**3. Chemical constituents**

298 Toxicity and Drug Testing

(Costa-Lotufo et al., 2002); it also induces DNA breaks, cytogenetic abnormalities in human peripheral blood leukocytes, and positive genotoxic effects in the liver, kidney and spleen of mice (Cavalcanti et al., 2010). In addition, kaurenoic acid has been shown dose-dependent

Isolated coumarin has been shown to be carcinogenic, especially in the liver and lungs of rats and mice (Lake, 1999). With long exposure, this substance may change biochemical and hematological parameters and cause ulcers and necrosis, fibrosis, and cytologic alterations in the liver (National Toxicology Program, 1993b). In humans, the majority of tests for mutagenicity and genotoxicity suggest that coumarin is not toxic. This low toxicity is attributed to the mechanism of the detoxification of coumarin, which occurs via the 7 hydroxylation pathway in humans. In rats and mice, the main route is by 3,4-epoxidation,

Considering that the toxic and therapeutic effects of these metabolites are dose dependent, understanding their mechanisms and scientific advances is a key point to validate their therapeutic indications without putting human health at risk. This chapter describes the scientific aspects of guaco, especially the pre-clinical and clinical studies, with a particular emphasis on the pharmacological and toxicological effects of the extracts, preparations and

**Keywords:** *Mikania laevigata, Mikania glomerata*, guaco, toxicity, pharmacological effects,

*Mikania glomerata* Sprengel and *M*. *laevigata* Schultz Bip. ex Baker, commonly known as guaco, are medicinal species used to treat several inflammatory and allergic conditions, particularly in the respiratory system due to their bronchodilator properties (Gasparetto et

Both species grow in the same regions and have similar morphological characteristics, which make them hard to distinguish. The leaves are similar, and both species have the characteristic odor of coumarin. The main difference between the species is the flowering period, which occurs in January for *Mikania glomerata* and September for *M. laevigata*. Therefore, humans use these plants without distinction (Lima, 2003; Ritter & Miotto, 2005). An similar chemical profile has also been described for these plants (Oliveira, 1986; Lima & Biasi, 2002). Therefore, detailed studies of their morphological and anatomical features are necessary to allow botanical identification and quality control of these medicinal species in

In folk medicine, these plants have a long history of use by rainforest inhabitants, especially by native peoples in South American, who have an ancient tradition of using guaco for the treatment of several diseases. Amazonian tribes have used the crushed leaf topically on skin eruptions and on snakebites. They also consume teas made from the leaves and/or stems against snake venom and to cure fevers, stomach disorders and rheumatism. South American tribes also believe that the aroma of the freshly crushed leaves left around

In recent decades, guaco has been used as a home and commercial remedy. In popular medicine, the leaves have been widely used due to their tonic, antipyretic, balsamic, antiophidic, appetite stimulant, neuralgia, antispasmodic, expectorant, and antimalarial properties and for the treatment of rheumatism, eczema, influenza, asthma and sore throat.

genotoxicity in Chinese hamster lung fibroblast cells (Cavalcanti et al., 2006).

resulting in the formation of toxic metabolites (Lake, 1999).

review, coumarin, *o*-coumaric acid and kaurenoic acid.

the absence of another way to make the distinction.

sleeping areas keeps snakes away (Napimoga & Yatsuda, 2010).

isolated metabolites.

**2. General overview** 

al., 2010).

Numerous studies have been conducted to evaluate the chemical composition of guaco species. Detailed screenings revealed the presence of alcohols, acids, esters, aldehydes, organic esters, terpenes, diterpenes, triterpenes and steroids, among other metabolites; some of them are associated with the therapeutic effects of guaco (Gasparetto et al., 2010).

A wide variation in metabolite content has been observed among different extracts and pharmaceutical preparations (Gasparetto et al., 2011). In fact, the geographic origins, agronomic aspects, extraction solvent and extraction techniques have been described as crucial factors to obtain a desirable substance. Thus, to maximize the yield of any metabolite and to standardize the extracts, these aspects must be considered (Gasparetto et al., 2010).

In the essential oil of guaco, a variety of compounds have been found, including α-acorenol, α-cadinol, α-copaene, α-humulene, α-muurolol, α-pinene, α-terpinol, β-pinene, β-farnesene, β-bourbonene, β-cubebene, β-elemene, β-caryophyllene, γ- elemene, (E)-β-ocimene, (E) nerolidol, *p*-cymene, α, β, γ and Δ cardinene, α and TAU- caudynol, epi-α-bisabolol, epi-αmuurolol, aromadendrene, bicyclogermacrene, caryophyllene oxid, citronellyl acetate, coumarin, cubebene, elemol, germacrene-B, germacrene-D, globulol, limonene, linalol, myrcene, nerolidol E, nonanal, sabinene, silvestrene, spathulenol, terpin 4-ol, *trans*-ocymene, *trans*-cariophyllene and 1,4-dimethoxybenzene (Radunz, 2004; Duarte et al., 2005; Rehder et al., 2006).

In hexanic and dichloromethane extracts, the presence of coumarin, *o*-coumaric acid, campesterol, kaurenoic acid, grandiforic acid, stigmasterol, lupeol, lupeol acetate, germacrene, sesquiterpenes, 11-methylbutanoic acid, *ent*-15β-benzoyloxykaur-16(17)-en-19 oic acid, 17-hydroxy-*ent*-kaur-15(16)-en-19-oic acid, β-sitosterol and peroxides has been described (Oliveira et al., 1984; Vilegas et al., 1997a; Vilegas et al., 1997b; Santos et al., 1999; Veneziani et al., 1999; Cabral et al., 2001; Schenkel et al., 2002; Contini et al., 2006).

Hydroalcoholic extracts are the most common preparations that have been commercialized for therapeutic purposes, and the majority of phytochemical assays that have been conducted have been to evaluate their chemical compositions. Thus, using different analytical procedures, the presence of a large number of compounds has been described, including stigmasterol, phytol, 1-ethoxy-1-phenylethanol, 4-hydroxy-3,5 dimethoxybenzaldehyde, hexanoic acid, ethyl hexadecanoate, ethyl linoleoate, kaurenol, an isomer of kaurenoic acid, spathulenol, hexadecanoic acid, 9,12,15-octadecatrienoic acid, cupressenic acid, isopropiloxigrandifloric acid, 2-5-ciclohexadiene-1,4-dione,2,6-bis, 1 octadecene, octadecanoic acid, ester diterpenic, caryophyllene oxide, 10,13-octadecadienoic acid, isobutiloxigrandifloric acid, *trans*-cariofileno, 8,11-octadecadienoic acid, lupeol, lupeol

*Mikania glomerata* and *M. laevigata*: Clinical and Toxicological Advances 301

The tea of guaco leaves, administered orally in mice, had analgesic and anti-inflammatory activities following the intra-peritoneal administration of 0.1 N acetic acid or the intravenous administration of 0.2 mL Evans blue dye solution. The number of contortions was measured, and after 30 min of acid administration, a reduction of 63% was reached following oral administration of 10 mg/kg of the extract. The inhibition of dye diffusion to the peritoneal cavity was 49%, indicating an anti-inflammatory activity, but this result was

The hydroalcoholic extract also affected the inflammatory and oxidative stress caused by a single coal dust intratracheal instillation in rat. Histopathological analyses revealed that animals pretreated subcutaneously with the hydroalcoholic extract (100 mg/kg) had a reduction in lung inflammation, with an additional decrease in protein thiol levels, suggesting that guaco has an important protective effect on the oxidation of thiol groups

With regard to the antiedema activity of guaco, *in vivo* studies conducted in rats treated orally with an extract made from leaves (400 mg/kg) showed a complete reduction in the paw edema induced by carrageenan. A 28.26% decrease in leukocyte migration at the lesion site was also observed (Suyenaga et al., 2002). In mice, the subcutaneous administration of the same extract (3 mg/kg) significantly reduced the vascular permeability and leukocyte adhesion to inflammed tissues with carrageenan-induced peritonitis. The antiedema activity of guaco species has been associated with the inhibition of the pro-inflammatory cytokine

The ability of guaco to decrease ulcerative lesions was also tested by treating rats orally with 1000 mg/kg crude hydroalcoholic extract. A 50% decrease in the ulcerative lesions produced by reserpine was achieved, with higher levels of reduction in lesions caused by hypothermic restraint stress (82%), indomethacin (85%) and ethanol (93%). The antisecretory mechanism was confirmed by measuring acid hypersecretion induced by histamine, pentagastrin and bethanechol. Duodenal administration of the hydroalcoholic extract inhibited only the gastric acid secretion induced by bethanechol, a selective agonist of the muscarinic receptors

The dichloromethane fraction obtained from the ethanolic extract was evaluated in rats for its anti-allergic and anti-inflammatory properties on ovalbumin-induced allergic pleurisy and in models of local inflammation induced by biogenic amines, carrageenan and Platelet-Activating Factor (PAF). The subcutaneous injection of 100 mg/kg of the dichloromethane fraction significantly reduced the plasma exudation, leukocyte infiltration and PAF. Because the pre-treatment of the animals did not alter the pleurisy induced by histamine, serotonin or carrageenan, the fraction was considered effective only for inhibiting immunologic inflammation and not the acute inflammatory response caused by other agents (Fierro et al.,

Guaco also has antidiarrheal effects by decreasing the propulsive movements of the intestinal contents in mice. The percentage distances of the small intestine (from the pylorus to the ceccum) traveled by the charcoal plug were determined. Oral administration of aqueous guaco extract (1000 mg/mL) produced a significant reduction in the distance of the charcoal marker in the animal feces (66.99 ±10.60%). This extract was considered to give an excellent outcome because the reduction was as effective as that produced by loperamide

The antiparasitic effects of lyophilized hydroalcoholic extracts on the growth of *Leishmania amazonensis* and *Trypanosoma cruzi* were also established. By inoculating the parasites in

not consistent with the analgesic effects (Ruppelt et al., 1991).

production at the inflammatory site (Alves et al., 2009).

of the parasympathetic nervous system (Bighetti et al., 2005).

(62.34 ±11.21%), a reference antidiarrheal drug (Salgado et al., 2005).

(Freitas et al., 2008).

1999).

acetate, benzoylgrandifloric and cinnamoylgrandifloric acids (Oliveira et al., 1993; Moura et al., 2002; Biavatti et al., 2004; Santos, 2005; Yatsuda et al., 2005; Bertolucci et al., 2008; Alves et al., 2009; Bolina et al., 2009; Muceneeki et al., 2009).

In quantitative terms, the most prevalent metabolites of hydroalcoholic extracts are coumarin (1,2-benzopyrone) (Biavatti et al., 2004; Bueno & Bastos, 2009), *o*-coumaric acid (Santos, 2005), dihydrocoumarin (Alves et al., 2009), syringaldehyde (Muceneeki *et al.*, 2009) and kaurenoic acid (Vilegas et al., 1997a; Vilegas et al., 1997b; Yatsuda et al., 2005; Bertolucci et al., 2008). These substances have been associated with the therapeutic effects of guaco because they have anti-inflammatory and bronchodilator properties. The chemical structures of each compound are shown in Figure 1:

Fig. 1. Chemical structures of the main substances associated with the therapeutic effects of guaco. Data: (A) coumarin, (B) *o*-coumaric acid, (C) kaurenoic acid, (D) syringaldehyde and (E) dihydrocoumarin.

#### **4. Pre-clinical and clinical trials**

In addition to the use of guaco in popular medicine, pre-clinical studies have justified the main therapeutic uses of guaco species. Aqueous extracts prepared from several plant parts efficiently inhibit the different toxic, pharmacological, and enzymatic effects induced by the venom of *Bothrops* and *Crotalus* snakes. For example, guaco root extracts reduced the hemorrhage zone stimulated by the intradermal injection of *Bothrops* venom by 80% in rats. This result suggests that there is an interaction between the components of guaco and metalloproteases involving the catalytic sites of these enzymes or essential metal ions, thereby inhibiting their hemorrhagic activities (Maiorano et al., 2005).

Guaco extracts have also been considered to be powerful inhibitors of clotting activity, probably due to the interaction with thrombin-like enzymes. Guaco leaves and stems significantly diminished the coagulant activity induced by C*rotalus* and *Bothrops* venoms, especially the root extract, which led to clotting times of more than 45 min. Root extracts (1:50 *w/w*) also neutralized the edema caused by *Crotalus durissus terrificus* venom by 40%, with additional phospholipase A2 activity inhibition (95%). Nevertheless, no significant inhibition was observed against *Bothrops jararacussu* venom by incubating different ratios of guaco extracts and snake venom (1:50, 1:100 and 1:200 *w/w*) (Maiorano et al., 2005).

acetate, benzoylgrandifloric and cinnamoylgrandifloric acids (Oliveira et al., 1993; Moura et al., 2002; Biavatti et al., 2004; Santos, 2005; Yatsuda et al., 2005; Bertolucci et al., 2008; Alves

In quantitative terms, the most prevalent metabolites of hydroalcoholic extracts are coumarin (1,2-benzopyrone) (Biavatti et al., 2004; Bueno & Bastos, 2009), *o*-coumaric acid (Santos, 2005), dihydrocoumarin (Alves et al., 2009), syringaldehyde (Muceneeki *et al.*, 2009) and kaurenoic acid (Vilegas et al., 1997a; Vilegas et al., 1997b; Yatsuda et al., 2005; Bertolucci et al., 2008). These substances have been associated with the therapeutic effects of guaco because they have anti-inflammatory and bronchodilator properties. The chemical

Fig. 1. Chemical structures of the main substances associated with the therapeutic effects of guaco. Data: (A) coumarin, (B) *o*-coumaric acid, (C) kaurenoic acid, (D) syringaldehyde and

In addition to the use of guaco in popular medicine, pre-clinical studies have justified the main therapeutic uses of guaco species. Aqueous extracts prepared from several plant parts efficiently inhibit the different toxic, pharmacological, and enzymatic effects induced by the venom of *Bothrops* and *Crotalus* snakes. For example, guaco root extracts reduced the hemorrhage zone stimulated by the intradermal injection of *Bothrops* venom by 80% in rats. This result suggests that there is an interaction between the components of guaco and metalloproteases involving the catalytic sites of these enzymes or essential metal ions,

Guaco extracts have also been considered to be powerful inhibitors of clotting activity, probably due to the interaction with thrombin-like enzymes. Guaco leaves and stems significantly diminished the coagulant activity induced by C*rotalus* and *Bothrops* venoms, especially the root extract, which led to clotting times of more than 45 min. Root extracts (1:50 *w/w*) also neutralized the edema caused by *Crotalus durissus terrificus* venom by 40%, with additional phospholipase A2 activity inhibition (95%). Nevertheless, no significant inhibition was observed against *Bothrops jararacussu* venom by incubating different ratios of

guaco extracts and snake venom (1:50, 1:100 and 1:200 *w/w*) (Maiorano et al., 2005).

thereby inhibiting their hemorrhagic activities (Maiorano et al., 2005).

et al., 2009; Bolina et al., 2009; Muceneeki et al., 2009).

structures of each compound are shown in Figure 1:

(E) dihydrocoumarin.

**4. Pre-clinical and clinical trials** 

The tea of guaco leaves, administered orally in mice, had analgesic and anti-inflammatory activities following the intra-peritoneal administration of 0.1 N acetic acid or the intravenous administration of 0.2 mL Evans blue dye solution. The number of contortions was measured, and after 30 min of acid administration, a reduction of 63% was reached following oral administration of 10 mg/kg of the extract. The inhibition of dye diffusion to the peritoneal cavity was 49%, indicating an anti-inflammatory activity, but this result was not consistent with the analgesic effects (Ruppelt et al., 1991).

The hydroalcoholic extract also affected the inflammatory and oxidative stress caused by a single coal dust intratracheal instillation in rat. Histopathological analyses revealed that animals pretreated subcutaneously with the hydroalcoholic extract (100 mg/kg) had a reduction in lung inflammation, with an additional decrease in protein thiol levels, suggesting that guaco has an important protective effect on the oxidation of thiol groups (Freitas et al., 2008).

With regard to the antiedema activity of guaco, *in vivo* studies conducted in rats treated orally with an extract made from leaves (400 mg/kg) showed a complete reduction in the paw edema induced by carrageenan. A 28.26% decrease in leukocyte migration at the lesion site was also observed (Suyenaga et al., 2002). In mice, the subcutaneous administration of the same extract (3 mg/kg) significantly reduced the vascular permeability and leukocyte adhesion to inflammed tissues with carrageenan-induced peritonitis. The antiedema activity of guaco species has been associated with the inhibition of the pro-inflammatory cytokine production at the inflammatory site (Alves et al., 2009).

The ability of guaco to decrease ulcerative lesions was also tested by treating rats orally with 1000 mg/kg crude hydroalcoholic extract. A 50% decrease in the ulcerative lesions produced by reserpine was achieved, with higher levels of reduction in lesions caused by hypothermic restraint stress (82%), indomethacin (85%) and ethanol (93%). The antisecretory mechanism was confirmed by measuring acid hypersecretion induced by histamine, pentagastrin and bethanechol. Duodenal administration of the hydroalcoholic extract inhibited only the gastric acid secretion induced by bethanechol, a selective agonist of the muscarinic receptors of the parasympathetic nervous system (Bighetti et al., 2005).

The dichloromethane fraction obtained from the ethanolic extract was evaluated in rats for its anti-allergic and anti-inflammatory properties on ovalbumin-induced allergic pleurisy and in models of local inflammation induced by biogenic amines, carrageenan and Platelet-Activating Factor (PAF). The subcutaneous injection of 100 mg/kg of the dichloromethane fraction significantly reduced the plasma exudation, leukocyte infiltration and PAF. Because the pre-treatment of the animals did not alter the pleurisy induced by histamine, serotonin or carrageenan, the fraction was considered effective only for inhibiting immunologic inflammation and not the acute inflammatory response caused by other agents (Fierro et al., 1999).

Guaco also has antidiarrheal effects by decreasing the propulsive movements of the intestinal contents in mice. The percentage distances of the small intestine (from the pylorus to the ceccum) traveled by the charcoal plug were determined. Oral administration of aqueous guaco extract (1000 mg/mL) produced a significant reduction in the distance of the charcoal marker in the animal feces (66.99 ±10.60%). This extract was considered to give an excellent outcome because the reduction was as effective as that produced by loperamide (62.34 ±11.21%), a reference antidiarrheal drug (Salgado et al., 2005).

The antiparasitic effects of lyophilized hydroalcoholic extracts on the growth of *Leishmania amazonensis* and *Trypanosoma cruzi* were also established. By inoculating the parasites in

*Mikania glomerata* and *M. laevigata*: Clinical and Toxicological Advances 303

However, no antimicrobial action has been reported against Gram negative bacteria such as

Using the microculture tetrazolium assay (MTT), it was shown that 78 μM kaurenoic acid led to a 95% growth inhibition of CEM leukemic cells and a 45% growth inhibition of MCF-7 breast and HCT-8 colon cancer cells (Costa-Lotufo et al., 2002). In experiments conducted by the trypan blue dye-exclusion method, 70 µM kaurenoic acid reduced the viability of MCF7 and SKBR3 cells by 40% and 25%, respectively. However, resistance to treatment was observed in the HB4A cell line, demonstrating a selective activity in cancerous cells (Peria et

Kaurenoic acid also contributes to the anti-inflammatory activity of guaco. To determine this effect, lipopolysaccharide (LPS)-induced RAW264.7 macrophages were treated with different concentrations of kaurenoic acid, and a dose-dependent inhibition of nitric oxide production (IC50 = 51.73 μM) and prostaglandin E2 release (IC50 = 106.09 μM) was observed. A reduction in the protein levels of COX-2 and the expression of inducible nitric oxide synthase was also seen. Additionally, kaurenoic acid dose-dependently inhibited the LPSinduced activation of the NF-kB mediator as assayed by electrophoretic mobility shift assay (EMSA), and it almost abolished the binding affinity of NF-kB for at 100.0 μM (Choi et al.,

The anti-inflammatory effect of kaurenoic acid on acetic acid-induced colitis in rats has also been proven. Colitis was induced by intracolonic instillation of 2 ml of a 4% (*v/v*) acetic acid solution; 24 h later, the colonic mucosal damage was analyzed microscopically for the severity of mucosal damage, myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels in the colon segments. A significant reduction in the gross damage score (52% and 42%) and wet weight of damaged colon tissue (39% and 32%) were observed in rats that received 100 mg/kg kaurenoic acid by rectal and oral routes, respectively. This effect was confirmed biochemically by a two- to three-fold reduction of the colitis-associated increase in MPO activity, a marker of neutrophilic infiltration, and by a marked decrease in the level of MDA, an indicator of lipoperoxidation in colon tissue. Furthermore, light microscopy revealed a marked decrease of inflammatory cell infiltration and submucosal edema formation in the colon segments of rats treated with kaurenoic acid (Paiva et al., 2002). The *in vivo* anti-inflammatory effect of 50 mg/kg kaurenoic acid was examined in carrageenan-induced paw edema in mice. Kaurenoic acid dose-dependently reduced paw swelling up to 34.4% 5 h post-induction, demonstrating inhibition in an acute inflammation model. Taken together, the action of kaurenoic acid on COX-2 and inducible nitric oxide synthase expression is one of the mechanisms responsible for its anti-inflammatory

At 160 μM, kaurenoic acid significantly decreased the contraction of rat uterine muscle precontracted with oxytocin (*E*max = 83%) and acetylcholine (*E*max = 91%) (Cunha et al., 2003). At 10 μM and above, kaurenoic acid also had concentration-dependent activity on vascular smooth muscle (endothelium-intact or denuded rat aortic rings) precontracted with phenylephrine and potassium chloride (Tirapelli et al., 2002, 2004). The mechanism of the vasorelaxant action involves the block of extracellular Ca2+ influx, but it is partly mediated by the activation of the nitric oxide cyclic GMP pathway and the opening of K+ channels sensitive to charybdotoxin and 4-aminopyridine. Activation of the endothelial and neuronal nitric oxide synthase isoforms is also required for the relaxant effect induced by kaurenoic

*E. coli* and *P. aeruginosa* (Silva et al., 2002; Zgoda-Pols et al., 2002).

al., 2010).

2011).

acid.

properties (Choi et al., 2011).

medium containing 100 μg/mL of the extract, approximately 50% growth inhibition was observed for the *Trypanosoma* epimastigote and *Leishmania* promastigote forms. Additionally, under the tested concentration, a nearly complete reduction was achieved for the *Leishmania* amastigote form (97.5 ± 2.6%) (Luize et al., 2005).

Different guaco extracts were also tested for their antimicrobial properties. Using the minimal inhibitory concentration (MIC) assay, the essential oil obtained from guaco leaves had only limited action (MIC values from 300 to >1000 μg/mL) against *Candida albicans* and different serotypes of *Escherichia coli* (Duarte et al., 2005, 2007). Lyophilized hydroalcoholic extracts showed some degree of antibacterial activity, with MIC values of 500 μg/mL for *Staphylococcus aureus,* 250 μg/mL for *Bacillus subtilis,* 500 μg/mL for *Escherichia coli,* >1000 μg/mL for *Pseudomonas aeruginosa,* 500 μg/mL for *Candida krusei* and *C. tropicalis*, and >1000 μg/mL for *C. albicans* and *C. parapsilosis* (Holetz et al., 2002).

The antimicrobial activities of the hexane, ethanolic and ethyl acetate fractions from the ethanolic extract of both guaco species were also evaluated by the MIC and minimum bactericidal concentration (MBC) assays. Negligible activity was observed using ethyl acetate fractions against strains of *Streptococcus mutans, S. cricetus and S. sobrinus*. The ethanolic extract fraction had moderate activity (MIC and MBC values from 25 to > 800 μg/mL) against different strains of *S. cricetus*, *S. sobrinus* and *S. mutans* but no bactericidal activity (MIC and MBC values > 800 μg/mL) against *S. mutans* D1 and P6 strains. Only the hexane fraction showed remarkable antibacterial activity, having the lowest MIC (12.5–100 mg/ml) and MBC (12.5–400 mg/ml) values (Yatsuda et al., 2005).

Regarding the use of guaco for the treatment of respiratory diseases, *in vitro* studies revealed that the hydroalcoholic extract produced dose-dependent relaxation in denuded and intact rat epithelium tracheal precontracted with acetylcholine, with a median effective concentration (EC50) of 1400 μg/mL and a maximum effect (Emax) of 95%. The mechanism of relaxation has also been established, leading to the conclusion that the antispasmodic activity of guaco does not depend on epithelium-derived substances but instead involves changes in the cellular mobilization of calcium (Graça et al., 2007a). A dose-dependent relaxation was also observed in human bronchi precontracted with potassium, with a median inhibitory concentration (IC50) of 0.34 mg/mL, supporting the indication that guaco is effective for the treatment of respiratory diseases in which bronchoconstriction is present (Moura et al., 2002).

In addition to guaco extracts, isolated compounds, especially the main metabolites, also have substantial pharmacological effects. Studies conducted in a mouse model of allergy pneumonitis recognized that both coumarin and *o*-coumaric acid are part of the phytocomplex responsible for the therapeutic activities of guaco species because a reduction in the influx of total leukocytes and eosinophils in lung tissue was observed upon treatment with these substances. Anti-inflammatory and antioxidant properties have been described for dihydrocoumarin, reported to be one of the major compounds in hydroalcoholic extracts. Syringaldehyde has been shown to have a moderate antioxidant activity (Bortolomeazzi et al., 2007) and a dose-dependent inhibition of cyclooxygenase-2 (COX-2) activity (IC50 = 3.5 μg/mL), thereby contributing to the anti-inflammatory properties of guaco extracts (Farah & Samuelsson, 1992; Stanikunaite et al., 2009).

Kaurenoic acid (*ent*-kaur-16-en-19-oic acid) has lately been of considerable interest relating to the pharmacological activities of guaco species. At a concentration of 0.69 mg/mL, it has *in vitro* activity against trypomastigote forms of *T. cruzi*. It also has a moderate antimicrobial activity against strains of *S. aureus*, *S. epidermidis, Mycobacterium smegmatis* and *B. cereus*.

medium containing 100 μg/mL of the extract, approximately 50% growth inhibition was observed for the *Trypanosoma* epimastigote and *Leishmania* promastigote forms. Additionally, under the tested concentration, a nearly complete reduction was achieved for

Different guaco extracts were also tested for their antimicrobial properties. Using the minimal inhibitory concentration (MIC) assay, the essential oil obtained from guaco leaves had only limited action (MIC values from 300 to >1000 μg/mL) against *Candida albicans* and different serotypes of *Escherichia coli* (Duarte et al., 2005, 2007). Lyophilized hydroalcoholic extracts showed some degree of antibacterial activity, with MIC values of 500 μg/mL for *Staphylococcus aureus,* 250 μg/mL for *Bacillus subtilis,* 500 μg/mL for *Escherichia coli,* >1000 μg/mL for *Pseudomonas aeruginosa,* 500 μg/mL for *Candida krusei* and *C. tropicalis*, and >1000

The antimicrobial activities of the hexane, ethanolic and ethyl acetate fractions from the ethanolic extract of both guaco species were also evaluated by the MIC and minimum bactericidal concentration (MBC) assays. Negligible activity was observed using ethyl acetate fractions against strains of *Streptococcus mutans, S. cricetus and S. sobrinus*. The ethanolic extract fraction had moderate activity (MIC and MBC values from 25 to > 800 μg/mL) against different strains of *S. cricetus*, *S. sobrinus* and *S. mutans* but no bactericidal activity (MIC and MBC values > 800 μg/mL) against *S. mutans* D1 and P6 strains. Only the hexane fraction showed remarkable antibacterial activity, having the lowest MIC (12.5–100

Regarding the use of guaco for the treatment of respiratory diseases, *in vitro* studies revealed that the hydroalcoholic extract produced dose-dependent relaxation in denuded and intact rat epithelium tracheal precontracted with acetylcholine, with a median effective concentration (EC50) of 1400 μg/mL and a maximum effect (Emax) of 95%. The mechanism of relaxation has also been established, leading to the conclusion that the antispasmodic activity of guaco does not depend on epithelium-derived substances but instead involves changes in the cellular mobilization of calcium (Graça et al., 2007a). A dose-dependent relaxation was also observed in human bronchi precontracted with potassium, with a median inhibitory concentration (IC50) of 0.34 mg/mL, supporting the indication that guaco is effective for the treatment of respiratory diseases in which bronchoconstriction is present

In addition to guaco extracts, isolated compounds, especially the main metabolites, also have substantial pharmacological effects. Studies conducted in a mouse model of allergy pneumonitis recognized that both coumarin and *o*-coumaric acid are part of the phytocomplex responsible for the therapeutic activities of guaco species because a reduction in the influx of total leukocytes and eosinophils in lung tissue was observed upon treatment with these substances. Anti-inflammatory and antioxidant properties have been described for dihydrocoumarin, reported to be one of the major compounds in hydroalcoholic extracts. Syringaldehyde has been shown to have a moderate antioxidant activity (Bortolomeazzi et al., 2007) and a dose-dependent inhibition of cyclooxygenase-2 (COX-2) activity (IC50 = 3.5 μg/mL), thereby contributing to the anti-inflammatory properties of guaco extracts (Farah

Kaurenoic acid (*ent*-kaur-16-en-19-oic acid) has lately been of considerable interest relating to the pharmacological activities of guaco species. At a concentration of 0.69 mg/mL, it has *in vitro* activity against trypomastigote forms of *T. cruzi*. It also has a moderate antimicrobial activity against strains of *S. aureus*, *S. epidermidis, Mycobacterium smegmatis* and *B. cereus*.

the *Leishmania* amastigote form (97.5 ± 2.6%) (Luize et al., 2005).

μg/mL for *C. albicans* and *C. parapsilosis* (Holetz et al., 2002).

mg/ml) and MBC (12.5–400 mg/ml) values (Yatsuda et al., 2005).

(Moura et al., 2002).

& Samuelsson, 1992; Stanikunaite et al., 2009).

However, no antimicrobial action has been reported against Gram negative bacteria such as *E. coli* and *P. aeruginosa* (Silva et al., 2002; Zgoda-Pols et al., 2002).

Using the microculture tetrazolium assay (MTT), it was shown that 78 μM kaurenoic acid led to a 95% growth inhibition of CEM leukemic cells and a 45% growth inhibition of MCF-7 breast and HCT-8 colon cancer cells (Costa-Lotufo et al., 2002). In experiments conducted by the trypan blue dye-exclusion method, 70 µM kaurenoic acid reduced the viability of MCF7 and SKBR3 cells by 40% and 25%, respectively. However, resistance to treatment was observed in the HB4A cell line, demonstrating a selective activity in cancerous cells (Peria et al., 2010).

Kaurenoic acid also contributes to the anti-inflammatory activity of guaco. To determine this effect, lipopolysaccharide (LPS)-induced RAW264.7 macrophages were treated with different concentrations of kaurenoic acid, and a dose-dependent inhibition of nitric oxide production (IC50 = 51.73 μM) and prostaglandin E2 release (IC50 = 106.09 μM) was observed. A reduction in the protein levels of COX-2 and the expression of inducible nitric oxide synthase was also seen. Additionally, kaurenoic acid dose-dependently inhibited the LPSinduced activation of the NF-kB mediator as assayed by electrophoretic mobility shift assay (EMSA), and it almost abolished the binding affinity of NF-kB for at 100.0 μM (Choi et al., 2011).

The anti-inflammatory effect of kaurenoic acid on acetic acid-induced colitis in rats has also been proven. Colitis was induced by intracolonic instillation of 2 ml of a 4% (*v/v*) acetic acid solution; 24 h later, the colonic mucosal damage was analyzed microscopically for the severity of mucosal damage, myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels in the colon segments. A significant reduction in the gross damage score (52% and 42%) and wet weight of damaged colon tissue (39% and 32%) were observed in rats that received 100 mg/kg kaurenoic acid by rectal and oral routes, respectively. This effect was confirmed biochemically by a two- to three-fold reduction of the colitis-associated increase in MPO activity, a marker of neutrophilic infiltration, and by a marked decrease in the level of MDA, an indicator of lipoperoxidation in colon tissue. Furthermore, light microscopy revealed a marked decrease of inflammatory cell infiltration and submucosal edema formation in the colon segments of rats treated with kaurenoic acid (Paiva et al., 2002).

The *in vivo* anti-inflammatory effect of 50 mg/kg kaurenoic acid was examined in carrageenan-induced paw edema in mice. Kaurenoic acid dose-dependently reduced paw swelling up to 34.4% 5 h post-induction, demonstrating inhibition in an acute inflammation model. Taken together, the action of kaurenoic acid on COX-2 and inducible nitric oxide synthase expression is one of the mechanisms responsible for its anti-inflammatory properties (Choi et al., 2011).

At 160 μM, kaurenoic acid significantly decreased the contraction of rat uterine muscle precontracted with oxytocin (*E*max = 83%) and acetylcholine (*E*max = 91%) (Cunha et al., 2003). At 10 μM and above, kaurenoic acid also had concentration-dependent activity on vascular smooth muscle (endothelium-intact or denuded rat aortic rings) precontracted with phenylephrine and potassium chloride (Tirapelli et al., 2002, 2004). The mechanism of the vasorelaxant action involves the block of extracellular Ca2+ influx, but it is partly mediated by the activation of the nitric oxide cyclic GMP pathway and the opening of K+ channels sensitive to charybdotoxin and 4-aminopyridine. Activation of the endothelial and neuronal nitric oxide synthase isoforms is also required for the relaxant effect induced by kaurenoic acid.

*Mikania glomerata* and *M. laevigata*: Clinical and Toxicological Advances 305

Following oral administration, coumarin is rapidly absorbed from the gastrointestinal tract and distributed throughout the body (Egan et al., 1990; O´Kennedy & Thornes, 1997). The quick absorption is related to its non-polar characteristics and high partition coefficient (21.5%), which are considered favorable for rapid absorption, suggesting that coumarin

In systemic circulation, only 2 to 6% of coumarin molecules remain intact (Ritschel et al., 1979). In the liver, coumarin is converted to 7-hydroxycoumarin by a specific cytochrome P-450-linked mono-oxygenase enzyme (CYP2A6). Then, 7-hydroxycoumarin undergoes a phase II reaction, a glucuronide conjugation, that results in the formation of 7 hydroxycoumarin glucuronide, which is subsequently eliminated in the urine (O´Kennedy

In addition to 7-hydroxycoumarin, the formation of other metabolites is possible, and the metabolic pathways are species-specific. Thus, coumarin may be hydroxylated at one of the other five possible positions, carbons 3, 4, 5, 6, and 8, to yield 3-, 4-, 5-, 6- and 8 hydroxycoumarin, respectively. In addition, the lactone ring can also open and lead to the formation of a variety of metabolites, including *o*-hydroxyphenylacetaldehyde, *o*hydroxyphenylethanol, *o*-hydroxyphenylacetic acid and *o*-hydroxyphenylacetic acid. The formation of 6,7-dihydroxycoumarin, *o*-coumaric acid, *o*-hydroxyphenylpropionic acid and

In humans, the half-life of intravenously administered coumarin can vary slightly according to the dosage (Ritschel et al., 1976), but its metabolism is usually fast. The low availability along with the short half-life (1.02 hrs peroneal vs. 0.8 hrs intravenous) lead coumarin to be considered as a pro-drug and 7-hydroxycoumarin as the substance with more therapeutic relevance (Lacy & O'Kennedy, 2004). In other species, the half-life of coumarin can vary

The mechanism of the excretion of coumarin and its metabolites also depends of the species. For example, a large amount of biliary excretion has been described followed by a considerable elimination via feces in rats. In the Syrian hamster, rabbit and baboon, elimination is via urine. In humans, the rapid and total excretion via urine suggests that

Regarding dermal application, coumarin is amply absorbed, distributed and excreted in the urine and feces of humans and rats. Following the applied dose of 0.02 mg/cm2, the total absorption was 60% in humans and 72% in rats with a 6-h exposure. The mean plasma halflife of coumarin and its metabolites was approximately 1.7 h for humans and 5 h for rats. As in oral administration, the dermal application of coumarin resulted in the formation of 7 hydroxycoumarin and excretion in the urine as 7-hydroxycoumarin glucuronide. In rats, at least twenty metabolites were found, but only *o-*hydroxyphenylacetic acid was identified

In summary, the 7-hydroxylation pathway is characteristic for human and a minor route for rat and mouse, which primarily use the 3,4-epoxidation pathway (Lacy & O'Kennedy, 2004). Another possible route in rat, Syrian hamster, gerbil and human is the 3 hydroxylation pathway leading to the formation of 3-hydroxycoumarin (Lake et al., 1992). The 3-hydroxycoumarin is a minor *in vivo* metabolite in rat and human and a major urinary metabolite in rabbit. The possible metabolic pathways of coumarin are shown in

should easily cross the lipid bilayer by passive diffusion (Lacy & O'Kennedy, 2004).

& Thornes, 1997; Wang et al., 2005).

dihydrocoumarin has also been described (Lake, 1999).

there is little or no biliary excretion (Shilling et al., 1969).

(Ford et al., 2001).

Figure 2.

from 1 to 4 hours and is quickly eliminated from systemic circulation.

Although several guaco metabolites have been described as having therapeutic relevance, the simple coumarin (1,2 benzopyrone) has been considered to be the main component, and it has been used for the treatment of various clinical conditions. For example, in Brazil, the daily uptake (0.5–5 mg) of this substance has been assured by regulatory agencies (Brasil, 2008), but the recommended doses for the treatment of several diseases can vary largely according to the therapy (Lacy & O´Kennedy, 2004).

Coumarin is an anticoagulant and antithrombotic agent. It has been widely used in combination with troxerrutine to improve peripheral venous and lymphatic circulation and is also used to reduce the swelling caused by lymphatic and venous vessel problems. Preclinical studies also revealed that coumarin administered to the rat duodenum (100 mg/kg) produces antiulcerogenic activity by inhibiting the acid secretion mediated by the parasympathetic system (Bighetti et al., 2005).

In clinical trials, coumarin had *in vivo* macrophage-derived actions and has been used as an adjuvant in melanoma therapy and for recurrence prevention (Thornes et al., 1994). In carcinoma, coumarin (100 mg/day) in combination with cimetidine (1200 mg/day) led to metastatic reduction without toxic side effects (Thornes et al., 1982). Patients with metastatic prostate cancer were treated with 3 g of coumarin daily, and stable levels of prostate specific antigen (PSA) were maintained for over 7 years (Lacy & O´Kennedy, 2004).

Coumarin also activates macrophages and cells of the immune system (Hoult & Paya, 1996; Lacy & O´Kennedy, 2004). It has also been reported to reduce acute and chronic protein edema. In rodents, coumarin decreases the swelling caused by thermal damage; in humans, a significant reduction of lymphoedema was confirmed through a double-blind trial involving patients with elephantiasis and postmastectomy (Hoult & Paya, 1996).

Coumarin induces a concentration-dependent relaxation in guinea pig trachea precontracted with histamine (EC50 = 35.0 μg/mL) or carbachol (EC50 = 33.4 μg/mL) (Ramanitrahasimbola et al., 2005). However, this effect was not associated with the antispasmodic activity on rat jejunum and ileum cells isolated from guinea pig (Aboy et al., 2002). Coumarin was also less effective in guinea pig trachea (EC50 = 130.8 μg/mL) and endothelium-denuded trachea (EC50 = 153.4 μg/mL) pre-contracted with potassium chloride. When coumarin was combined with theophylline, a significant additive relaxing effect on pre-contracted trachea was observed, and this effect was not blocked by propranolol. These results indicate that the bronchodilator effect of coumarin is partly due to endothelium-dependent tracheal relaxation and also mediated through a non-specific tracheal relaxation (Ramanitrahasimbola et al., 2005).

#### **5. Absorption, distribution, metabolism and excretion of coumarin, the main substance of guaco**

Coumarin (1,2-benzopyrone) is a naturally occurring compound, which is present in a wide variety of plants, micro-organisms and in some animal species (Lake, 1999). In the 1990s, coumarin was widely used as a trial drug in cancer treatment and is still used to improve peripheral venous and lymphatic circulation, to stimulate the proteolytic effect of macrophages, and to treat edema. As a consequence, the metabolism of coumarin, including the excretion of some of its metabolites, has been widely studied in humans and other animal species.

Although several guaco metabolites have been described as having therapeutic relevance, the simple coumarin (1,2 benzopyrone) has been considered to be the main component, and it has been used for the treatment of various clinical conditions. For example, in Brazil, the daily uptake (0.5–5 mg) of this substance has been assured by regulatory agencies (Brasil, 2008), but the recommended doses for the treatment of several diseases can vary largely

Coumarin is an anticoagulant and antithrombotic agent. It has been widely used in combination with troxerrutine to improve peripheral venous and lymphatic circulation and is also used to reduce the swelling caused by lymphatic and venous vessel problems. Preclinical studies also revealed that coumarin administered to the rat duodenum (100 mg/kg) produces antiulcerogenic activity by inhibiting the acid secretion mediated by the

In clinical trials, coumarin had *in vivo* macrophage-derived actions and has been used as an adjuvant in melanoma therapy and for recurrence prevention (Thornes et al., 1994). In carcinoma, coumarin (100 mg/day) in combination with cimetidine (1200 mg/day) led to metastatic reduction without toxic side effects (Thornes et al., 1982). Patients with metastatic prostate cancer were treated with 3 g of coumarin daily, and stable levels of prostate specific

Coumarin also activates macrophages and cells of the immune system (Hoult & Paya, 1996; Lacy & O´Kennedy, 2004). It has also been reported to reduce acute and chronic protein edema. In rodents, coumarin decreases the swelling caused by thermal damage; in humans, a significant reduction of lymphoedema was confirmed through a double-blind trial

Coumarin induces a concentration-dependent relaxation in guinea pig trachea precontracted with histamine (EC50 = 35.0 μg/mL) or carbachol (EC50 = 33.4 μg/mL) (Ramanitrahasimbola et al., 2005). However, this effect was not associated with the antispasmodic activity on rat jejunum and ileum cells isolated from guinea pig (Aboy et al., 2002). Coumarin was also less effective in guinea pig trachea (EC50 = 130.8 μg/mL) and endothelium-denuded trachea (EC50 = 153.4 μg/mL) pre-contracted with potassium chloride. When coumarin was combined with theophylline, a significant additive relaxing effect on pre-contracted trachea was observed, and this effect was not blocked by propranolol. These results indicate that the bronchodilator effect of coumarin is partly due to endothelium-dependent tracheal relaxation and also mediated through a non-specific

**5. Absorption, distribution, metabolism and excretion of coumarin, the main** 

Coumarin (1,2-benzopyrone) is a naturally occurring compound, which is present in a wide variety of plants, micro-organisms and in some animal species (Lake, 1999). In the 1990s, coumarin was widely used as a trial drug in cancer treatment and is still used to improve peripheral venous and lymphatic circulation, to stimulate the proteolytic effect of macrophages, and to treat edema. As a consequence, the metabolism of coumarin, including the excretion of some of its metabolites, has been widely studied in humans and other

antigen (PSA) were maintained for over 7 years (Lacy & O´Kennedy, 2004).

involving patients with elephantiasis and postmastectomy (Hoult & Paya, 1996).

according to the therapy (Lacy & O´Kennedy, 2004).

parasympathetic system (Bighetti et al., 2005).

tracheal relaxation (Ramanitrahasimbola et al., 2005).

**substance of guaco** 

animal species.

Following oral administration, coumarin is rapidly absorbed from the gastrointestinal tract and distributed throughout the body (Egan et al., 1990; O´Kennedy & Thornes, 1997). The quick absorption is related to its non-polar characteristics and high partition coefficient (21.5%), which are considered favorable for rapid absorption, suggesting that coumarin should easily cross the lipid bilayer by passive diffusion (Lacy & O'Kennedy, 2004).

In systemic circulation, only 2 to 6% of coumarin molecules remain intact (Ritschel et al., 1979). In the liver, coumarin is converted to 7-hydroxycoumarin by a specific cytochrome P-450-linked mono-oxygenase enzyme (CYP2A6). Then, 7-hydroxycoumarin undergoes a phase II reaction, a glucuronide conjugation, that results in the formation of 7 hydroxycoumarin glucuronide, which is subsequently eliminated in the urine (O´Kennedy & Thornes, 1997; Wang et al., 2005).

In addition to 7-hydroxycoumarin, the formation of other metabolites is possible, and the metabolic pathways are species-specific. Thus, coumarin may be hydroxylated at one of the other five possible positions, carbons 3, 4, 5, 6, and 8, to yield 3-, 4-, 5-, 6- and 8 hydroxycoumarin, respectively. In addition, the lactone ring can also open and lead to the formation of a variety of metabolites, including *o*-hydroxyphenylacetaldehyde, *o*hydroxyphenylethanol, *o*-hydroxyphenylacetic acid and *o*-hydroxyphenylacetic acid. The formation of 6,7-dihydroxycoumarin, *o*-coumaric acid, *o*-hydroxyphenylpropionic acid and dihydrocoumarin has also been described (Lake, 1999).

In humans, the half-life of intravenously administered coumarin can vary slightly according to the dosage (Ritschel et al., 1976), but its metabolism is usually fast. The low availability along with the short half-life (1.02 hrs peroneal vs. 0.8 hrs intravenous) lead coumarin to be considered as a pro-drug and 7-hydroxycoumarin as the substance with more therapeutic relevance (Lacy & O'Kennedy, 2004). In other species, the half-life of coumarin can vary from 1 to 4 hours and is quickly eliminated from systemic circulation.

The mechanism of the excretion of coumarin and its metabolites also depends of the species. For example, a large amount of biliary excretion has been described followed by a considerable elimination via feces in rats. In the Syrian hamster, rabbit and baboon, elimination is via urine. In humans, the rapid and total excretion via urine suggests that there is little or no biliary excretion (Shilling et al., 1969).

Regarding dermal application, coumarin is amply absorbed, distributed and excreted in the urine and feces of humans and rats. Following the applied dose of 0.02 mg/cm2, the total absorption was 60% in humans and 72% in rats with a 6-h exposure. The mean plasma halflife of coumarin and its metabolites was approximately 1.7 h for humans and 5 h for rats. As in oral administration, the dermal application of coumarin resulted in the formation of 7 hydroxycoumarin and excretion in the urine as 7-hydroxycoumarin glucuronide. In rats, at least twenty metabolites were found, but only *o-*hydroxyphenylacetic acid was identified (Ford et al., 2001).

In summary, the 7-hydroxylation pathway is characteristic for human and a minor route for rat and mouse, which primarily use the 3,4-epoxidation pathway (Lacy & O'Kennedy, 2004). Another possible route in rat, Syrian hamster, gerbil and human is the 3 hydroxylation pathway leading to the formation of 3-hydroxycoumarin (Lake et al., 1992). The 3-hydroxycoumarin is a minor *in vivo* metabolite in rat and human and a major urinary metabolite in rabbit. The possible metabolic pathways of coumarin are shown in Figure 2.

*Mikania glomerata* and *M. laevigata*: Clinical and Toxicological Advances 307

sodium azide in assays with the TA100 strain, without exogenous metabolism (S9 fraction). A synergistic effect was also observed in frameshift mutagenic events, with direct action in the presence of 4-oxide-1-nitroquinoline and a tendency to a low percentage of action

In contrast to the outcomes from the Salmonella/microsome trials, studies conducted by the comet assay revealed that guaco extracts have deleterious effects. DNA damage was observed in rat hepatoma cells treated with hydroalcoholic maceration (10 and 20 μL/mL) and infusion (20 and 40 μL/mL) of the leaves. The genotoxic potential of the infusion was also observed by the micronucleous test at a very high concentration (40 μL/mL), suggesting a limitation in the phytotherapeutic use of guaco species (Costa et al., 2008). Caution is recommended for patients who use lyophilized extracts or medicines containing isolated compounds, such as coumarin and *o*-coumaric acid. Hemorrhaging lung tissue was observed in mice treated with these substances and with the extract. However, this effect was not observed in animals treated with the whole hydroalcoholic extract, leading to the conclusion that some protective effect of the whole extract can be lost during the

Because guaco showed an effect against *L. amazonensis* and *T. cruzi*, it was important to assess its toxic effects on mammalian host cells to determine the ratio of selectivity to biological activity. For this purpose, a test of cytotoxicity in sheep erythrocytes was performed using hydroalcoholic extracts of leaves at different concentrations and times of incubation. At 100, 500 and 1000 μg/mL, guaco extracts caused, respectively, 25, 50 and 75% hemolysis in erythrocytes incubated at 120 min. However, the hydroalcoholic extract was not considered cytotoxic to sheep erythrocytes because no significant hemolytic effect was

The hydroalcoholic extract did not impair the fertility of rats following 52 days of oral treatment with a chronic dose of 3.3 g/kg of animal. In females, no changes in mating, gestation, preimplantation loss, the number of implanted embryos or offspring, weaning and the implantation and resorption indexes were observed using this kind of extract (SÁ et al., 2006). In males, the treatment did not alter body and organ weights and did not interfere in gamete production, serum testosterone levels or food intake (SÁ et al., 2003). Following 90 days of treatment, no significant change was observed in body and organ weights, gamete concentration on the epididymis cauda, serum testosterone level or food consumption, suggesting the absence of toxicity or antifertility activity of the hydroalcoholic

The absence of any effect on body weight gain or behavioral patterns in mice subjected to a repeated-dose over 14-, 28- or 60-day treatments (3 mg/kg) indicated that the *M. laevigata*  ethanolic extract does not induce significant toxicity. The lack of alterations in hematological parameters, liver cell injury and serum aminotransferases (AST and ALT) was indicative of normal hepatic and biliary function. In addition, there was no change in urea levels, indicating the absence of alterations in the kidney. Additionally, the LD50 was found to be

The potential genotoxicity of the dichloromethane fraction of the hydroalcoholic extract was evaluated on plasmid DNA using an alkaline lysis procedure, in which plasmid DNA was treated with SnCl2 and the *M. glomerata* extract fraction. The role of reactive oxygen species in DNA damage was also evaluated by incubating the extract fraction with sodium benzoate, a hydroxyl radical scavenger. The results showed that the dichloromethane

almost 75-fold higher than the pharmacological dose tested (Alves et al., 2009).

observed at 100 and 500 μg/mL after 60 minutes of incubation (Luize et al., 2005).

enhancement in the presence of the 2AF mutagen (Fernandes & Vargas, 2003).

lyophilization process (Santos et al., 2006).

extract (SÁ et al., 2010).

Fig. 2. Pathways of coumarin metabolism. Dihydrocoumarin (DHC); *o*-hydroxyphenylpropionic acid (o-HPPA); *o*-coumaric acid (o-CA); 3, 4, 5, 6, 7 and 8 hydroxycoumarin (HC); 7-hydroxycoumarin glucoronide (7-HC-GLUC); 6,7 dihydroxycoumarin (6,7-diHC); *o*-hydroxyphenylacetic acid (o-HPLA); o-hydroxyphenylacetic acid (o-HPAA); *o*-hydroxyphenylacetaldehyde (o-HPA); *o*-hydroxyphenylethanol (o-HPE); 4-hydroxydihydrocoumarin-glutathione-conjugated (4- HDHC-GSH).
