**1. Introduction**

296 Toxicity and Drug Testing

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*Mikania laevigata* and *M. glomerata*, commonly known as guaco, are important medicinal plant species used in South America for the treatment of respiratory diseases. In folk medicine, their leaves have ample use due to their balsamic, antiophidic, appetite stimulant, antispasmodic, expectorant, and antimalarial properties, among others (Coimbra, 1942; Lucas, 1942; Neves & Sá, 1991; Alice et al., 1995; Gasparetto et al., 2010; Napimoga & Yatsuda, 2010).

There is also pre-clinical evidence of the anti-inflammatory, anti-allergy, and bronchodilation activities of these species (Fierro et al., 1999; Moura et al., 2002; Suyenaga et al., 2002; Graca et al., 2007a). Due to their important effects, pharmaceutical preparations, including syrup and oral solutions, are freely distributed through various government phytotherapy programs and, thus, are widely used by the population (Gasparetto et al., 2010).

The pharmacological effects of guaco are attributed mainly to the presence of coumarin (1,2 benzopyrone); however, other metabolites have been shown to produce significant pharmacological effects. Studies that have evaluated isolated markers in the mouse model of allergic pneumonitis have demonstrated that coumarin and *o*-coumaric acid are part of the phytocomplex that is responsible for the therapeutic activities of guaco species (Santos et al., 2006). In addition, dihydrocoumarin and syringaldehyde have antioxidant, immunologic and anti-inflammatory properties (Farah & Samuelsson, 1992; Hoult & Paya, 1996; Bortolomeazzi et al., 2007; Stanikunaite et al., 2009; Gu & Xue, 2010). Finally, kaurenoic acid, isolated in high quantities from both species (Fierro et al., 1999; Veneziani et al., 1999; Yatsuda et al., 2005), has been shown to contribute to the effects of guaco through its antimicrobial, antinociceptive, anti-inflammatory and smooth muscle relaxant activities (Block et al., 1998; Costa-Lotufo et al., 2002; Wilkens et al., 2002; Cunha et al., 2003; Cotoras et al., 2004; Tirapelli et al., 2004; Cavalcanti et al., 2006).

The presence of these metabolites is directly related to the benefits of guaco, but studies have shown them to be toxic. Dihydrocoumarin administered to groups of rodents led to carcinogenic activity, ulcers, forestomach inflammation, parathyroid gland hyperplasia and increased nephropathy (National Toxicology Program, 1993a). Kaurenoic acid has been shown to kill sea urchin embryos and to cause hemolysis in mouse and human erythrocytes

*Mikania glomerata* and *M. laevigata*: Clinical and Toxicological Advances 299

Guaco can be used as infusion and decoction, but it is most commonly used in the commercialization of crude extracts for medicinal purposes (Coimbra, 1942; Lucas, 1942; Neves & Sá, 1991; Ruppelt et al., 1991; Galvani & Barreneche, 1994; Alice et al., 1995; Matos,

Because of the therapeutic effects attributed to guaco species, syrups and oral solutions are widely used by the South American population and have been distributed in free government phytotherapy programs (Gasparetto et al., 2010). These preparations have been used as an effective natural bronchodilator, expectorant and cough suppressant in treatment of respiratory problems such as bronchitis, pleurisy, cold, flu, coughs and asthma, and sore

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

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

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;

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

Veneziani et al., 1999; Cabral et al., 2001; Schenkel et al., 2002; Contini et al., 2006).

of them are associated with the therapeutic effects of guaco (Gasparetto et al., 2010).

2000; Souza & Felfili, 2006; Botsaris, 2007).

**3. Chemical constituents** 

al., 2006).

throats, laryngitis and fever (Napimoga & Yatsuda, 2010).

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

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

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 isolated metabolites.

**Keywords:** *Mikania laevigata, Mikania glomerata*, guaco, toxicity, pharmacological effects, review, coumarin, *o*-coumaric acid and kaurenoic acid.
