Effects of Maytenus ilicifolia Mart . and Bauhinia candicans Benth infusions on onion root-tip and rat bone-marrow cells

Medicinal plants are widely used to treat various diseases, and in Brazil the plants Maytenus ilicifolia Mart. and Bauhinia candicans Benth are commonly used in popular medicine. However, there are a large number of compounds in plants which can produce alterations in genetic material, and this study was conducted to investigate any possible mutagenic and cytotoxic effects that M. ilicifolia and B. candicans infusions may have on the cell cycle and chromosomes. Infusions were prepared with in natura leaves to give two concentrations of infusions, one at the concentration normally used by the population in general and the other at 10 times this value (i.e. 3.5 and 35 mg/mL for M. ilicifolia and 0.465 and 4.65 mg/mL for B. candicans). Onion (Allium cepa L.) root-tip cells (RTC) and Wistar rat bone-marrow cells (BMC) were used as test systems in in vivo assays. The M. ilicifolia infusions at both concentrations, and the B. candicans infusion at the lower concentration, had no statistically significant depressive mitotic effect on RTC. A statistically significant depressive mitotic effect on RTC was found with the more concentrated (4.65 mg/mL) B. candicans infusion as compared with a negative control. In BMC, infusions of B. candicans and M. ilicifolia produced no statistically significant increase in the number of chromosome alterations or rates of cell division as compared to controls. The significance of these findings are discussed in the light of the use of these plants as therapeutic agents.


Introduction
Plants have always been used as a common source of medicines, both in traditional remedies and in industrialized products.In Brazil, the majority of the population uses traditional natural preparations derived from plant material for treating a variety of disease, and because of this it is extremely important that genotoxicity tests are applied to the active ingredients of these preparations in order to assess their mutagenic potential.The plants Maytenus ilicifolia Mart.(Celastraceae) and Bauhinia candicans Benth (Leguminosae) are commonly used in popular Brazilian medicine.The fresh or dry leaves of M. ilicifolia are used as an infusion to alleviate stomach pain and nausea and to treat ulcers and gastritis, this plant containing phenols, gallic tannins and other tannic compounds as well as epigallocatechinic derivatives.The leaves and flowers of B. candicans are used as an infusion to treat hypocholesterol and non-insulin dependent diabetes, the tissues of this plant containing sterols, flavonoids, pinetol, coline, trigoneline and citosterol.The research reported in this paper used onion (Allium cepa L.) root-tip cell and Wistar rat (Rattus norvegicus) bone-marrow cell assays to assess whether these plants have any effect on mitotic index or the occurrence of chromosome aberrations.

Maytenus ilicifolia Mart. and Bauhinia candicans
Benth were obtained from the Irenice Silva medicinal plant garden (State University of Maringá, Brazil).Infusions were prepared in the same way as is normally done when they are used by the general population.The M. ilicifolia leaves (in natura) were boiled for five minutes in tap water, and the infusion covered and allowed to cool.The B. candicans leaves (in natura) had boiling water poured over them and were left to stand for ten to fifteen minutes, strained and the infusion allowed to cool.The infusions from both plants were prepared at two concentrations, one corresponding to that normally used by the general population and the other at a concentration ten times higher, i.e. 3.5 and 35.0 mg/mL for M. ilicifolia and 0.465 and 4.65 mg/mL for B. candicans.

Root-tip cells
Onion bulbs (Allium cepa L.) were placed in flasks containing aerated water at room temperature until rooted, after which root sample were taken to act as time-zero (t zero ) controls.Some bulbs were then placed in the infusions prepared above (controls were put in water) for 24 h, after which more roots were removed and the bulbs returned to water for 24 to observe if there was recovery from any possible damage.The roots were fixed and stained using the Feulgen reaction and permanently mounted on slides.The slides were examined `blind' (i.e.without knowing their treatments) using an optical microscope with a 40X objective.For each bulb 1000 cells were analyzed, i.e. a total of 6000 cells each for the control, treatment and recovery groups.Cells with morphological structural alterations were recorded and the mitotic index (MI) of the cells calculated.The statistical evaluation was performed using the χ 2 test at a probability level of 0.05.

Bone marrow cells
Wistar rats (Rattus norvegicus) with a body weight (b.w.) of about 100 g were obtained from the Central Animal House at the State University of Maringá, three male and three female rats being used for each group (treatments and control).For each group, live rats were injected intraperitoneally for 24 h with 1 mL of one of the infusions prepared above, positive control animals being treated with 1.5 mg of cyclophosphamide (CP)/100g b.w.All rats were injected with 0.5 mL/100 g b.w. of a 0.16% colchicine solution an hour-and-a-half before sacrifice, bone marrow cells being obtained by modification of the method of Ford and Hamerton (1956).Chromosome analysis was carried out using optical microscopy and a 100X immersion lens.For each rat, 100 metaphases were examined in a `blind' design, giving a total of 600 metaphases each for the control and treatment groups.Mitotic index values were calculated for the 5000 cells by sex (a total of 10000 cells/group).Statistical evaluation was performed using he χ 2 test at a probability level of 0.05.

Root-tip cells
Table 1 shows the total number of cells analyzed, mean mitotic index values and the number of cells at the different phases of the cell cycle (interphase, prophase, metaphase, anaphase and telophase) in root-tip cells treated with M. ilicifolia and B. candicans infusions.
In the case of M. ilicifolia the mitotic index of the root-tip cells decreased after 24 h in each concentration of extract, with a lower mean mitotic index occurring at the higher concentration, this effect remaining even after the bulbs were subjected to a 24 h recovery period in water, although the results were not significant according to the χ 2 test.
For B. candicans the mitotic index of the root-tip cells also decreased after 24 h in each concentration of extract with the difference being statistically significant only for the higher concentration of 4.65 mg/mL (χ 2 = 4.86).After recovery in water for 24 h there was a small increase in mitotic index, which although not significant, was well below the mitotic index of the t zero controls (χ 2 = 3.38).
Neither of the M. ilicifolia infusions, nor the 0.465 mg/mL B. candicans infusion, produced a permanent significant depressive mitotic effect on the root-tip cells, although there was a statistically significant temporary inhibition of cellular division in the more concentrated M. ilicifolia infusion (35.0 mg/mL), but this effect was reversible.

Rat bone-marrow cells
Table 2 shows the mean total mitotic index, total analyzed metaphases and the number of chromosome alterations observed in male and female Wistar-rats treated with infusions of M. ilicifolia and B. candicans and in positive and negative control rats.Compared with the negative controls, there was no statistically significant increase in the number of chromosome alterations in bone-marrow cells from animals treated with any of the infusions prepared from either M. ilicifolia or B. candicans, nor was there any alteration in the cellular division index.

Discussion
Although frequently used by the general population as medicines, plant infusions are phyto-complexes of varying composition and contain alkaloids, flavonoids, tannins and other complex compounds that are produced by plants as protective mechanisms and which may be toxic or non-toxic when isolated in pure form.
In spite of the infusions having caused a decrease in cellular division in root-tip cells compared with non-treated controls only the higher concentration of B. candicans showed a statistically significant inhibitory effect, although this cytotoxic effect was reversible with slight recovery in cell division after 24 h recovery in water.It is possible that a high concentration of any chemical will have an effect (inhibitory or stimulatory) on the cell cycle, as has been shown for caffeine in Drosophila prosaltans (Itoyama et al., 1997), mefloquine in human blood lymphocytes (Grisolia et al., 1995), Alpinia mutans and Pogostemun heyneanus extracts in A. cepa root-tip cells (Dias and Takahashi, 1994) and glaucolide B extracted from Vemonia eremphila Mart. in human lymphocytes (Burim et al., 1999).Yen and Chen (1994) studied the relationship between the chemical composition of tea leaves and their extracts and their antimutagenic activity.They found that the principal components of tea leaves and their extracts are catechins, which seem to be responsible for antimutagenic activity, which varied from 4.3% in black tea to 26.7% for green tea.Green tea is produced from non-fermented Thea In vivo assay of the medicinal plants 87 The number of cells analyzed in each group was 6000.Concentration of infusion in mg/mL in parenthesis.
The number of cells analyzed in each group was 600.Positive control rats were injected with 1.5 mg cyclophosphamide/100 g body weight.Concentration of infusion in mg/mL in parenthesis.
sinensis leaves (the most popular beverage in the orient) much used in Japan as an antipyretic, diuretic and antioxidant, and which has been shown to have antimutagenic and antitumor effects in vitro and in vivo (Kada et al., 1985;Shimoi et al., 1986;Jain et al., 1989;Wang et al., 1989).It has also been reported that the mortality rate due to human cancers in areas of tea cultivation is significantly lower than in areas where tea is not grown (Sasaki et al., 1993), and that the catechin present in green tea suppresses the action of many environmental mutagens (Nakamura et al., 1997).
Black tea also has high antimutagenic activity in vitro, which varies according to the extent of fermentation of the tea during the manufacturing process (Yen and Chen, 1994;Apostolides et al., 1996).It has also been reported that black and green teas, without caffeine, have a chemo-preventive dose-dependent effect in preventing liver and lung cancer in rats (Cao et al., 1996).Horikawa et al. (1994) assessed the activity of six Chinese medicinal herbs on Salmonella and found that tannin and catechin compounds were responsible for the inhibition of mutagenicity caused by benzo[a]pirene.
Bauhinia candicans contains flavonoids (the second most common group of metabolites in the vegetable kingdom) and their very low toxicity makes them attractive compounds for use as therapeutic agents (Martins et al., 1995).In a study involving the mutagenic effects of 2-(2-furyl)-3-(5-nitro-2-furyl) (AF-2), Ohtsuka et al. (1995) investigated the antimutagenic effects of nine active compounds from the Chinese medicinal herb, sho-saiko-to and found that the main active antimutagenic compounds were the saponins and the flavonoids.According to Bu-Abbas et al. (1996) the high concentration of flavonoids in green tea compared with black tea may mean that these compounds are one of those responsible for the antimutagenic and (possibly) anticarcinogenic properties of tea and its fermented products.
It may be that the presence of flavonoids and tannins in M. ilicifolia and B. candicans was the reason why these medicinal plants did not show cytotoxic and clastogenic effects when tested in our system.It is also possible that extracts of these plants may have antimutagenic effects in different test systems, since the literature cited above indicates that plants containing flavonoids and tannin-like compounds can have such an effect.
Our results indicate that the consumption of infusions made from M. ilicifolia and B. candicans can be continued, although they should be used with caution always exactly following the traditional methods of preparation, especially with regards to the concentration of the infusions and the duration of treatment, so that the infusions have the desired pharmacological effects without toxicity.Medicinal plants can be very useful, but it is still necessary for the general population to take care not to use such plants indiscriminately.

Table I -
Mitotic index of onion root-tip cells treated with Maytenus ilicifolia (Mi) and Bauhinia candicans (Bc) infusions.

Table II -
Mitotic index and chromosome aberrations of bone-marrow cells of Wistar rats treated with Maytenus ilicifolia (Mi) and Bauhinia candicans (Bc) infusions.