Toxicological and cytotoxic evaluation of standardized extracts of Galphimia glauca

Journal of Ethnopharmacology 109 (2007) 35–40

Toxicological and cytotoxic evaluation of standardized extracts of Galphimia glauca Luc´ıa Aguilar-Santamar´ıa a , Guillermo Ram´ırez a , Armando Herrera-Arellano a , Alejandro Zamilpa a , Jes´us E. Jim´enez a , Daniel Alonso-Cort´es a , Elva I. Cort´es-Guti´errez b , Nestor Ledesma c , Jaime Tortoriello a,∗ a

Centro de Investigaci´on Biom´edica del Sur, IMSS, Argentina 1, Xochitepec, Morelos CP 62790, M´exico b Centro de Investigaci´ on Biom´edica del Noreste, IMSS, Monterrey, Nuevo Le´on, M´exico c Facultad de Medicina Veterinaria y Zootecnia, UNAM, DF, M´ exico Received 7 November 2005; received in revised form 22 June 2006; accepted 30 June 2006 Available online 8 July 2006

Abstract Galphimia glauca Cav (Malpighiaceae) has been widely used in Mexican traditional medicine as a remedy for the treatment of mental disorders, principally as a sedative and tranquilizer. The sedative activity of extracts obtained from this plant has been demonstrated with different neuropharmacological models. Different triterpenes, known as galphimines, have been identified from the active extract. Galphimine-B (G-B) possesses anxiolytic activity and is able to selectively inhibit discharges of dopaminergic neurons in the ventral tegmental area in rats. Nevertheless, there have been no toxicological investigations carried out with products obtained from this plant. In this work three different extracts (aqueous, ethanolic, and methanolic) of Galphimia glauca, standardized in the content of three galphimines, were evaluated for their behavioral and pharmaco-toxicological effects. After administering the extracts to mice for 28 days (2.5 g/kg, p.o.), no deaths were found and the histopathological analysis of different organs did not show alterations; only the behavioral parameters analyzed showed a diminution of spontaneous activity. The administration of these extracts for 56 days (same doses and route) in mice did not cause any changes in the biochemical parameters that evaluate liver function. On the other hand, no cytotoxic effects were found on KB, UISO, and OVCAR-5 transformed cell lines, but all extracts specifically inhibited colon cancer cell line growth with an ED50 lower than 2 ␮g/ml. The extracts were also evaluated in genotoxicity tests in vitro (250, 100 and 50 ␮g/ml), which demonstrate that none of the three extracts from Galphimia glauca showed a genotoxic effect. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Galphimia glauca; Malpighiaceae; Anxiolytic; Toxicology; Galphimine-B; Complementary and alternative medicine; Citotoxicity; Genotoxicity

1. Introduction An infusion prepared with the aerial parts from Galphimia glauca Cav., commonly known as “calderona amarilla”, has been used in Mexican traditional medicine for the treatment of mental disorders and for diminishing nervous excitement

Abbreviations: CNS, central nervous system; G-B, galphimine-B; G-A, galphimine-A; G-E, galphimine-E; VTA, ventral tegmental area; HPLC, high precision liquid chromatography; NMR, nuclear magnetic resonance; ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ANOVA, analysis of variance; MN, micronuclei; SCE, sister chromatid exchange ∗ Corresponding author. Tel.: +52 777 3612155; fax: +52 777 3612155. E-mail addresses: [email protected], [email protected] (J. Tortoriello). 0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.06.013

(Estrada, 1985). Previous reports demonstrated the sedative and anticonvulsant effects produced by the methanolic extract of the aerial parts from this plant (Tortoriello and Lozoya, 1992). A bio-guided chemical separation allowed the identification of one of the active compounds, the nor,seco-triterpene known as galphimine-B (G-B) (Toscano et al., 1993). This compound was able to reproduce the sedative effects displayed by the methanolic extract, but in this case, it was necessary to use lower doses. However, this compound was unable to produce any anticonvulsant effect (Tortoriello and Ortega, 1993). In electrophysiological experiments, local and systemic administration of G-B on rats produced modifications of the extracellular spiking activity records in the ventral tegmental area (VTA) neurons (Tortoriello et al., 1998). Later, it was proved that this effect is not a result of the interaction with the GABAergic system (Prieto-G´omez et al., 2003).

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by water (17 l, at 90 ◦ C for 50 min), or exhaustive maceration with methanol, or ethanol. The aqueous (420 g, 24.7%), methanol (451 g, 26.5%) and, ethanol (179 g, 10.5%) extracts were filtered and the solvent was evaporated under vacuum to dryness. The residue of each extract was re-dissolved in water for its successively lyophilized process. The lyophilized extracts were subjected to toxicological tests and quantitative analysis. 2.3. HPLC analysis

Fig. 1. Chemical structure of galphimine-B, galphimine-A and galphimine-E.

Since the VTA is a region with high density of dopaminergic neurons responsible for the innervation of the prefrontal cortex, nucleus accumbens and entorhinal region, and because these areas are targets for the action of antipsychotic drugs, the pharmacological effects produced by G-B have greater importance. With this background, the extract of Galphimia glauca (standardized for G-B content) was evaluated in behavioral animal models, and showed interesting anxiolytic effects in ICR mice (Herrera-Ruiz et al., 2006). More recently, other galphimines (Fig. 1) with similar structures and with spasmolytic effect, were identified in this species (Gonz´alez-Cortazar et al., 2005). In these galphimines, G-B and G-A also showed significant anxiolytic properties, and thus are considered the active compounds in the methanolic extract (Herrera-Ruiz et al., 2006b). Despite the interesting results obtained from the pharmacological and chemical studies and their potential therapeutic usefulness, no toxicological investigation of this species has been reported. In this work, based on the demonstrated pharmacological effects, three different extracts were prepared and submitted to different in vivo and in vitro toxicological tests. Previously, these extracts were monitored by the HPLC analytical method in order to quantify the G-B, G-A and G-E concentrations.

Galphimia glauca extracts were analyzed on a Merck-Hitachi modular HPLC system, consisting of an L-6200A intelligent pump, connected with a L-4500 photodiode array detector, equipped with an AS-2000 auto sampler and Chromatography data station software (Merck-Hitachi). Acquisition was set at 232 nm (spectral acquisition in the range 200–400 nm). The analyses were carried out on a Chromolith Performance RP-18e column (4.6 mm × 100 mm, Merck). The eluent was an isocratic acetonitrile/water 35:65 system. The mobile phase flow-rate was 1.7 ml/min for 12 min. 2.4. Quantitative determination of galphimines The galphimines G-A, G-B, and G-E, used as standards, were previously isolated from Galphimia glauca leaves. Their identity and purity were confirmed by comparison with published spectral data (IR, 1D and 2D NMR). Calibration curves were constructed using four points dilutions of each compound: 50, 100, 200 and 400 ␮g/ml in methanol (R2 = 0.99, 0.98 and 0.99 for G-A, G-B and G-E, respectively). All compounds were detected at 232 nm (Fig. 2). A volume of 50 ␮l was injected. The calibrations curves were based on the peak areas of the HPLC chromatograms. The experiments were performed in triplicate. The values were expressed in terms of percent on the basis of the dry weight in grams. Methanol and ethanol samples extracts were each prepared by elution of 10 mg of the extract on LC-18 Supelclean cartridge with 5 ml of methanol. The aqueous extract (10 g) was partitioned by CHCl3 /H2 O for obtaining the water sample. The organic fraction (10 mg) was evaporated to dryness and dissolved in 5 ml of methanol. Samples of 70 ␮l of each solution (2 mg/ml) were injected in triplicate.

2. Materials and methods 2.5. In vivo toxicological evaluation 2.1. Plant material Leaves of Galphimia glauca Cav. (Malpighiaceae) were collected in an experimental field in Xochitepec, Morelos, Mexico on August 18th, of 2000 and identified by M.Sc. Abigail Aguilar, Director of the IMSSM Herbarium, where a botanical voucher was deposited for reference. 2.2. Preparation of the extracts Plant material was dried under dark conditions during 10 days and milled to obtain 2–5 mm particles. This material was divided into three portions (1.7 kg, each), which were extracted

2.5.1. Animals Animal experimentation was performed observing “official regulations of experimental animal care” (NOM-062-ZOO1999), and in accordance with the internationally accepted principles for laboratory animal use and care as found in the US guidelines (NIH publication #85-23 revised in 1985). The experimental protocol was approved by the Institutional Research and Ethics Committee. Male and female Balb-C mice (18 ± 2 g) were used. Animals were maintained under regular husbandry conditions; 23 ± 2 ◦ C, 12 h light-dark cycle with ad libitum access to water and standard rodent chow (2018S, Harlan Teklad).

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2.5.3. Evaluation of hepatotoxicity Following the procedure described above, the animal groups (8 each) were treated orally, but in this case for 56 days, with the Galphimia glauca extracts. At the beginning of the experiment and every 2 weeks, blood samples were collected in 1.5 ml tubes without any additives, and centrifuged (955 × g, 10 min). Serum samples were immediately subjected to enzymatic activity determination. The enzymatic activity profiles of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were assayed according to the methods described by Otto et al. (1946), Bergmeyer and Horder (1980) and Bergmeyer et al. (1976), respectively. Results were expressed as means ± S.D. The differences between media were statistically analyzed by one-way ANOVA and a post hoc Bonferrioni test with SPSS-11.0 software. Values for p < 0.05 were considered statistically significant. 2.6. In vitro toxicological evaluation

Fig. 2. Chromatographic profile of different extracts from Galphimia glauca leaves acquired by HPLC-DAD at l = 232 nm. For HPLC run conditions see experimental procedures.

2.5.2. Subchronic toxicity Animals were organized in groups of five mice each, and introduced in translucent animal cages. In order to become accustomed to environmental and handling conditions, all animals were handled daily for a week before experiments. Different groups received a daily dose (2.5 g/kg, p.o.) of methanolic, ethanolic or aqueous extract from Galphimia glauca for 28 days. Dry extracts were prepared every day by solving in distilled water and were administered in a constant volume of 0.8 ml/kg. A control group was used and treated only with the vehicle by the same route and volume. All the animals were observed for the appearance of signs of toxicity or behavioral alterations during the experimental period and for one week after the extract administration. On day 35 all animals were killed under diethyl ether anesthesia and organs were carefully collected (brain, liver, pancreas, heart, kidney, lung, and stomach), fixed in 10% neutral buffered formalin, and prepared for histopatological analysis.

2.6.1. Cytotoxicity In accordance with international methods and following the regulations of the NCI (Suffnes and Pezzuto, 1991; Villarreal et al., 1992), HCT-15 (colon), UISO (uterus), KB (nasopharyngeal) and OVCAR-5 (ovarian) cell lines were maintained in an RPMI medium with 10% fetal bovine serum, and cultured at 37 ◦ C in a 5% CO2 –air atmosphere (100% humidity). The cells at the log phase of growth were treated in triplicate with different concentrations of the extracts (1, 10 and 100 ␮g/ml), and incubated for 72 h. The cell concentration was determined by protein analysis (Oyama and Eagle, 1956). Results were expressed as the concentration that inhibits 50% of control growth after the incubation period (ED50 ). The values were estimated from a semi-log plot of the drug concentration (␮g/ml) against the percentage of viable cells. 2.6.2. Genotoxicity Sister chromatid exchange (SCE) in vitro: peripheral blood samples of five masculine volunteers (27–34 years old) were collected in sterile heparinized vacutainers. Each sample was cultured in 11 series: a negative control, a positive control, and nine test culture cells that were treated with different concentrations (250, 100, and 50 ␮g/ml) of three different extracts from Galphimia glauca. The negative and positive controls were treated with isotonic saline solution (NaCl 0.9%), and cyclophosphamide (at a final concentration of 0.025 ␮l/ml), respectively. Lymphocytes were cultured for 72 h in McCoy 5A medium supplemented with 2% phytohemagglutinin, 15% fetal calf serum and antibiotics (streptomycin–penicillin). Bromodeoxyuridine (BrdU) was added after 24 h of incubation at a final concentration of 10 ␮g/ml. Colchicine was added 45 min prior to harvesting, and slides were prepared according to the standard procedure. Sister chromatid differentiation was achieved by the Fluorescence Plus Giemsa technique (Perry and Wolff, 1974). For each treatment, 250 metaphases were studied by blind analysis.

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Table 1 Behavioral parameters observed during 35 days, in female (F) and male (M) mice treated daily for 28 days (2.5 g/Kg, p.o.) with Galphimia glauca ethanolic (Et-OH), methanolic (Me-OH), or aqueous extracts Control

Hindlimbs grip strength Piloerection Righting reflex Equilibrium Dehydration Ataxia Alarm reaction Lethargy Defecation/Urination

Et-OH

Me-OH

Aqueous

F

M

F

M

F

M

F

M

N NP N N NP NP N NP N

N NP N N NP NP N NP N

(−) 2 (+) 11 (−) 17 (−) 6 (+) 16 NP N (+) 33 N

(−) 2 (+) 10 (−) 16 (−) 5 (+) 16 NP N (+) 33 N

N (+) 3 (−) 14 N (+) 12 NP N (+) 33 N

N (+) 3 (−) 13 N (+) 12 NP N (+) 33 N

N (+) 3 (−) 14 N (+) 11 NP N (+) 33 N

N (+) 4 (−) 13 N (+) 11 NP N (+) 33 N

Number of days with modifications are shown. N: normal; NP: non-present; (−): diminished; (+): increased.

Micronuclei (MN) in vivo: for MN analysis, the same population and treatments described previously were used. Lymphocytes were cultured for 72 h in McCoy 5A medium supplemented with 15% fetal calf serum and antibiotics (streptomycinpenicillin). Cytochalasin-B (0.6 ␮g/ml) was added for the final 24 h of culture. Binucleated lymphocytes were obtained through standard procedures (Fenech, 2000). The slides were analyzed using Wright’s stain 0.28%. For each treatment, 10,000 peripheral lymphocytes were studied under a light microscope using the criteria described by Fenech (2000). Data were analyzed by one-way ANOVA using the Newman–Keuls test for multiple comparisons. A value of p < 0.05 was considered statistically significant. 3. Results The total content of G-A (rt = 5.50 min), G-B (rt = 7.59 min), and G-E (rt = 10.00 min) in Galphimia glauca extracts were as follows: aqueous extract 0.6, 1.034 and 1.12, methanol extract 7.29, 17.47 and 13.6, ethanol extract 5.35, 18.8 and 17.49 mg/g of the extract, respectively (Fig. 2). 3.1. Subchronic toxicity study in mice After 28 days of administration (2.5 g/kg, p.o.) of the different extracts prepared with aerial parts of Galphimia glauca no deaths were observed in mice. Independently of the extract utilized, the histophatological analysis did not show alterations or differences when compared with the control group (data not shown). The body weight of each animal was evaluated weekly until the 35th day observation, showing a normal increment without differences between groups (data not shown). As illustrated in Table 1, animals of the control group showed normal and healthy activity, without evidence of dehydration after 35 days of treatment. The groups treated with the Galphimia glauca extracts showed, from the second day of treatment throught the last day of observation, a diminution of activity and dehydration. These animals were usually in groups, and became more sensitive to manipulation.

3.2. Evaluation of hepatotoxicity Table 2 illustrates the enzymatic activity of ALP, ALT and AST evaluated in samples of blood obtained every 14 days from mice treated daily for 56 days with different extracts of Galphimia glauca. There was no statistical difference between the values obtained before, or after administering the extracts. All rates were in the normal range. This result suggests the absence of hepatotoxic effects of the utilized extracts. 3.3. Cytotoxicity All extracts tested showed an ED50 higher than 20 ␮g/ml on KB, UISO, and OVCAR-5 cell lines. Nevertheless, all extracts produced a cytotoxic effect in the COLON cell line (HCT-15), in which the ED50 for all three extracts were lower than 2 ␮g/ml (Table 3). 3.4. Genotoxicity The average number of SCE/metaphase and the number of cells with MN/2000 lymphocites in three different concentrations of Galphimia glauca are showed in Tables 4 and 5, respectively. The ANOVA test showed that the negative control group was not different from the other treated groups, while all the treatments were different from the positive control (p < 0.05). These results demonstrate that the evaluated extracts from Galphimia glauca at a concentration of 250, 100 and 50 ␮g/ml did not show a genotoxic effect. 4. Discussion The contemporary search for novel molecules has taken a new direction in which ethnobotany and ethnopharmacognosy are being used as guides to lead the chemist to different sources and classes of compounds (Gurib-Fakim, 2006). Based on ethnomedical knowledge, some phytopharmaceuticals have been developed, among which those related to the prevention and treatment of psychiatric disorders constitute the market area with highest growth (Costa et al., 2004). In the last two decades, numerous publications have described the great importance

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Table 2 Enzymatic activity of ALP, ALT, and AST evaluated every 14 days in mice treated daily (2.5 g/kg, P.O.) for 56 days with Galphimia glauca ethanolic (Et-OH), methanolic (Me-OH), or aqueous extracts Day 1

14

28

42

56

ALP Control Et-OH Me-OH Aqueous

41.82 38.56 64.13 54.17

± ± ± ±

13.66 9.58 23.60 26.26

29.43 34.76 40.51 37.67

± ± ± ±

9.07 11.17 15.44 17.41

48.91 38.58 42.70 55.52

± ± ± ±

14.67 11.17 21.39 18.70

40.66 37.68 44.95 40.44

± ± ± ±

9.35 12.56 14.56 12.83

34.17 29.04 31.81 38.38

± ± ± ±

14.99 9.75 13.84 17.77

ALT Control Et-OH Me-OH Aqueous

3.37 5.96 8.31 5.27

± ± ± ±

1.80 3.59 6.13 3.23

11.36 9.12 10.6 7.61

± ± ± ±

2.48 4.1 6.94 4.71

6.66 19.78 10.6 7.38

± ± ± ±

3.92 6.64 2.62 4.98

10.27 15.91 10.56 6.47

± ± ± ±

3.02 6.89 5.24 3.07

8.39 10.62 31.81 5.68

± ± ± ±

3.25 5.51 15.74 2.14

AST Control Et-OH Me-OH Aqueous

8.39 7.29 7.97 7.61

± ± ± ±

3.25 4.55 3.72 4.71

21.77 31.66 7.56 7.38

± ± ± ±

8.93 20.79 4.16 4.98

13.85 6.47 17.81 22.76

± ± ± ±

7.19 3.07 5.93 8.72

19.00 15.83 19.79 7.02

± ± ± ±

6.5 4.84 6.71 5.71

4.89 16.11 9.89 3.95

± ± ± ±

2.26 4.48 6.85 1.71

Results are means ± S.D., n = 8. *p<0.05, **p<0.001, on Bonferrioni test.

that natural products have in the development of new pharmaceuticals or phytopharmaceuticals (Gilani and Rahman, 2005; Patwardhan, 2005). Consequently, numerous works have been published signaling the potential toxicological effects that these products could also possess (Marcus and Snodgrass, 2005; Pak et al., 2004). Galphimia glauca, based on experimentally demonstrated properties, is an interesting candidate for developing new phytotherapeutics, and it is necessary to characterize its effects on bio-systems, including its toxicological activities. In the present work, when high doses of the standardized extracts of Galphimia glauca were administered orally in mice, no toxicological effects were demonstrated on the liver function. Moreover the absence of damage or structural modification shown in the histological evaluation of important organs such as the liver, kidney, and heart; the absence of genetic alterations in peripheral blood lymphocytes; and the absence of mortality, including after the administration of 2.5 g/kg of extract by a 56 days treatment, allow us to conclude that the extracts obtained form this plant species, by the method described, possess very low risk of toxicity and genotoxicity within a reasonable margin of therapeutic safety. The observed behavior modifications in the experimental animals, particularly when the methanolic extract was used, could be due to a potent depressant activity on the CNS produced by the large doses utilized. These modifications include some autonomic effects as piloerection, lack of equilibrium, and Table 3 Cytotoxic effect produced by different concentrations (1, 10 and 100 ␮g/ml) of aqueous, ethanol (Et-OH) and methanol (Me-OH) extract from Galphimia glauca on growth cycle of cancer cell lines Cell lines

KB (␮g/ml)

UISO (␮g/ml)

OVCAR-5 (␮g/ml)

COLON (␮g/ml)

Et-OH Me-OH Aqueous

>20 >20 >20

>20 >20 >20

>20 >20 >20

0.63 0.5 1.99

Results are ED50 obtained by triplicate. Values <20 ␮g/ml are considered active.

diminution of righting reflex, while other signs were not found (shedding tears, modifications of pupil diameter, or respiratory movements, paralysis or diminution of prehensile activity). Piloerection could be produced by a vasorelaxation, as has already reported by Perusquia et al. (1995) in the aqueous extract from Galphimia glauca flowers. Observed dehydratation could be produced by an unidentified diuretic effect produced by the administered extracts, because other signs, such as soft stool or diarrhea, were not identified. According to the traditional use as anxiolytic, a 0.5 L of infusion prepared with 3 g of Galphimia glauca leaves is consumed through the day. Such preparation has a yield of 1.31 g of extract, and contains 2.45 mg of G-B. This dose is administered in a subject with 70 kg of body weight. The 2.5 g/kg dose, evaluTable 4 Average number of SCE/metaphase in lymphocytes treated with different concentrations of Galphimia glauca ethanolic (Et-OH), methanolic (Me-OH), or aqueous extracts Groupa

X ± S.D.

Negative controlb

5.14 ± 1.27

250 ␮g/ml Et-OH 250 ␮g/ml Me-OH 250 ␮g/ml aqueous

6.44 ± 1.76 6.58 ± 0.95 5.82 ± 0.84

100 ␮g/ml Et-OH 100 ␮g/ml Me-OH 100 ␮g/ml aqueous

4.97 ± 0.79 5.56 ± 1.67 5.28 ± 1.35

50 ␮g/ml Et-OH 50 ␮g/ml Me-OH 50 ␮g/ml aqueous

6.14 ± 0.77 6.03 ± 1.32 5.72 ± 0.98

Positive controlc

14.20 ± 0.78

ANOVA test: negative control = 250 ␮g/ml = 100 ␮g/ml = 50 ␮g/ml = positive control (for the ethanol, methanol, and aqueous extracts). a For each group 250 metaphases were studied. b Treated with saline solution (NaCl 0.9%). c Treated with cyclophosphamide at a final concentration of 0.025 ␮l/ml.

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Table 5 Number of cells with MN in lymphocytes treated with 50, 100 or 250 ␮g/ml of ethanolic (Et-OH), methanolic (Me-OH), or aqueous extracts of Galphimia glauca Group

MN/10,000 cells

MN/cell

50

0.0050

250 ␮g/ml Et-OH 250 ␮g/ml Me-OH 250 ␮g/ml Aqueous

48 51 57

0.0048 0.0051 0.0057

100 ␮g/ml Et-OH 100 ␮g/ml Me-OH 100 ␮g/ml aqueous

49 56 59

0.0049 0.0056 0.0059

50 ␮g/ml Et-OH 50 ␮g/ml Me-OH 50 ␮g/ml aqueous extract

54 49 53

0.0054 0.0049 0.0053

Positive controlb

95

0.0095

Negative

controla

ANOVA test: negative control = 250 ␮g/ml = 100 ␮g/ml = 50 ␮g/ml = positive control (for the ethanol, methanol, and aqueous extracts). a Treated with saline solution (NaCl 0.9%). b Treated with cyclophosphamide at a final concentration of 0.025 ␮l/ml.

ated in this study, is considerably larger than the average human dose. The interesting cytotoxic activity observed on HCT-15, a colon cell line, was produced by the three evaluated extracts; however, this activity could not be directly related to the G-B, G-A and G-E content, because of their different concentration in the extracts. Middleton et al. (2000) have reported that the flavonoid quercetine, present in Galphimia glauca, possesses an antiproliferative effect that is exerted by binding with the Type II nuclear estrogens receptor (EBS II). Nevertheless, quercetine has proven to be inactive on KB cells (IC50 = 295.8 ␮M) (del Rayo-Camacho et al., 2002). A chemical study, guided by a cytotoxic test, is necessary in order to identify the compounds that inhibit the growth of HCT-15 cells. In conclusion, the subchronic administration of high doses (2.5 g/kg, p.o) of ethanol, methanol and aqueous extract of Galphimia glauca, prepared under standardized conditions here described, did not demonstrate toxicological or genotoxic effects. No cytotoxic effects were found upon KB, UISO, and OVCAR-5 transformed cell lines, but all extracts did inhibit the HCT-15 cells (colon cancer, ED50 lower than 2 ␮g/ml). Acknowledgements This work was supported by the Research Council of Health of the Mexican Institute of Social Security, FOFOI 2005/1/I/097, and by CONACYT-SALUD 2004-1059. The authors wish to thank Dr. Corinne Hayden for reviewing the article. References Bergmeyer, H.U., Bowers, G.N., Horder, M., Moss, D.W., 1976. Provisional recommendations on OFCC methods for the measurement of catalytic concentrations of enzymes. Part 2. IFCC method aspartate aminotransferase. Clinica Chimica Acta 70, F19–F29. Bergmeyer, H.B., Horder, M., 1980. IFCC methods for the measurement of catalytic concentration of enzymes. Part 3. IFCC. Method for alanine amino-

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