Changes in chromosome structure, mitotic activity and nuclear DNA content from cells of Allium Test induced by bark water extract of Uncaria tomentosa (Willd.) DC

Journal of Ethnopharmacology 107 (2006) 211–221

Changes in chromosome structure, mitotic activity and nuclear DNA content from cells of Allium Test induced by bark water extract of Uncaria tomentosa (Willd.) DC ´ Mieczysław Kura´s a,∗ , Julita Nowakowska a , Elwira Sliwi´ nska b , Radosław Pilarski d , Renata Ilasz a , Teresa Tykarska a , Alicja Zobel c , Krzysztof Gulewicz d b

a Department of Ecotoxicology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland Department of Genetics and Plant Breeding, University of Technology and Agriculture, Kaliskiego 7, 85-796 Bydgoszcz, Poland c Department of Biochemistry, Trent University, Peterborough, Ont., Canada K9J 7B8 d Laboratory of Phytochemistry, Institute of Bioorganic Chemistry PAS, Z, Noskowskiego 12/14, 61-704 Pozna´ n, Poland

Received 17 September 2003; received in revised form 17 February 2006; accepted 9 March 2006 Available online 27 March 2006

Abstract The influence of water extract of Uncaria tomentosa (Willd.) DC bark on the meristematic cells of the root tips of Allium cepa L., e.g. cells of Allium Test, was investigated. The experiment was carried out in two variants: (1) continuous incubation at different concentrations (2, 4, 8 and 16 mg/ml) of the extract for 3, 6, 12, 24, 48 and 72 h; and (2) 24-h incubation in three concentrations of the extract (4, 8 or 16 mg/ml), followed by post-incubation in distilled water for 3, 6, 12, 24 and 48 h. During the continuous incubation, the mitotic activity was reduced (2 and 4 mg/ml) or totally inhibited (8 and 16 mg/ml), depending on the concentration of the extract. All the concentrations resulted in gradual reduction of the mitotic activity. In the concentration of 2 mg/ml, the mitotic activity reached its lowest value after 12 h (2 mg/ml) and after 24 h in 4 mg/ml, followed by spontaneous intensification of divisions during further incubation. Instead, in higher concentrations of the extracts (8 and 16 mg/ml), the mitotic activity was totally inhibited within 24 h and did not resume even after 72 h. Incubation caused changes in the phase index, mainly as an increase in the number of prophases. After 24 h of incubation, in all phases, condensation and contraction of chromosomes were observed. During post-incubation, divisions resumed in all concentrations, reaching even higher values than the control. Cytometric analysis showed that the extract caused inhibition of the cell cycle at the border between gap2 and beginning of mitosis (G2 /M). © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Uncaria tomentosa; Allium Test; Activity of mitosis; Chromosome condensation and chromosome contraction; Blockade of G2 /M cycle; Nuclear DNA content

1. Introduction Uncaria tomentosa (Rubiaceae) water extracts were use in folk medicine (De Jong et al., 1999; Heitzman et al., 2005; Keplinger, 1982; Reinhard, 1997; Reinhard, 1999). Their use is raising more and more interest worldwide, mainly connected with the phytotherapy of cancer. Apart from alkaloids, which intensively affect the metabolism of live organisms, Uncaria tomentosa has been found to contain numerous glycosides of quinovic acid (Aquino et al., 1989; Cerri et al., 1988), triter-

∗

Corresponding author. Tel.: +48 22 554 2007; fax: +48 22 554 2022. E-mail address: [email protected] (M. Kura´s).

0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.03.018

penes such as ursolic and oleanolic acid (Aquino et al., 1997) and numerous sterols (Senatore et al., 1989) as well as polyphenolic and uncarine acids (Lee et al., 2000; Wirth and Wagner, 1997). The results obtained so far indicate that the compounds isolated from Uncaria tomentosa accelerate phagocytosis (Wagner et al., 1985), show anti-inflammatory activity (Aguilar et al., 2002; Aquino et al., 1991; Krowicka et al., 1998; Reinhard, 1997; Sandoval-Chacon et al., 1998; Senatore et al., 1989), antimutagenic action (Keplinger et al., 1999; Sheng et al., 2000), antiviral activity (Keplinger et al., 1999) and contraceptive action (Salazar and Jayme, 1998). It has also been shown that the extracts have a cytoprotective effect against factors inducing oxidative stress in the human body (Deschmarchelier et al., 1997; Sandoval et al., 2000). They act as immunostimulators, as evidenced by raised

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production of interleukin-1 and -6 in macrophages of alveoli (Lemaire et al., 1999). However, the most important fact is the ˚ antiproliferative effect of these extracts (Akesson et al., 2003). They induce delayed-type apoptosis and, depending on concentration, strongly inhibited proliferation in vitro of human cancer cells: HL-60 leukemia, lymphoma line (Raji) from B cells transformed with the EBV virus (Sheng et al., 1998), as well as a breast cancer cell line (Riva et al., 2001). Simultaneously, the same preparation applied to rats increased leukocytosis in vivo, stimulated in vitro the proliferation of healthy lymphocytes isolated from the animals, and also induced higher leukocytosis in healthy humans (Sheng et al., 2000; Wurm et al., 1998). Additional toxicological research carried out on experimental animals showed that Uncaria tomentosa extracts were not toxic (SantaMaria et al., 1997; Sheng et al., 2000). These results are very promising since they indicate that the extracts have a selective antimitotic effect only with respect to damaged cells. In order to study the mechanism and broad range of action of the water extracts of Uncaria tomentosa bark, the objective of this work was to determine the influence of the extract on meristematic cells of root tips of Allium cepa L. This model root system of plant cells is commonly used as a test for investigating environmental pollution factors, toxicity of chemical compounds and evaluating potential anticancer properties (Keightley et al., 1996; Kupidłowska et al., 1994; Kura´s and Malinowska, 1978; Majewska et al., 2003; Podbielkowska et al., 1981, 1995). It has been used since 1938 (Levan, 1938). It is very comfortable as it is easy to make preparations of onion roots. They contain rather homogenous meristematic cells, having only 16 chromosomes, which are very long, well visible and get stained easily. The test is a fast and inexpensive method, allowing the investigation of universal mechanisms for meristematic plant cells and extrapolation on animal cells. The comparison of results obtained with animal and human test systems to those obtained using a model plant system (Allium) could bring additional information (mainly cariological) on the biological activity of Uncaria tomentosa extracts and could contribute to explaining the mechanisms of their action on cells. Our work dealt with the influence of different concentrations of the bark extract on cell morphology and the intensity of cell divisions. Flow cytophotometry was useful for evaluating the relative amount of DNA in the interphase nuclei (Otto, 1990) and ratio of G1 and G2 during mitotic cycle under treatment. We have used these methods in more complex investigations on the influence of the Uncaria tomentosa extract on human cancer cells in vitro (not published). The aim of this work was therefore to find concentrations of water extract of Uncaria tomentosa bark which inhibited mitoses and to investigate the fate of the cells when they were removed from the extract and transferred into water for post-incubation, to find out if the changes were reversible. Changes in structure of the interphase chromatin were analyzed, and phases of mitosis as well as chromosomal aberrations counted, which would indicate possible dangerous effects of treatment. Flow cytometry was used to investigate changes in the G2 to G1 ratio. Chromosomal changes, in particular mitotic phases, whose elevation would indicate possible dangerous effects of the treatment, were counted.

2. Material and methods 2.1. Preparation of the extracts Bark of Uncaria tomentosa originated from Laborations Induquimica, Lima, Peru was supplied by A-Z Medica Company, Gda´nsk. The voucher material is deposited at the Laboratory of Phytochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Pozna´n, Poland. A dry sample of ground bark (1 g) was added to 10 ml of Milli-Q water at room temperature and mixed in a shaker for 12 h. The extract was then sonicated (4 impulses × 1 min.) (Sonics and Materials Inc., Vibra Cell) and centrifuged (17,092 × g for 20 min.). The supernatant was used to prepare an extract, containing 80 mg/ml of soluble substances, which was then used as a stock to prepare the solutions of all concentrations used in the experiments. 2.2. HPLC-fingerprint analysis of alkaloids To 625 ␮l of stock solution of extract containing 50 mg of soluble substances, 15 ml 2% sulphuric acid solution were added and sonified for 15 min in an ultrasonic bath (Bandelin Sonorex RK 103H). The mixture was then centrifuged at 17,092 g for 10 min and extracted three times with 10 ml ethylacetate. Next, the aqueous phase was separated and adjusted to pH 10 with 10% NH4 OH and extracted three times with 10 ml of ethylacetate each. The organic extracts were combined, evaporated to dryness and the residue dissolved in 1 ml of methanol. To 100 ␮l of methanol solution corresponding to 5 mg of dry mass of water extract, 50 ␮l of caffeine (1 mg/ml) were added as internal standard. Next, this solution was adjusted with methanol to a volume of 500 ␮l. The qualitative and quantitative content of alkaloids was determined by the HPLC fingerprint analysis [HPLC: L7100 Intelligent Pump (Merck-Hitachi), L-7200 Autosampler (Merck-Hitachi), L-7450 Diode Array Detector (MerckHitachi); Software: D-7000 Chromatography Data Station Software Version 4.0; Column: LiChrospher® 100 RP-18 (250 mm × 4 mm, Merck); Precolumn: LiChrospher® 100 RP18 (4 mm × 4 mm, Merck); solvents: A, phosphate buffer solution (10 mM, pH, 6.6), B, methanol: acetonitrile (1:1); gradient: (60% A and 40% B) to (30% A and 70% B); injection 10 ␮l of sample; time: 35 min; washing: 20% solvent A and 80% solvent B; temp: 21 ◦ C; flow rate: 1.0 ml/min.; detection: 245 nm] (Sheng et al., 2000; Stuppner et al., 1992). The results of this analysis are presented in Fig. 1 and Table 1. 2.3. Allium cepa Test Root tips of onion (Allium cepa L. var. Dawidowska). e.g. cells of Allium cepa Test (Fiskesj¨o, 1985) were used for the experiments. The roots were grown in distilled water in 250-ml Erlenmeyer flasks under laboratory conditions. After reaching a length of 3 cm (±0.5 cm), the roots were treated with the extracts, 2, 4, 8 or 16 mg/ml. The treatment (incubation) of roots was carried out in two variants. The first variant was a continuous

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Table 2 Mean mitotic index of control (water) and during continuous incubation at different concentrations (2, 4, 8, 16 mg/ml) of the Uncaria tomentosa bark extract, counted as percentage of time (h) 0 + 0 Time (h)

Concentration Water

Fig. 1. HPLC chromatogram of oxindole-alkaloid in a water extract of Uncaria tomentosa bark: (1) caffeine (internal standard), (2) uncarine F, (3) speciophylline, (4) mitraphylline, (5) isomitraphylline + pteropodine and (6) isopteropodine.

incubation during 72 h, with samples collected after 3, 6, 12, 24, 48 and 72 h. The second variant was of 24 h incubation with dilutions of 4, 8 and 16 mg/ml of the extract, followed by rinsing the roots several times in distilled water within a period of 30 min and then putting them into pure distilled water for post-incubation to check the possibility of reversal of action. Post-incubation lasted for 3, 6, 12, 24 or 48 h. In order to study the influence of Uncaria tomentosa bark extract on the mitotic activity and phase index of meristematic cells during the experiment, three 2 mm-long root tips from three different onions were cut off, stained and macerated in 2% acetoorceine with the addition of HCl (in the proportion of 9:1) and used for squash microscope preparations. The mitotic and phase indexes were counted according to the method of Lopez-Saez and Fernandez-Gomez (1965). For each variant of the experiment, nine onion roots tips were taken. Average results and standard errors are presented in Tables 2–5. The average results were calculated in proportion to the initial level (time 0 + 0), which was treated as 100%. All values are expressed as the mean ± S.E.M. Statistical analyses were performed with the paired Student’s test. Changes in chromosome morphology were photographed under a light microscope (NU Zeiss) with a Nikon photographic camera. 2.4. Flow cytometry

0+0 3+0 6+0 12 + 0 24 + 0 48 + 0 72 + 0

100 104 105 107 110 103 100

± ± ± ± ± ± ±

2 mg/ml 6.1 6.3 5.8 7.2 8.5 5.2 3.7

100 94.5 74.5 47 55 95.2 127.4

± ± ± ± ± ± ±

4 mg/ml 6.1 5.4 7.2 6.2 4.4 8.6 7.2

100 78.3 43.8 21.3 10.1 57.8 69.3

± ± ± ± ± ± ±

8 mg/ml 100 ± 6.3 59.8 ± 4.3 13.3 ± 4.1 9.5 ± 4.3 0 0 0

6.2 7.5 8.3 8.5 4.7 8.2 7.1

16 mg/ml 100 ± 6.1 55 ± 4.2 8 ± 4.3 2 ± 1.2 0 0 0

Table 3 Mean mitotic index in 48 h post-incubation following incubation of 24 h in the extract from Uncaria tomentosa bark of 4, 8 and 16 mg/ml (counted as percentage of control time (0 + 0) Time (h)

Concentration 4 mg/ml

0+0 3+0 6+0 12 + 0 24 + 0 24 + 3 24 + 6 24 + 12 24 + 24 24 + 48

100 80 55.1 26 4.6 73.2 144 115.5 80.9 101.8

± ± ± ± ± ± ± ± ± ±

6.3 4.9 6.7 5.3 3.2 5.9 6.8 9.3 10.1 12.2

8 mg/ml

16 mg/ml

100 ± 6.2 72.4 ± 3.3 40 ± 6.2 16 ± 7.3 0 16.9 ± 3.6 47.1 ± 8.3 146 ± 9.2 89.5 ± 7.3 159.6 ± 8.6

100 ± 6.2 70.4 ± 4.1 38.5 ± 6.1 7.6 ± 4.2 0 0 0 158 ± 8.1 118.9 ± 4.3 144.2 ± 6.2

Table 4 Change of G2 /G1 (% of control) in nuclei of root tip cells during incubation at different concentrations of the extract Time (h)

Concentration 2 mg/ml

0+0 24 + 0 48 + 0 72 + 0

100 120 83.7 100.2

± ± ± ±

4 mg/ml 4.4 8.2 7.3 6.3

100 165 83.3 98.4

± ± ± ±

8 mg/ml 7.1 5.3 3.4 5.5

100 155 130 141

± ± ± ±

6.1 5.3 4.2 5.3

16 mg/ml 100 159.9 142 160

± ± ± ±

5.1 7.1 6.3 4.2

An analysis of the contents of nuclear DNA was performed in meristematic root cells, which were incubated with the conTable 1 Content of alkaloids expressed in mg/100 g of water extract and their percentage participation in total

Table 5 Change of G2 /G1 (counted in proportion to control) in nuclei of root tip cells during incubation at different concentrations of the (4, 8, 16 mg/ml) extract then post-incubation

Peak no.

Compound

mg

%

Time (h)

1 2 3 4 5 6 2–6

Caffeinea Uncarine F Speciophylline Mitraphylline Isomitraphylline/pteropodine Isopteropodine Total

– 28 68 33 230 70 430

6.59 15.89 7.74 53.44 16.34 100

a

Internal standard.

Concentration 4 mg/ml

0+0 6+0 12 + 0 24 + 0 24 + 12 24 + 24

100 126 157 174 163.6 122

± ± ± ± ± ±

8 mg/ml 7.1 2.2 6.6 5.2 5.1 4.3

100 117.7 121.7 160 164.8 141.6

± ± ± ± ± ±

16 mg/ml 6.1 8.2 8.3 5.3 6.1 2.2

100 108 110.9 164.2 139.1 133.5

± ± ± ± ± ±

5.1 6.3 7.2 4.3 8.2 3.6

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centrations of 2, 4, 8 or 16 mg/ml for 6, 12, 24, 48 and 72 h (variant 1), or after 24 h incubation then 48 h of post-incubation in distilled water (variant 2). In the experiment, three onions were used for every concentration of the extract. For flow cytometric analysis, a two-step procedure was used (Otto, 1990). Root tips about 2 mm long were cut off after 6, 12 or 24 h of incubation in a 1:8 dilution of the extract, as well as after 6, 12 and 24 h of post-incubation in water. Three root tips from each onion (nine altogether) were chopped into small pieces in a Petri dish with 250 ␮l of pretreatment solution of 0.1 M citric acid monohydrate + 0.5% Tween 20, then incubated for 3 min to isolate the nuclei. Subsequently, the suspension was passed through a 50 ␮m mesh nylon filter, and 1 ml of staining solution of 0.4 M Na2 HPO4 ·12H2 O + 2 ␮g/ml 4 ,6diamidino-2-phenylindole (DAPI)) was added. For each sample, the DNA content of 8000–10,000 nuclei was measured with a Partec CCA (M¨unster, Germany) Flow Cytometer. From the obtained histograms, the average G2/G1 ratio was calculated. The results were presented in charts compared to 100% of control. 3. Results 3.1. Phytochemical analysis As shown in Fig. 1 and Table 1, the total alkaloid content in the bark water extract was 430 mg/100 g of dry weight. Under the applied chromatographic conditions, isomitraphylline and pteropodine are not separated and have been previously observed in chromatograms of the oxindole-alkaloid standards (not published data). These alkaloids have the highest percentage contribution (53.44%) in the extract, whereas the lowest one belongs to uncarine F and mitraphilline (6.59 and 7.74%, respectively). The tetracyclic oxindole alkaloids were not detected in the analyzed extract. 3.2. Structure of the interphase nuclei and chromosomes of control and treated root cells Meristematic cells of control and treated onion roots showed differences. Control cells showed typical morphological and structural variations resulting from different phases of mitosis and interphase cells (Fig. 2A). The interphase cells were a distinctly prevalent group (85–95%). They showed variations: the first group consisted of distinctly smaller post-telophase cells with chromosomal territories still visible in their nuclei (Fig. 2I, arrow), probably entering the G1 phase. The second group consisted of small cells with despiralized nuclear chromatin, most likely finishing the G1 phase and at the beginning of phase S (Fig. 2B, white arrows); the third and the largest group were large cells, probably at the late S and G2 phase cells (Fig. 2B, black arrow). The remaining cells (5–15%) were at different stages of mitosis. Their appearance showed a typical variation of the Allium genus. The mitosis started from chromatin condensation within the nucleus and formation of chromosomal territories (Fig. 2C, black arrows, early prophase). At the middle prophase stage, the chromatin

appeared as thin, tangled threads (Fig. 2C, white arrow), which were slightly shortened and thickened at the end of this stage (prometaphase, Fig. 2D). Metaphase chromosomes were thickened and started forming the metaphase plate (Fig. 2E, early metaphase; Fig. 2F, middle metaphase). Their two components, the chromatids, are usually visible at this stage. During the next phase of mitosis, anaphase, chromatids moved towards the cell poles in an orderly way (Fig. 2G, anaphase; Fig. 2H, late anaphase, white arrow) and later, till reaching the pools in telophase (Fig. 2H, black arrow, early telophase; Fig. 2H, double arrow, late telophase). The nuclei of newly formed cells were oval, with cell wall forming between them (Fig. 2I, arrow). Incubation of roots in the bark extract (Figs. 3 and 4) changed the structure both of the chromosomes and of cell nuclei. The structure of interphase nuclei, causing uniformity of their size and structure was most distinct after 24 h of incubation at higher concentrations of the extract, e.g. in 8 and 16 mg/ml (Fig. 4A). The longest period (72 h) of incubation at the higher concentration (16 mg/ml), with total inhibition following 24 h of incubation, induced deformation of cells shape and strong vacuolation of cytoplasm (Fig. 4C), with distinctly shrunk or even deformed nuclei. Some cells of the upper part of the meristem contain scarce dense bodies connected with the cell nucleus, probably apoptotic bodies (Fig. 4D, arrow). Incubation of Allium cepa roots in the bark extract caused changes in structure of chromosomes at the lowest concentration, as well as over short periods at higher concentrations (Fig. 3A–F). This was manifested by strong condensation and contraction of the chromosomes, not leading, however, to chromosomal aberrations. We shall refer to such changed mitoses as cc divisions (condensed and contracted). There were numerous thickened prophases, with strongly condensed chromosomes showing clear patches of karyolymph between them (Fig. 3B, arrow). Changed metaphases had distinctly shortened chromosomes, usually forming a closely packed metaphase plate (Fig. 3C), or sometimes dispersed typically for C-metaphases (Fig. 3D). The cc changes were also seen in anaphases and telophases (Fig. 3E and F, arrow). Detailed analysis of cc mitoses and their quantitative proportion to the normal mitoses is presented in Section 3.4. During post-incubation following 24 h incubation, the structures of metaphase nuclei and chromosomes were similar to those of the control cells, even in high concentrations (Fig. 4B). 3.3. Mitotic activity in control root-tips During the continuous experiment (variant I), the mean mitotic index in control onions (Table 2) was higher at subsequent times of collecting samples than in the initial control by up to 10% (Table 2 after 24 h). Meanwhile, the phase index showed no marked changes. The proportions of particular phases are similar after different periods of the experiment. The mean percentage values of particular mitotic phases of control in the experiment were: 50.2% ± 3.7% for prophase, 17.2% ± 2.5% for metaphase, 9.9% ± 2.5% for anaphase and 22% ± 3.9% for telophase (Fig. 5A).

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Fig. 2. Microphotographs of control meristematic cells of root tips of Allium cepa. Squash preparations, stained in 2% acetoorcein and observed in light microscope; bar = 10 ␮m. All photographs and preparations were made this way (Tab. 3–6). (A) A typical view of different size, shape and basophility of nuclei and in interphase and mitotic phases; (B) cells with small, strongly dyed nuclei of dense structure, probably at the end of G1 phase (white arrows) and ones containing large nuclei of loosened structure, probably at late S and G2 phase (black arrows); (C) pre-prophase cells (black arrows), a cell at the early prophase stage (no arrow) and in typical prophase (white arrow); (D) early prometaphase; (E) late prometaphase; (F) metaphase (chromosomes arrayed in equatorial plate); (G) anaphase (regular alignment of anaphase chromosomes); (H) typical anaphase (the first cell on the left, white arrow), early telophase (the middle cell, arrow) and telophase cell (the one on the right, double arrow); (I) post-telophase cells with chromosomal territories (arrow) still visible.

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Fig. 3. Cell structure and forms of changed mitoses in Allium cepa root tip cells following different variation of incubation in the Uncaria tomentosa bark extract. (A) Low mitotic activity and changed divisions (cc prophases and metaphases) following 12 h of incubation in the extract at the concentration of 8 mg/ml; (B) changed prophase with strongly condensed chromosomes showing clear patches of karyolymph between them (arrow), after 6 h incubation in the extract at the concentration of 4 mg/ml; (C) cc metaphases after 6 h of incubation in the extract at the concentration of 8 mg/ml; (D) typical C-metaphase with “sky” chromosomes (with the set of 16 chromosomes, typical for onion), following 12 h of incubation at the concentration of 4 mg/ml; (E) changed anaphase with disturbed chromosome system following 12 h of incubation at the concentration of 8 mg/ml; (F) changed telophase (arrow) with slant alignment of chromosome groups, after 12 h of incubation in the extract at the concentration of 4 mg/ml.

3.4. Mitotic activity in root tips during continuous (0–72 h) incubation in the extracts It was found during the first variant of the experiment (Table 2), that the lowest concentrations of the extract 2 mg/ml caused a gradual decrease of the mitotic activity, not leading, however, to its total inhibition. During the incubation of the roots in this extract a minimal value of mitotic activity was observed after 12 h of incubation (about 50% of the initial value), but it started rising as early as after 24 h of incubation and ultimately reached its maximal value after 72 h ca. 130%. Incubation in an extract of 4 mg/ml caused, as in the previous concentration, a gradual decrease of mitotic activity, but the lowest value (10%) was observed after 24 h. After the further incubation period

(72 h) the mitotic activity reached only about 70% its initial level. The two highest concentrations of the extract (8 and 16 mg/ml) lowered the mitotic activity more strongly and faster. Its level decreased below 60% after 3 h incubation, finally leading to a total inhibition of cell division after 24 h (Table 2). 3.5. Mitotic activity in root tips treated 24 h in the extracts and followed by post-incubation in distilled water during next 48 h The aim of the second variant of the experiment (Table 3) was to establish the degree of toxicity of concentrations causing total inhibition of mitotic activity after 24 h and establish if the inhibition is lethal or can be reversed by post-incubation

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Fig. 4. Structure of meristematic cells of Allium cepa root tips after incubation in solutions of Uncaria tomentosa bark (A, C, D) and during post-incubation in water (B); bar = 10 ␮m. (A) A general view of meristematic cells following a 24-h incubation in the extract at the concentration of 8 mg/ml; inhibition of divisions, homogenization of cell nuclei structure. (B) Resumed cell divisions (prevalence of prophases) after 24-h post-incubation following a 24-h incubation in the extract at the concentration of 8 mg/ml; (C) disorganization and shrinkage of cells, strong vacuolation of cytoplasm and marked contraction of cell nuclei after 72-hour-long incubation in the extract at the concentration of 16 mg/ml; (D) cells of the upper part of the meristem, showing atypical, probably apoptotic bodies, connected with cell nuclei (arrow), after 72 h of incubation in the extract at the concentration of 16 mg/ml.

in water. The experiment was carried out only with concentrations causing total or near-total inhibition of mitotic activity, i.e., 4, 8 or 16 mg/ml. During the 24-h incubation in these extracts, similar changes of mitotic activity were observed as in the first variant (continuous incubation): the dilution of 4 mg/ml induced a decrease of the mitotic activity down to ca. 5% followed by a spontaneous increase (higher than in I variant) of the mitotic index. After 6 h, the index increased up to nearly 150% of the control level, but it returned to the control value after 48 h postincubation. Incubation in the two highest concentrations, as in the first variant of the experiment, caused total inhibition of mitoses after 24 h. At a concentration of 8 mg/ml, the mitotic index after the inhibition reached about 50% of the control val-

ues after 6 h in the water post-incubation, then after 12 h climbed to nearly 150%. A decrease at then 50% occurred after 24 h of incubation; however, the index reached 160% of the control value after 48 h.The highest used concentration used (16 mg/ml) caused strong inhibition of mitoses which still persisted after 6 h of post-incubation. Intensive cell divisions reappeared only after 12 h of post-incubation, soon reaching about 150% of the control value and then remaining at the same level as at both previously described concentrations, despite a slight decrease after 24 h of post-incubation (Table 3). The stage of the reappearance of divisions, after the roots had been placed in clean water for post-incubation, was distinctly reflected in the course of the phase index.

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Fig. 5. Mean phase index in root meristematic cells kept in: (A) water, 72 h (control); (B) 2 mg/ml bark extract (incubation); (C) 4 mg/ml (incubation); (D) 4 mg/ml incubation then 24 h in water (post-incubation); (E) 8 mg/ml then in water (post-incubation) and (F) 16 mg/ml then in water (post-incubation).

3.6. Changes of phase index The above-described incubation in the bark extracts also changed the mutual proportions of phases of mitosis (Fig. 5) depending on the duration of exposure and concentration of the extract. In the control (Fig. 5A), they maintained a comparatively constant level. During the initial 12 h of incubation in the extract at concentrations of 2 (Fig. 5B) and 4 mg/ml (Fig. 5C), the percentage of prophases increased gradually but distinctly, while the percentage of the remaining phases was lowered. Higher concentrations (Fig. 5E and F) of the extract caused an increase of the prophase index, lasting up to 12 h of incubation for 8 mg/ml and up to 6 h for 16 mg/ml. In post-incubation the proportions of the phase index returned to those of the control.

The percentage of cc mitoses (Fig. 5, black part of the bar) during constant incubation (72 h) proportionally increased with time in the first 12 h for concentrations of 2, 4 and 8 mg/ml and for 16 mg/ml, only up to 6 h, and then it decreased in small concentrations (Fig. 5B and C). The divisions showing changes during 24-h incubation rapidly disappeared after only 3 h of postincubation, for all concentrations used (Fig. 5D and E). 3.7. DNA content in the nuclei of control cells, treated and in post-incubation Cytometric analysis (Fig. 6) showed that cell nuclei of control cells showed typically differentiated content of DNA, depending on the phase of the division cycle. The most numerous were diploidal nuclei with 2C content of DNA, at the G1 /G0 phase.

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Fig. 6. Histograms of frequency of nuclei occurrence with different DNA levels in meristematic cells: (A) control in water; (B) treated in 4 mg/ml for 12 h; (C) treated in 4 mg/ml for 24 h and (D) treated in 4 mg/ml for 24 h then 24 h in water (post-incubation).

In the control, the proportion of this nuclei of the G2 /M phase, containing 4C DNA was almost constant and oscillated around 0.5 (Tables 4 and 5, time 0 + 0). In variant I, during constant incubation, the proportion of G2 /G1 (Table 4) dose depending on concentration of the extract and varied during incubation. After 24 h at all concentrations values were higher than in the control. After a 48-h incubation there was a decrease of the number of 4C nuclei, which in turn resulted in a lowering of the G2 /G1 proportion, falling below the control level at 2 and 4 mg/ml. After 72 h, this proportion for lower concentrations increased to reach the control level, and for higher concentrations (8 and 16 mg/ml) it increased to 140 and 160% of the control value, respectively (Table 4). In the second variant of the experiment, after a 24-h incubation, when a significant increase of the G2 /G1 was noted at all concentrations, the roots were transferred for post-incubation. At the time of post-incubation, for concentrations of 4 and 16 mg/ml, the value of the G2 /G1 proportion decreased after 12 h of post-incubation. Only at a concentration of 8 mg/ml after 12 h of post-incubation did the value rose slightly, to decrease again after 24 h (Fig. 6). The characteristics of changes in the DNA contents in the meristematic nuclei of control cells, as well as after incubation in the extract at the concentration of 4 mg/ml and during post-incubation, are presented in histograms (Fig. 6). 4. Discussion Investigations have been carried out in many laboratories aimed at finding effective treatment against a major disease of our civilization—cancer. Uncaria tomentosa is one of the plants a long used in folk medicine (Lemaire et al., 1999; Riva et al., 2001). Extracts of its bark show a very complex chemical composition and have a wide range of biological properties, from imunostimulation to immunosuppression (inhibition). Large amount of pentacyclic oxindole alkaloids, triterpenes and polyphenolics contribute to the high biological activity of extracts of that plant (Aquino et al., 1997; Keplinger et al., 1999; Laus et al., 1997). Moreover Stuppner et al. (1993) and Sheng et al. (1998) showed activity agains leukemia cell lines: HL 60

and U-937, when water extracts induced delayed-type apoptosis. Kim et al. (1998) showed that ursolic acid (triterpene) isolated from oleander, occurring also in Uncaria tomentosa bark extract (Falkiewicz and Łukasiak, 2001), has very strong antiproliferative properties and induces apoptosis in human cancer cell lines: A549 (lung cancer), SK-OV-3 (ovary cancer), SK-MEL-2 (skin cancer), XF498 (brain tumor), HCT-15 (leukemia) and B16-F-0 (melanoma). In the present work we confirmed the antimitotic activities of the Uncaria tomentosa bark extract with using a simple inexpensive test Allium root meristem, and showed its influence on the particular phases of mitosis. The treatment of root apical meristems of onion with the extract caused reduction or inhibition of meristematic cell divisions confirms which confirms its antimitotic properties also with respect to non-animal cells. A distinct increase in the prophase percentage was noted, especially at higher concentrations of the extract (8 and 16 mg/ml), whereas the numbers of other phases of mitosis were lowered. The G2 :G1 ratio may indicate a blockage of mitosis at the control point between prophase/metaphase, i.e. so-called Chfr point, as described by Scolnic and Halazonetis (2000) in normal and cancerous human cells treated with noctodasol, a compound inducing depolymerisation of microtubules. The strongly shortened and thickened chromosomes observed in prophases and metaphases (cc divisions) indicate an effect of the Uncaria tomentosa extract on the organization of chromatin, which may be related to a disturbed balance of the quantity of histones or other proteins responsible for controlling the proper structure of nuclear chromatin (Stryer, 1997). Similar changes in the chromatin structure were caused by water extract of yew (Taxus baccata) needles (Majewska et al., 2000). Cytometric measurements of the contents of nuclear DNA in root apical cells showed a general increase in the number of cells containing 4C DNA during treatment with the extract. So in spite of a lowered mitotic index, DNA replication did occur and the structures of all cell nuclei were similar to those of interphase nuclei. A typical feature of the effect of the extract was that, in its higher concentrations, cell divisions were totally inhibited after 24 h of incubation and the structures of all cell

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nuclei at that time were similar to that of interphase nuclei. This fact indicated blockage of the cell cycle at the G2 /M stage so in the initial period of mitosis. Besides the effective inhibition of cell division, a crucial requirement for anticancer drugs is minimal toxicity for healthy cells of the treated organism. We observed that all divisions of meristematic root cells of onion during post-incubation were unchanged, indicating that the extract did not cause permanent chromosomal aberrations, and that the cells of onions were not killed. In experiments on animal cells Uncaria tomentosa extract caused a strong proliferation of healthy lymphocytes along with simultaneous inhibition of cancer cells (Keplinger et al., 1999; Sheng et al., 2000; Wurm et al., 1998). The inhibition of division is a very promising result for anticancer therapy, as it leads first to block the development of cancer; additionally, the extract allows induction of DNA repair mechanisms. Such ability of Uncaria tomentosa extracts for DNA repair was discovered in murine cells exposed to irradiation (Sheng et al., 2000). All these results suggest that the anti-oxidative effect of the Uncaria tomentosa extract, preventing aberrations and triggering self-repair abilities in dividing cells, may give hope for complementary (traditional and natural therapy) treatment of human cancer. Acknowledgments This work was done with support of A-Z Medica, Gda´nsk. Authors are grateful for Prof. Steward Brown, Trent university, Peterborough, Canada for scientific and language remarks. References ˚ Akesson, C., Lindgren, H., Pero, R.W., Leanderson, T., Ivars, F., 2003. An extract of Uncaria tomentosa inhibiting cell division and NF-␬B activity without inducing cell death. International Immunopharmacology 3, 1889–1900. Aguilar, J.L., Rojas, P., Marcelo, A., Plaza, A., Bauer, R., Reininger, E., Klaas, A.Ch., Merfort, I., 2002. Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae). Journal of Ethnopharmacology 81, 271–276. Aquino, R., De Simone, F., Pizza, C., Conti, C., Stein, M.L., 1989. Plant metabolites. Structure and in vitro antiviral activity of quinovic acid glycosides from Uncaria tomentosa and Guettarda platypoda. Journal of Natural Products 52, 679–685. Aquino, R., De Feo, V., De Simone, F., Pizza, C., Cirino, G., 1991. Plant metabolites. New compounds and antiinflammatory activity of Uncaria tomentosa. Journal of Natural Products 54, 453–459. Aquino, R., De Tommasi, N., De Simone, F., Pizza, C., 1997. Triterpenes and quinovic acid glycosides from Uncaria tomentosa. Phytochemistry 45, 1035–1040. Cerri, R., Aquino, R., De Simone, F., Pizza, C., 1988. New quinovic acid glycosides from Uncaria tomentosa. Journal of Natural Products 51, 257–261. De Jong, W., Melnyk, M., Lozano, L.A., Rosales, M., Garcia, M., 1999. Una de gato: fate and future of a Peruvian forest resource. Center for International Forestry Research, Indonesia, 22, pp. 1–14. Deschmarchelier, C., Mongolli, E., Coussio, J., Ciccia, G., 1997. Evaluation of the in vitro antioxidant activity in extracts of Uncaria tomentosa (Willd.) DC. Phytotherapy Research 11, 254–256. Falkiewicz, B., Łukasiak, J., 2001. Vilcacora [Uncaria tomentosa (Willd.) DC i Uncaria guianansis (Aublet) Gemell. Case Reports & Clinical Practice Review n2, 310–322. Fiskesj¨o, G., 1985. The Allium test as a standard in environmental monitoring. Hereditas 102, 99–112.

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