bromat 3-2009 127

BROMAT. CHEM. TOKSYKOL. – XLII, 2009, 3, str. 1063 – 1066
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 sativa, Uncaria tomentosa, and a mixture of surface compounds from Taxus wallichiana 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 first for 1-2 seconds by dipping them into
almost-boiling water (Zobel and Brown, 1988) to remove melted waxes and natural products deposited in and on the wax. A standard method was then used to
remove flavonoids and other phenolics from the rest of the tissue, with prolongation of the extraction 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 coumarin 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 surface 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 fixed in glacial acetic acid: absolute ethanol (1:3 v/v). After 1 and 3 h of incubation the changes observed within
the first mitotic phases were evaluated and, after 24 h of incubation, those observed
over the whole cell cycle were evaluated. The specific 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 individual squash preparations of 1 mm root tips stained with acetoorceine (Levan, 1938).
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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 treatment of A. cepa root promeristem with surface and interior compounds of R. graveolens, 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 interior 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 synergistic 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 mitoses over twice as much as the interior extracts. Both extracts of R. graveolens, which
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Surface compounds extruded by food and feed plants affect mitoses
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cause photophytodermatitis (Zobel and Brown, 1990), retarded mitoses, but the surface ones were twice as active. In continuous postincubation 24 h/48 h interior extracts 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
Streszczenie
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 stopniu 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 flawonoidów i kwasów
fenolowych z modyfikacją 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 synergistyczny mechanizm zadziałał.
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Adress: Peterborough, ON, Canada K9J 7B8.