZCHNIOWE ZWIĄZKI WYDZIELANE PRZEZ ŻYWIENIOWE I PASTEWNE ROŚLINY INHIBOWAŁY MITOZY
S t r e s z c z e n i e
Powierzchniowe naturalne produkty z Ruta graveolens, Brassica oleracea, Medicago sativa, Uncaria
tomentosa i mikstura powierzchniowych związków z Taxus wallichiana z kumaryną inhibowały mitozy.
Dlatego mogłyby działać antymikrobialnie i jako substancje antyrakowe. Substancje z wnętrza tych roślin albo stymulowaly mitozy jak w przypadku Brassica i Medicago, albo inhibowały, ale w mniejszym stop-niu niż powierzchniowe związki. Liście lub pędy były najpierw zanużane w prawie wrzącej wodzie na 1–2 sekundy, by usunąć topiący się wosk i w nim lub na nim występujące naturalne produkty. Następnie w celu usunięcia substancji ze środka używano standardowej metody do usuwania fl awonoidów i kwasów fenolowych z modyfi kacją przedłużenia ekstrakcji przez noc. W Ruta, Brassica, Medicago kumaryn była największa koncentracja. W Uncaria alkaloidy I tanniny dominowały. W Taxus taksoidy, ale ponieważ są one mutagenne kumaryna była dodana, by uniknąć chromosomowych aberacji, dlatego tylko 10-15 ppm
taksoidów z 50 ppm kumaryny uznano za najlepszą miksturę do inhibowania mitoz. Możliwe, że syner-gistyczny mechanizm zadziałał.
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