Journal of Ethnopharmacology 110 (2007) 334342

Acute toxicity and mutagenic activity of Mexican plants used in traditional medicine
Myrna D ciga-Campos a,b , Isabel Rivero-Cruz b , Myriam Arriaga-Alba c , e Gabriela Casta eda-Corral b , Guadalupe E. Angeles-L pez b , n o Andr s Navarrete b, , Rachel Mata b, e
a Facultad de Farmacia, Universidad Aut noma del Estado de Morelos, Cuernavaca, Morelos, Mexico o Departamento de Farmacia, Facultad de Qumica, Universidad Nacional Aut noma de M xico, M xico D.F. 04510, Mexico o e e c Laboratorio de Investigaci n en Microbiologa, Direcci n de Investigaci n y Ense anza, Hospital Ju rez de M xico. Av. Instituto Polit cnico Nacional, o o o n a e e 5160 M xico D.F. 07760, Mexico e b

Received 24 May 2006; received in revised form 21 September 2006; accepted 2 October 2006 Available online 13 October 2006

Abstract The present work was undertaken to determine safety parameters of selected Mexican medicinal plants chosen on the basis of their frequency of medicinal use and commercial importance. The medicinal herbs included Amphipteryngium adstringens, Hintonia standleyana, Hintonia latiora, Piper sanctum, Haemathoxylon brasiletto, Iostephane heterophylla, Valeriana procera, Arracacia tolucensis, Brickellia veronicaefolia, Scaphyglottis livida, Exostema caribaeum, Hippocratea excelsa, Ligusticum porteri, Poliomintha longiora and Gnaphalium sp. In the acute toxicity studies in mice performed according to the Lorke procedure, Exostema caribaeum, Hippocratea excelsa, Ligusticum porteri and Poliomintha longiora were the most toxic with LD50 values between 1085 and 2 mg/kg. The Ames test revealed that Gnaphalium sp. and Valeriana procera extracts induced mutations of S. typhimurium TA98 with or without the S9 microsomal fraction, and TA100 in the presence of the enzymatic fraction, respectively. The tincture of Valeriana procera, however, was non-mutagenic. Finally, in the Artemia salina lethality test Brickellia veronicaefolia, Arracacia tolucensis, Poliomintha longiora and Piper sanctum caused signicant mortality of the crustacean larvae with LC50 in the range of 37227 g/mL. 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mexican medicinal plants; Acute toxicity; Lorke method; Ames test; Brine shrimp test; Mexican traditional medicine

1. Introduction According to a recent inventory carried out by Instituto Nacional Indigenista (INI) there are more than 3015 medicinal plant species commonly used for the treatment of common diseases in Mexico (Argueta et al., 1994). Most of these species have not been subjected to chemical, toxicological, pharmacological or clinical investigations and have been ignored by national health authorities for many decades. However, Mexican health authorities, following WHO guidelines, are now interested in those herbs employed for the relief of common ailments. The government has recognized the need to study
Corresponding authors. Tel.: +52 55 5622 5289; fax: +52 55 5622 5329. E-mail addresses: anavarrt@servidor.unam.mx (A. Navarrete), rachel@servidor.unam.mx (R. Mata). 0378-8741/$ see front matter 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.10.001

herbal products in order to generate ofcial monographs according to WHO criteria. Those monographs are intended to provide scientic information on the safety, efcacy and quality control of widely used Mexican medicinal plants. The best testimony of this initiative has been the publication in Mexico of the rst Herbal Pharmacopoeia (FHEUM) by the General Health Ofce in 2001 (Comisi n Permanenete FEUM, o 2001). In this context, this work was undertaken to determine safety parameters of selected Mexican medicinal plants. The results of this investigation will be useful for the elaboration of ofcial monographs of 14 species chosen on the basis of their frequency of medicinal use and commercial importance. Table 1 includes scientic names, common uses as well as pertinent references of previous chemical, pharmacological and/or clinical investigations, if any, on these plants.

Table 1 Mexican medicinal plants tested on Lorke, Ames and Artemia salina toxicity tests Botanical species Amphipteryngium adstringens (Julianiaceae) Pharmacological activities Antimycobacterial, hipocholesterolemic, gastroprotective and anti-inamatory Smooth muscle relaxing, hypoglycemic and anti-inammatory Hypoglycemic and antimalarial Antibacterial Antibacterial Hypoglycemic Antihyperglycemic and antinociceptive Gastroprotective, antibacterial and anti-inammatory activity Phytochemicals Anacardic acids, anacardic aldehydes, alkyl naphtalenes, triterpenoids and sterols References Mata et al. (1991), Mata (1993), Makino et al. (2004), Oviedo-Ch vez et al. (2004), Rivero-Cruz et al. (2005a) and a Navarrete et al. (2005) M. D ciga-Campos et al. / Journal of Ethnopharmacology 110 (2007) 334342 e Calderon et al. (1983), Roberts et al. (1980, 1984), P rez et al. e (2000) and Rivero-Cruz et al. (2005b) Sanchez-Viesca (1969), Mata et al. (1987, 1988), Noster and Kraus (1990), Pinto et al. (1997) and Slijepcevic et al. (1997). Torrenegra et al. (1992) and Villag mez-Ibarra et al. (2001) o Czako and Marton (2001), Yasunaka et al. (2005), Escobar et al. (2005) and Heredia et al. (2005) Mata and Cervera (1990), Mata and Camacho (1992), Mata et al. (1992) and Korec et al. (2000) Gonz lez-Ch vez et al. (2000), Guerrero-Analco et al. (2005) and a e D ciga-Campos et al. (2006). e Mata et al. (1990) and Calzada et al. (1991), P rez et al. (1995), e Liao et al. (2001), Furukawa et al. (2002), Navarrete et al. (2002), Reyes-Chilpa et al. (2003), Alanis et al. (2005) and Aguilar-Gonz lez et al. (2005). a Aguilar and Delgado (1995), Campos et al. (2000), Mata et al. (2001) and Aguilar et al. (2001) Delgado et al. (1992), Delgado (1996) and Cegiela-Carlioz et al. (2005) Rudolf and Jutta (1982a,b) and Mata et al. (2004)

Brickellia veronicaefolia (Asteraceae) Exostema caribaeum (Rubiaceae) Gnaphalium sp. (Asteraceae) Haemathoylon brasiletto (Leguminosae) Hintonia latiora (Rubiaceae) Hintonia standleyana (Rubiaceae) Hippocratea excelsa (Hippocrateaceae)

Flavonoids, diterpenoids, benzylbenzoates, sesquiterpenoids and salycilic acid derivatives 4-Phenylcoumarins Flavonoids, diterpenoids, acetylenic compounds and carotenoids Isoavonoids 4-Phenylcoumarins, tanins phenylstyrene 4-Phenylcoumarins, cucurbitacins, and alkaloids Sesquiterpene evoninoate alkaloids, friedelanes and triterpenoid quinone methides Bisabolene type sesquiterpenoids, diterpenoids and coumarins Phtalides Apornic alkaloids, kawapyrones, piperolides, furanes, pyranes, aromatic compounds. Phenolic compounds Stilbenoids, triterpenoids Valepotriates, avonoids, valerenic acids, lignans and monoterpenoids

Iostephane heterophylla (Asteraceae) Ligusticum porteri (Apiaceae) Piper sanctum (Piperaceae)

Antibacterial, vasorelaxation Antibacterial Antimycobacterial

Poliomintha longiora (Lamiaceae) Scaphyglottis livida (Orchidaceae) Valeriana procera (Valerianeaceae)

Antioxidant Spasmolytic Sedative, antispasmodic, insomnia, anticonvulsant and antidepressant

Zheng and Wang (2001) Estrada et al. (1999, 2001, 2006) Hazelhoff et al. (1982), Houghton (1988, 1997), Herrera-Arellano et al. (2001), Oliva et al. (2004) and Ugalde et al. (2005)

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2. Materials and methods 2.1. Plant materials The stem bark of Amphipteryngium adstringens (voucher: NPC0701) was collected in Colima, Mexico in June 2004. The aerial parts of Arracacia tolucensis var. multida (voucher: Bye & Morales 27040) were collected on September 1999 in the Valley of Mexico, D.F., Mexico. The aerial parts of Brickellia veronicaefolia were collected in Municipio de la Soledad, Aculco, State of Mexico, Mexico, on December 10, 2000 (voucher: BRC-1M). The roots of Iostephane heterophylla were collected in San Luis Potos, San Luis Potosi, Mexico, on October 25, 1995 (voucher: R. Bye, E. Linares, G. Morales, M. Mendoza 20528). The aerial parts of Polimintha longiora were collected in Real de Catorce, San Luis Potos, Mexico, on November 2005 (voucher: Bye & Linares 33925). The leaves of Piper sanctum were collected in May 1999 in San Andr s Tuxtla, State of Veracruz, Mexico (voucher: e GM 423-h). Scaphyglottis livida (whole plant) was collected in Catemaco, State of Veracruz, in May 1995 (voucher: G. Carmona Daz 116 AMO). Roots and rhizomes of Valeriana procera were obtained for micropropagation and were provided by Laboratorios Mixim. Stem bark of Hintonia latiora (voucher: Bye 33437) were collected in Chihuahua, Mexico in January 2004. Roots of Ligusticum porteri (voucher Bye 33434) and stem bark of Hintonia latiora (voucher: Bye 33437) were collected in Chihuahua, Mexico, in January 2004. Stem bark of Exostema caribaeum (voucher: Marchena 1167098) and stem bark of Hintonia standleyana (voucher: P. Hersh 824) were collected in Atenango del Rio Guerrero, Mexico, in January 2003. Stem bark of Haemathoxylon brasiletto (voucher: Bye 20339), inorescences of Gnaphalium sp. and root of Hippocratea excelsa were purchased in the Mercado de Sonora, Mexico City. The identity of these species was corroborated by Dr. Robert Bye, and all references but Scaphyglottis livida and Valeriana procera were deposited at the National Herbarium (MEXU), Mexico City. The voucher specimen of Scaphyglottis livida was deposited at the Herbarium of Instituto de Ecologa A.C. (XAL), Xalapa, Veracruz, Mexico and that of Amphipteryngium adstringens at the National Center for Natural Product Research, University of Mississippi, USA. The air-dried plant material in all cases was ground into a powder and extracted exhaustively by maceration, at room temperature, with a mixture of MeOHCH2 Cl2 (1:1). The resulting extracts were concentrated to dryness in vacuo. It is important to point out that the extracts used throughout the investigation were prepared using this solvent mixture in order to assure the extraction of both polar and non-polar compounds. To prepare the tincture of Valeriana procera; the dried and powdered roots (100 g) were extracted with 70% (v/v) ethanol (one part of drug to ve parts solvent) in accordance with a standard pharmacopoeia procedure (List and Schmidt, 1989). The resulting tincture was ltered by gravity and concentrated through air current at room temperature (approximately 22 C) yielding 20 g of extract.

2.2. Reagents Dimethyl sulfoxide (DMSO), 2-aminoanthracene (2AA), picrolonic acid (PA), N-ethyl-N -nitro-N-nitrosoguanidine (ENNG), cyclophosphamide (CP), colchicine, and mitomycin C were from SigmaAldrich Co. (St. Louis, MO, USA). S. typhimurium strains were kindly given by Professor Bruce Ames, University of California at Berkley, USA. 2.3. Acute toxicity study in mice Experiments were performed on male mice ICR (body weight range, 2530 g), obtained from Centro UNAM/Harlan (Harlan Mexico, S.A. de C.V). All experiments were performed following the Mexican Ofcial Norm for Animal Care and Handing (NOM-062-ZOO-1999). Mice were housed in a climate and light controlled room with a 12 h light/dark cycle. Twelve hours before experiments, food was withheld, but animals had free access to drinking water. The extracts were suspended in vehicle (Tween-80, 0.2% in saline). The concentrations were adjusted to orally administrate 0.2 mL/10 g body weight. Mice were treated in two phases. In the rst, intragastric doses of 10, 100 and 1000 mg/kg of crude extracts were administered. On the second, the doses were administered according to Lorke method (1983) as shown in the Table 2. In both phases, mice were observed daily in a period of 14 days for mortality, toxic effects and/or changes in behavioral pattern. At the end of the experiments the animals were sacriced in a CO2 chamber. 2.4. Artemia salina lethality test The extracts were evaluated for lethality to brine shrimp larvae (BST) according to the procedure described by Anderson et al. (1991). Briey, dried brine shrimp eggs were bred in saline medium (Instant Ocean ). After 48 h a few shrimps hatched and were ready for testing. One-day-old larvae (10 per vial) were transferred into 5 mL vials containing the dry extract and saline solution. The extracts were tested at 10, 100 and 1000 g/mL. In each case three replicates of each concentration were assayed. After 24 h the numbers of survivors were counted and percentage of death calculated. From these percentages, the LC50 s in g/mL were calculated using probit analysis. Criterion of activity: LC50 values of <1000 g/mL for extracts. Colchicine (LC50 = 25 g/mL) was used as a positive control. 2.5. Mutagenicity test The extracts were assayed as potential mutagens in the Ames tube-incorporation test. S. typhimurium (His ) strains TA98, TA100 and TA102, were grown in nutrient broth (NB) liquid medium for 16 h at 37 C in agitation (90 rpm). A suspension of 100 L of TA-98, TA100 or TA102 strains was transferred to sterile screw-top tubes with 2 mL of 0.6% soft-agar and the extracts were added at different concentrations (250, 500 and 1000 g/plate) dissolved in DMS. The assay was performed with or without 500 L of an enzymatic liver fraction (S-9 mix)

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Table 2 Results of the potential toxic effect of selected Mexican medicinal plants using the Lorke approach in mice and the lethality test against Artemia salina of the crude extracts Extract Phase I dose (mg/kg) 10 Amphipteryngium adstringens Haemathoxylon brasiletto Hintonia standleyana Piper sanctum Iostephane heterophylla Valeriana procera Arracacia tolucensis Brickellia veronicaefolia Gnaphalium sp. Hintonia latiora Scaphyglottis livida Extract 0/3a 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 100 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 1000 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 Phase II dose (mg/kg) 1600 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 Phase II dose (mg/kg) 1000 2/3 2/3 3/3 200 0/3 0/3 0/3 400 0/3 1/3 0/3 800 0/3 2/3 0/3 1600 1/3 3/3 1/3 1085 700 605 LD50 (mg/kg) 16 3/3 <2 2900 0/3 0/3 0/3 0/3 0/3 0/3 1/3 1/3 2/3 2/3 0/3 5000 0/3 0/3 0/3 0/3 1/3 1/3 3/3 3/3 1/3 3/3 1/3 >5000 >5000 >5000 >5000 3807 3807 2852 2852 2852 2852 2852 LD50 (mg/kg) LD50 (mg/kg) Artemia salina lethality CL50 b ( g/mL) >1000 630.95 >1000 227.50 >1000 439.33 71.94 37.15 >1000 719.45 436.51 Artemia salina lethality CL50 b ( g/mL) 777.98 >1000 >1000 Artemia salina lethality CL50 b ( g/mL) 169.04

Phase I dose (mg/kg) 10 100 0/3 0/3 0/3

Ligusticum porteri Exostema caribaeum Hippocratea excelsa Extract

0/3 0/3 0/3

Phase I dose (mg/kg) 10 100 3/3 1000 3/3

Phase II dose (mg/kg) 2 3/3 4 3/3 8 3/3

Poliomintha longiora
a b

3/3

Number of animals dead/number of animals used. LD50 was determined from the geometric mean for which 0/3 and 3/3 were found. In Artemia salina lethality test, colchicine (CL50 = 25 g/mL) was used as positive control.

obtained from male Wistar rats treated with 10% of Aroclor1254 (Ames et al., 1973). The total tube content was spread immediately into plates with VogelBonner medium and incubated for 48 h at 37 C. All experiments were performed three times by triplicate. The number of revertant colonies was determined in a Fisher colony counter. The reversion rate was compared to control plates with and without mutagen. The mutagens for TA98, TA100, TA102 when enzymatic fraction S9 was added were PA (50 g/plate), ENNG (10 g/plate) and Mytomicine-C (20 ng/plate), respectively. In the absence of S9 mutations, however, were induced with 2AA (20 g/plate), CP (500 g/plate) and 2AA (10 g/plate), respectively. The mutagenic index (MI) was calculated as the average number of revertants divided into the average number of revertants in the control. A sample was considered mutagenic when MI was higher than 2 for at least one of the tested concentrations (Bernstein et al., 1982; Margolin et al., 1981). In the case of Gnaphalium sp. and Valeriana procera the experiments were repeated preincubating TA98 (31500 g/plate) and TA100 (311000 g/plate), respectively, with the extracts during 60 min (Maron and Ames, 1983). 3. Results and discussion 3.1. Acute oral toxicity study Investigation of the acute toxicity is the rst step in the toxicological analysis of herbal drugs. In this study the Lorke procedure (Lorke, 1983) was selected because it offers the advantage that

when the doses are appropriately chosen, adequate information is obtained using only 13 animals, irrespective of the type of material tested and the route of administration selected. Table 2 showed the results of the acute toxicity of the crude extracts of fourteen Mexican medicinal plants widely used for treatment of all kind of illness. According to the DL50 values calculated using the geometric mean of the doses for which 0/3 and 3/3 deaths were found, the extracts can be classied into three groups. The rst group included Amphipteryngium adstringens, Hintonia standleyana, Piper sanctum and Haemathoxylon brasiletto with LD50 > 5000 mg/kg; they were regarded as non-toxic plants. The second group comprises slightly toxic plants whose LD50 values ranged from 3807 to 2852 mg/kg. In the last group were found Exostema caribaeum (LD50 = 700 mg/kg), Hippocratea excelsa (LD50 = 605 mg/kg), Ligusticum porteri (LD50 = 1085 mg/kg) and Poliomintha longiora which was particularly toxic with a LD50 < 2.0 mg/kg. During the rst phase of the evaluation, all animals administrated with 10, 100 or 1000 mg/kg of the extract of Poliomintha longiora died; the treatments provoked also brous nodules in the back and neck of the animals which died 2 or 3 days after the administration of the crude extract; during the second phase of the experiment, the same trend of effects were observed. Exostema caribaeum generated tremor, respiratory distress as well as decrease in the motor activity and body weight by 27.1% with respect to the vehicle treated animals. These effects were only observed during the rst phase of the experiments. The other species evaluated did not show important changes on behavior (i.e. ataxia, hyperactivity, hypoactivity),

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body weight or macroscopic morphology of heart, liver, kidney and lung. 3.2. Brine shrimp test The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. In addition, the method is rapid, simple, reproducible and economical. A wide variety of biologically active chemical compounds, in particular cytotoxic agents, are toxic to brine shrimp; the death of this organism when exposed to varying concentrations of these compounds forms the basis of a toxicity test. Bioactive compounds are nearly always toxic in high concentrations and, as toxicology can be described as pharmacology at higher doses, this premise has been applied to the screening of medicinal plant extracts in the brine shrimp toxicity test. The test has also been used for monitoring mycotoxins, wastewater and marine pollutants, detergents and surfactants, petroleum products, food dyes, antifouling paints for ships, heavy metals and pesticides (Sam, 1993). The results of Artemia salina testing are summarized in Table 2. The results revealed that Amphipteryngium adstringens, Iostephane heterophylla, Exostema caribaeum, Hintonia standleyana, Hippocratea excelsa and Gnaphalium sp. were not toxic to Artemia salina since the LC50 of the extracts were higher than 1000 g/mL. However Scaphyglottis livida, Valeriana procera, Hintonia latiora, Haemathoxylon brasiletto and Ligusticum porteri exhibited weak toxicity (LC50 values ranged from 436 to 778 g/mL). Finally, Brickellia veronicaefolia, Arracacia tolucensis, Poliomintha longiora and Piper sanctum showed signicant toxic effect against Artemia salina with LC50 ranging from 37 to 227 g/mL. 3.3. Mutagenicity Ames test Mutagenic compounds are potential hazards for man and should be scrutinized for benet, risk, in particular because it has been found in almost every instance that chemicals able to mutate bacteria are also carcinogenic to animals. A very useful procedure to detect mutagenic substances is the standard plate incorporation test developed by Ames et al. (1973). This assay identify if any sample provoke the mutation of genetically modied DNA of selected S. typhimurium strains (Ames et al., 1973). Since in some instances, chemicals are not mutagenic by themselves, but become mutagens as they are metabolized in the liver, the Ames assay includes also an evaluation in the presence of a mammalian mixture of liver enzymes, better known as the S9 microsomal fraction, to mimic this in vivo activation process (Ames et al., 1973). In some instances some mutagens are poorly detected in the standard plate incorporation assay; therefore, a modication of the method consisting in pre-incubating the potential mutagen, S9 mix and the bacteria during 2060 min at 37 C before adding the top agar is also carried out (Maron and Ames, 1983). The latter procedure increases the sensitivity of the method due to a more efcient interaction between the bacteria and the mutagens. Furthermore, the preincubation method allows testing of lower concentrations of mutagens.

Table 3 shows the results of the Ames assay of the crude extracts of the plants analyzed. According to these, Gnaphalium sp extract induced mutations on S. typhimurium TA98 with or without the S9 fraction; whereas Valeriana procera increased the number of revertants on TA100 in the presence of the enzymatic fraction. The remaining extracts did not provoke mutagenic effect on the three strains of S. typhimurium tested under the different assay conditions. In the initial experiments (Table 3), the extract of Gnaphalium at the concentration of 1000 g per plate, enhanced the number of revertants (His His+ ) on TA98; the MIs calculated were of 2.6 and 2.7 above the spontaneous background level, in presence or absence of S9 fraction, respectively. At 500 g per plate, the MIs were 1.9 and 2.4, respectively. Finally, at 250 g per plate no effect was observed in absence of S9 (MI = 1.7) but in the presence of S9 the MI was 2.1. The mutagenicity provoked by Gnaphalium extract on TA98 was concentration dependent (Fig. 1) in the presence or absence of the enzymes mixture. At the concentration range of 30500 g per plate no mutagenic effect was observed in the absence of the microsomal mixture. Altogether these results revealed that the Gnaphalium extract tested contains substances which evoked the reversion of TA98 S. typhymurum strain in vivo, since the effects are more important in the presence of the enzymes mixture (Fig. 1). It is important to point out that S. typhimurium strain TA98 is useful for detecting various frameshift mutagens such as aatoxins, dimethylbenzanthracene, daunomycin and avonoids. Thus, the presence of quercetin, with known mutagenic activity against S. typhimurium TA98 (Czeczot et al., 1990), in several Gnaphalium species (Villag mez-Ibarra et al., 2001), including the one o tested in the present investigation, can account for the mutagenic action demonstrated in this study. The crude CH2 Cl2 MeOH (1:1) extract of Valeriana procera at the concentration of 1000 g per plate, in the presence of the enzymatic fraction, signicantly accelerated bacterial growth. Therefore, the experiments were repeated with lower concentrations (30500 g per plate) using the pre-incubation method. According to the data summarized in Table 4, in this experiment a marginal mutagenic effect was observed (MI = 2).

Fig. 1. Mutagenic effect induced by the extract of Gnaphalium sp. on S. typhimurium TA98, employing the modied preincubation Ames test in the presence of S-9. Each value represents the mean of nine Petri dishes on three independent assays. TA98 spontaneous reversion: 38.1 2.0. PA: 382.0 37.5 and 2AA: 5416.89 726.91.

Table 3 Mutagenic activity expressed as the mean and standard deviation of the number of revertants/plate and mutagenic index (MI) in bacterial strains TA98, TA100 and TA102 exposed to the plant crude extracts (1000 g/plate) of selected Mexican medicinal plants without or with metabolic activation (S9) Number of revertants/plate and MIa TA 98 S9, X 0 DMSO Positive control Extract (1000 g/plate) Amphipteryngium adstringens Arracacia tolucensis Brickellia veronicaefolia Exostema caribaeum Gnaphalium sp. Haemathoxylon brasiletto Hippocratea excelsa Hintonia latiora Hintonia standleyana Iostephane heterophylla Ligusticum porteri Poliomintha longiora Piper sanctum Scaphyglottis livida Valeriana procera
a b

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TA 100 +S9, X 33.9 4.1 48.4 7.0 5416.9 726.9 43.3 9.0 36.6 9.0 35.4 6.8 36.4 5.6 132. 6 11.6* (2.7) 29.9 7.7 34.1 4.7 35.4 5.4 32.7 4.2 37.1 8.8 NT 62.6 12.4 37.1 7.0 38.0 8.4 39.4 8.0 S9, X 251.2 13.1 285.6 7.6 4921.9 1877.5 253.0 11.7 235.7 20 293.1 26.8 249.2 23.3 265 8.0 158.0 21.3 244.9 10.6 259.9 20.0 245.4 16.9 210.6 22.5 NT 314.2 29.0 203.6 21.3 237.4 27.4 159.6 28.1 +S9, X 263.3 19.4 286.6 7.6 826.1 47.6 238.1 24.9 267.2 24.4 261.8 26.3 262.3 24.2 280.1 27.5 144.9 28.3 243.1 17.0 250.0 12.0 374.0 50.1 184.0 21.6 NT 282.1 31.2 219.9 24.6 251.4 26.1 Toxic

TA 102 S9, X 305.3 15.8 336.1 23.8 4719.7 608.2 246.8 33.4 209.2 22.1 173.8 17.0 218.3 21.0 275.1 31.2 104.9 19.3 171.4 21.2 212.9 24.5 191.3 9.6 219.1 27.2 NT 312.0 36.5 133.1 21.4 228.9 29.7 251.6 21.3 +S9, X 270.4 19.6 351.4 24.5 3204.2 359.5 (1), 3873.6 186.0 (2) 289.9 32.0 163.2 26.0 197.4 19.5 225.2 25.9 310.6 30.3 218.8 18.5 209.4 19.4 205.2 21.1 254.3 21.7 272.3 27.5 NT 268.9 24.7 267.6 29.0 261.6 22.5 223.7 8.5

35.7 3.6 39.1 4.7 382 37.5 49.7 8.8 43.3 6.3 33.4 3.6 41.4 8.5 101.0 14.0* (2.6) 40.1 6.1 33.7 5.2 42.1 8.4 32.4 6.0 30.2 7.6 NTb 36.4 5.5 46.3 8.2 36.7 4.3 34.4 6.0

MI: mutagenic index. NT: not tested. Positive controls: +S9, 2-AA (10 g/plate) for TA98 strain; CP (500 g/plate) for TA100 strain and (1) 2-AA (10 g/plate) and (2) mitomycin C (20 ng/plate) for TA102 strains. S9, PA (50 g/plate) for TA98; ENNG (10 g/plate) for TA100 and mitomycin C (20 ng/plate) for TA102. The results are reported as means S.D. Each value means the average of nine petri dishes on three independent studies. Italicised values mean positive controls. * P < 0.05 respect to the control treated with DMSO.

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Table 4 Mutagenic action of a CH2 Cl2 MeOH extract and a tincture of Valeriana procera on S. typhimurium TA100, using the standard and preincubation Ames tests Number of revertants/plate and (MI)a , TA100 S9 Standard 0 DMSO Positive control 251.2 13.1 285.6 7.6 4921.9 1877.5 +S9 263.3 19.4 286.6 7.6 826.1 47.6 300.2 20.8 336.4 16.8 Toxic 295.8 24.5 238.9 26.2 346.2 27.4 263.8 16.0 242.6 18.2 822.6 55.4 250.2 17.7 266.0 24.5 324.6 33.7 374.0 43.6 500.9 52.0* (2.0)

CH2 Cl2 MeOH Valeriana extract 250 240.2 28.0 500 229.0 21.8 1000 256.2 17.8 Tincture EtOH Valeriana extract 250 300.4 20.5 500 290.3 31.2 1000 272.0 23.3 Preincubation 0 DMSO Positive control 238.1 19.6 252.8 27.8 3764.1 711.0

of 2501000 g per plate. The tincture was selected for testing because its lower content of valeprotriates (Navarrete et al., 2006). The decrement of these components in this hydroalcoholic mixture has been attributed to their low solubility and instability in the conditions tinctures are prepared (Navarrete et al., 2006). According to the data summarized in Table 4 the tincture of Valeriana procera tested was not mutagenic in S. typhimurium TA100 strain. Poliomintha longiora was not mutagenic in any of the studied strains but a toxic background was observed on the plates having 1000 g/Petri dish of this extract in all tested strains. This effect correlates with the toxic effect observed in the Lorke test. 4. Conclusions Based on the results from the brine shrimp and Lorke tests, Poliomintha longiora seems to be the most toxic species. This plant is widely commercialized as substitute of Lippia graveolens, regarded as Mexican oregano, and widely used for culinary and medicinal purposes. Therefore it is important that National Health Authorities notify local consumers as well as national and international traders about the risk of using this plant, at least until further studies are carried out. Piper sanctum contains several bioactive apornic alkaloids, kawapyrones and piperolides with known CNS and cytotoxic effects (Mata et al., 2004). Arracacia tolucensis is a species from the Apiaceae family and as some members of the family, this species might contain coumarins toxic to the crustacean larvae. In this regard, Ojala et al. (1999) demonstrated the ability of the brine shrimp test as a tool for detecting phototoxic coumarins in plants of the Apiaceae family. Further work is in progress to demonstrate this hypothesis. The ndings of this study corroborated the need for safety studies on plants extensively used for primary health care in Mexico. Such studies must be carried out before continued widespread use of some species provokes long term and irreversible damages. The use of Gnaphalium should be regulated by health authorities, since many herbal preparations contain organic extract made up with the crude drug. In the case of Valeriana procera it should strongly recommended the use of tinctures or extracts free of valepotriates. Chronic, carcinogenic, cytotoxic, neurotoxicity and teratogenic studies must be accomplished with Poliomintha longiora, Exostema caribaeum, Hippocratea excelsa, and Ligusticum porteri. Also, the toxic compounds of these species must be determined. Acknowledgements This research was supported by a CONACyT grant (C01-018) and DGAPA (IN212005). Myrna D ciga-Campos is a DGAPA e postdoctoral fellow. References
Aguilar, M.I., Delgado, G., 1995. Novel bisabolene glycoside and other constituents from the root of the medicinal plant Iostephane heterophylla (Asteraceae). Natatural Product Letters 7, 155162.

CH2 Cl2 MeOH Valeriana extract 31 253.2 13.7 62 270.5 12.3 125 319.4 22.1 250 309.4 27.1 500 323.2 36.3
a

MI: mutagenic index; positive controls: CP (500 g /plate) for S9 and +S9 TA100 strain. Each value means the average of nine replicates on three independent studies. Italicised values mean positive controls. The results are reported as means S.D. * P < 0.05 respect to the control treated with DMSO.

The initial increment of revertants provoked by the crude CH2 Cl2 MeOH (1:1) extract of Valeriana procera could be due to the presence valepotriates which are soluble in this system (Stahl and Schuetz, 1980). Indeed, Andreatini and Leite (1994) found that the valepotriates (notably valtrate and dihydrovaltrate) developed mutagenic activity in the Salmonella/microsome test and the SOS-chromotest in the presence of S9 mix. In addition, Von der Hude et al. (1986) found that the decomposition products of valepotriates, namely baldrinal and homobaldrinal, showed mutagenic effects in the same tests with and without metabolic activation with the S9 fraction. In addition, the valepotriates (Bos et al., 1998) are cytotoxic on several cell lines due to their ability to interact with thiolcontaining enzymes. This effect, in turn, has been attributed to the alkylating properties of the epoxide group present in most of this type of compounds (Houghton, 1988). More recently, it has been also reported that a dicloromethane extract of Valeriana procera (560 g/mL) generated DNA damage in human endothelial cell line ECV304 through epigenetic mechanisms, i.e., reactive oxygen species mediated oxidative DNA damage (Hui-lian et al., 2003). To nd out if the mutagenic effect of Valeriana procera could be due to the presence of valepotriates, a tincture of the plant was assayed using the standard Ames test at the concentrations

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