Author's Personal Copy Montanoa Tomentosa Glandular Trichomes

Author's personal copy

Fitoterapia 80 (2009) 12–17

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Fitoterapia

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Montanoa tomentosa glandular trichomes containing kaurenoic acids chemical proﬁle and distribution

Ramón E. Robles-Zepeda a, Edmundo Lozoya-Gloria b, Mercedes G. López c, María L. Villarreal d, Enrique Ramírez-Chávez c, Jorge Molina-Torres c,⁎ a Departamento de Ciencias Químico Biológicas, Universidad de Sonora, Hermosillo, Sonora, Mexico b Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Irapuato, Guanajuato, Mexico c Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Irapuato, Guanajuato, Mexico d Departamento de Productos Naturales, Centro de Investigación en Biotecnología, UAEM, Cuernavaca, Morelos, Mexico article info abstract

Article history: Montanoa tomentosa has been used in traditional medicine in Mexico to treat diverse female Received 20 August 2008 health disorders; it is particularly useful in inducing childbirth. Microscopic analysis of leaf Accepted in revised form 3 September 2008 surfaces of M. tomentosa revealed the presence of glandular trichomes. The chemical profile Available online 14 September 2008 and distribution of glandular trichomes from different developmental stages of M. tomentosa leaves were investigated. Two diterpenic acids, kaurenoic and grandiflorenic were detected in Keywords: glandular trichomes through the glandular microsampling technique and GC/MS analysis. In Montanoa tomentosa the glandular trichomes of the leaves also up to twenty-six volatile terpenes were identified, Asteraceae β Zoapatle where -eudesmol and valencene were the most abundant terpenes. Glandular trichomes © 2008 Elsevier B.V. All rights reserved. Volatile terpenes Kaurenoic acid Grandiflorenic acid

1. Introduction of their exudates throughout leaf development may have important consequences for plant adaptation to abiotic and The aerial surface of most plants contains trichomes, which biotic factors [6]. can be divided into glandular (GT) and non-glandular (NGT). Montanoa tomentosa Cerv. (Asteraceae), a perennial shrub GTs are secretory structures varying in size, form, location, known as zoapatle or cihuapatli, has been used in traditional and function in plants of different species. Peltate and capitate medicine in Mexico to treat diverse female health disorders glandular trichomes are common in plants [1] and are [7,8], as for example inducing childbirth [9] and some authors constituted of four to eight cells in a single disc [2]. Glandular attribute theses pharmacological properties of M. tomentosa trichomes have a spherical morphology after reaching the to two diterpenic acids: grandiflorenic (GA) [kaura-9 (11), 16- mature stage. Their characteristic morphology develops as a dien-18-oic acid] and kaurenoic (KA) [kaur-16-en-18-oic acid] result of the accumulation of secretory products [3]. They [10]. These diterpenic acids are present in other plants and contain chemical compounds with diverse functions, includ- have been fully chemically characterized previously [11,12] ing defense [4]. Secondary metabolites in trichomes are (Fig. 1). ecologically bioactive compounds of interest as pesticides, Stereomicroscopic observation of the surface of leaves of pharmaceuticals, flavors, and fragrances [5]. Changes in M. tomentosa confirmed the presence of abundant glandular number of trichomes and in composition and concentrations trichomes. In the present work, we studied the distribution and chemical profiles of glandular trichomes of leaves of M. tomentosa at different development stages. Emphasis is ⁎ Corresponding author. Tel.: +52 462 6239 645; fax: +52 462 6245 996. put in the presence of kaurenoic acids, putative bioactive com- E-mail address: [email protected] (J. Molina-Torres). ponents responsible for the ethnomedicinal use of this species.

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2. Experimental from about 700 glands, was dissolved in 500 μl of hexane: ethyl acetate (85:15). The solvent was evaporated to dryness 2.1. Plant under nitrogen and the residue re-suspended in 100 μlof ethanol. Samples were kept at 4 °C for further analysis. M. tomentosa leaves were collected from ﬁeld-grown mature one-year-old plants cultivated at CINVESTAV-IPN, 2.4. Glandular trichomes terpenes content Unidad Irapuato (N 20° 43′ 8.9″, W 101° 19′ 42.6″ altitude 1740 m), Guanajuato, Mexico. Samples were collected form A more general procedure was used to evaluate the September to December after the rainy season. Dr. Jerzy terpenes content of the GTs. First, a surface extraction was Rzedowski from the Instituto de Ecología, Centro Regional del carried out with dichloromethane as previously described Bajío in Pátzcuaro, Michoacán, México performed species [13]. Brieﬂy, each leaf section used for determination of GTS authentication. A voucher specimen (IEB-57689) was depos- density was subsequently submerged in 20 ml dichloro- ited in this Institute. methane for 20 s. Then, the solvent was evaporated under a nitrogen stream and the residue re-suspended in 1 ml of the 2.2. Leaf glandular trichomes distribution same solvent and kept at 4 °C for further analysis.

Leaves were collected in four size ranges: 7 to 7.5 cm, 9 to 2.4.1. Grandiflorenic and kaurenoic acids evaluation 9.5 cm, 14 to 14.5 cm, and 15 to 15.5 cm. These were classified The identification and quantification of these acids was as new, young, mature and old leaves, respectively. Within accomplished as follows: 100 μl of each surface extract was these size ranges, the smaller leaves were found in a higher dissolved in hexane:ethyl acetate (85:15), and the diterpenic position in the plant, whereas the older leaves were found at acids present derivatized adding100 μl of a mixture of BSTFA lower level. In order to evaluate the glandular trichomes (GT) [bis-(trimethyl) sililtrifluoroacetamide from Sigma Co.] and distribution density, each leaf was divided in three zones TMCS (trimethylchlorosilane from Alltech) in a 100:5 v/v characterized as basal, middle and apical. relation and heating for 30 min at 60 °C. The derivatized The evaluation of GTs density in the different leaf zones compounds were analyzed using a gas chromatograph was performed with the aid of a stereomicroscope (Nikon coupled to a mass selective detector (GC-MSD Hewlett- SMZ-2B, Schott KL500 lamp). The average number of GTs was Packard 6890 Series II) equipped with a capillary column calculated. Three replicas were evaluated for each leaf size. HP-1 (30 m long, 0.25 mm internal diameter and 0.25 μm film thickness). Helium was used as the carrier gas, at a 2.3. Glandular trichomes microsampling constant flow rate of 1 ml/min. The injector temperature was maintained at 200 °C. The oven temperature profile was the Three replicas of GTs direct extraction (microsampling) following: 3 min at 150 °C, followed by a temperature were performed with micro capillaries (prepared from 10 μl increment rate of 4 °C/min up to 300 °C, then kept at 300 °C capillary tubes by heat stretching) under the stereomicro- for 20 min. The mass selective detector consisted of a scope at a 100x magnification. The glandular fluid collected quadrupole analyzer with the ionization source set at 70 eV. Scan parameters were: mass range from 30.0 to 550 uam with 5.36 scans per second, and the temperatures of source and quadrupole were 230 °C and 150 °C respectively. The mass spectra identification and calibration curves for these compounds were made by comparison with authenticated standards obtained as a gift from Dr. Raúl Enríquez (Instituto de Química, UNAM, México).

2.4.2. Terpenic compounds profile The GTs dichloromethane extract of basal, middle, and apical zones, from leaves of different sizes were analyzed by GC-MSD. One microliter of each extract was injected onto a GC (Hewlett-Packard 5890 series II) coupled to a mass selective quadrupole detector (Hewlett-Packard/MSD 5972 series) for the characterization of components present in the samples. Compound separation was carried out using a capillary column HP-FFAP (25 m long, 0.32 mm internal diameter and 0.52 μm film thickness from Hewlett-Packard). The carrier gas was helium at a constant flow rate of 1 ml/min. The injector temperature was maintained at 180 °C. The oven temperature program was: 3 min at 40 °C, followed by a temperature increment rate of 3 °C/min up to 120 °C and then at a rate of 5 °C/min up to 200 °C. This temperature was maintained for 13 min. Peaks were identified by comparison Fig. 1. Structure of diterpenic acids in this study: A) Kaurenoic acid; with the NIST 98 mass spectral database. Results are presented B) Grandiflorenic acid. as a percent of the total area produced by the compounds Author's personal copy

14 R.E. Robles-Zepeda et al. / Fitoterapia 80 (2009) 12–17 identiﬁed in each leaf zone. The values presented are the average of three replicas.

3. Results and discussion

3.1. Leaf glandular trichomes distribution

The abaxial surface presented numerous GTs and Non- Glandular trichomes (NGT). On the adaxial surface only NGTs were observed. Based on observations using a stereomicroscope, the GTs were distributed on the leaf lamina, on secondary, tertiary, and quaternary veins with the exception of the main vein, where only NGTs were observed. The GTs have a green-yellowish color and a spherical shape. NGTs in both surfaces were multicellular and erect varying in length. As the GTs were restricted to the abaxial side of the leaf, the results presented here on the density of GTs are exclusively related to the abaxial surface of the leaves. GTs density did not vary significantly between different zones of the new, mature, and old leaves, but in young leaves, a higher GTs density was observed throughout this surface, as shown in the graph presented in Fig. 2. For the apical section of leaves, an average density of 38, 76, 41, and 45 GTs/mm2 was observed in new, young, mature, and old leaves, respectively. A similar pattern was observed for the other two (middle and basal) zones of the leaves evaluated. In young leaves, the GTs density Fig. 3. Montanoa tomentosa: Number of glandular trichomes in relation to the was unexpectedly higher than in any of the other three leaf leaf zone and the whole leaf. A. Number of GT per leaf zone. The bars in each developmental stages evaluated. This behavior was observed group from left to right corresponds to basal ■, middle and apical □ in all zones of young leaves. zones of the leaves at different developmental stages. B. Total number GT per leaf in the abaxial surface of different developmental stages leaves. Results The total amount of trichomes per leaf zone was estimated are the average of three independent counts. and is shown in Fig. 3A. In the apical zone, the average number of GTs was about 10,800 for new leaves, 28,100 for young leaves, 34,100 for mature, and 47,000 for old leaves. Within and old leaves presented an average number of 84,200, the leaf, a significant difference was observed between the 247,200, 403,700, and 477,500 GTs, respectively. basal, middle, and apical zones in all the evaluated leaves. This The presence of glandular and non-glandular trichomes difference was more evident as the leaf developed; thus, in old is common in Asteraceae species [14–16]. The density of leaves the total amount of GTs in the apical zone was 47,000, GTs found in M. tomentosa was similar to other species re- whereas the middle and basal sections averaged to 170,000, ported. The values ranged from 35–75 GT/mm2, depending on and 260,500 respectively (Fig. 3A). the leaf age. These are similar to 30–33 GT/mm2 reported The total amount of GTs on leaves of different develop- for Chrysothamnus nauseous [17] and Canabbis sativa [18], mental stages was calculated (Fig. 3B There was a propor- but clearly higher that those reported for Mentha x piperita, tional increase in total GTs as plants aged. New, young, mature 0.12 GT/mm2 [19], Gossypium hirsitum, 0.145 GT/mm2 [20], tobacco, 8.3 GT/mm2 and Pelargonium graveolens, 6.2 GT/mm2 [21].InM. tomentosa, the density of GT is homogenous throughout the whole abaxial side although an increase was observed during the young leaf stage where the highest GT density was observed. This may reflect a higher GT production at this stage to store an increased amount of terpenes. These results differ from those reported for M. x piperita [3] and other Lamiaceae [22], where the density of GT is not uniformly distributed on the surface of the leaf, and a higher density is usually found on the basal and middle zones of the abaxial side. In contra- diction to the proposed in Artemisia annua, where formed leaves achieved higher concentrations of artemisinin than earlier formed leaves, because of a higher trichome density and a higher capacity per trichome [23],inBetula species the density diminishes as the leaf is expanding, suggesting that the number is determined of origin [6]. The number of GTs found on leaves of different species Fig. 2. Average glandular trichomes density on the abaxial surface of basal, middle and apical zones of Montanoa tomentosa leaves. New □, young , is related to the GTs density and total leaf area. There are mature and ■ old leaves. Results are the average of three independent counts. not many reports in which the number of GT per leaf has been Author's personal copy

R.E. Robles-Zepeda et al. / Fitoterapia 80 (2009) 12–17 15 established. In Nepeta racemosa leaves, about 1500 GT/cm known [33,34]. Additionally, KA has demonstrated inhibitory were found [24] and in M. x piperita, 3500, 6800, and 7500 GT activity on vascular contractility [35]. Isolated KA has also been were counted in 0.75; 1.5 and 2.5 cm-long leaves, respectively shown to have antibiotic activity against Gram-positive and [3] resulting in a density close to 4700, 4500 and 3000 Gram-negative bacteria [36–38], Trypanosoma cruzi [39],as respectively. In M. tomentosa, we observed a similar number, well as cyto and embryotoxicity [40]. Also, theirs genotoxicity, with an average GT density of around 4300, 7600, 4400, and anti-inflammatory, hypotensive, and diuretic effects in vivo, 4500 per cm2 for new, young, mature and old leaves respec- smooth muscle relaxant and cytotoxic actions in vitro have tively. However, this species has a larger foliar area. Addi- reported [41]. Inclusive has been proposed that its analgesic tionally, a positive correlation was observed between the total properties are even higher than some commercial drugs [42]. number of GT and the leaf area progressing from the apical to For GA fewer biological activities have been proposed: basal zone. Similar results were observed in M. x piperita [3]. inducting effect on childbirth [9,43], discrete in vitro anti- It is not known if GT are degraded, reused, or if they are bacterial activity [44] and herbicide effect [45]. retained during the leaf's lifetime. According to the number of GT observed in all leaf ages, it seems that GTs are preserved 3.3. Glandular trichomes terpenes content during the entire life of the leaf. The distribution and number of GT in M. tomentosa suggests a continuous generation of Twenty-six volatile compounds were identified in GTs these glands during leaf expansion. This is similar to what extracts from the different leaf zones (Table 1). Most of them was observed in M. x piperita, where it was reported that the were terpenes, predominantly mono and sesquiterpenes. number of GT is not fixed at the emergence of the leaf, but The results in Table 1 are presented as the percentage of the increases with leaf development [19]. total compounds detected in each leaf zone. The type and abundance of these compounds varied between the leaf zones 3.2. Glandular trichomes microsampling: grandiflorenic and evaluated. However, it was difficult to compare the individual kaurenoic acids evaluation concentration directly in each zone. Valencene and β-eudesmol were the most abundant Several studies have demonstrated that GTs can accumu- compounds and were ubiquitously found in all leaves late organic compounds such as terpenes, which are either irrespectively of the age or section studied. However, these synthesized or stored in these organelles. In M. x piperita, two compounds were more abundant in the basal section, and isolated trichome cells were able to synthesize monoterpenes decreased towards the apical zone. This pattern appeared to be from exogenously administered sugar [25]. Enzymatic activ- independent of leaf age as it was observed in all develop- ities of the monoterpene biosynthetic pathway have been mental stages studied. Moreover, in the apical part of new detected in GTs employing cell-free enzyme assays [26,27]. leaves, β-eudesmol and valencene were the only compounds GTs extraction was performed with microcapillaries. The found in the extract. collected fluid was derivatized and analyzed by GC-MS. Under Similarly, agarospirol and cis-β-terpineol were mainly these conditions the grandiflorenic acid trimethylsilane located in the basal zone of most leaves. The percentage of and kaurenoic acid trimethylsilane had retention times of agarospirol increased concomitantly with the age of the 20.7 min and 22.07 min, respectively. The presence of the leaves. Cis-β-terpineol was mainly present in the basal zone grandiflorenic acid trimethylsilane and kaurenoic acid tri- of young, mature, and old leaves, but less abundantly than methylsilane was compared with authenticated standards. agarospirol. Nerol acetate was found in all the zones evaluated The mass spectra obtained were in agreement with those with the exception of the apical part of new leaves. Higher produced by standards. concentrations were found in mature leaves and lower con- Similar to the observed in M. tomentosa, the kaurenoic acid, centrations in new and young leaves. Germacrene D was also together with other kaurene derivatives have been reported found in all zones of young, mature, and old leaves, whereas, in in the GTs of Polymnia sonchifolia, an Asteraceae [28]. The new leaves, it was observed only in the basal zone. In all leaves presence of diterpenic acids in the capillary gland extracts evaluated, the content of germacrene D increased basipetally. confirms that the detection of GA and KA in GT was not the Variable levels of caryophyllene were observed in most zones result of contamination. It is possible that in M. tomentosa, of all leaves, with a gradual decrease occurring from basal to these diterpenic acids are biosynthesized by GT cells and apical zones. α-Thujene was found in old and mature leaves, accumulated in the glandular space. The lipophilic nature of and in most leaf zones. Linaool and α-muurolene were found KA and GA supports this possibility since their transport from in low concentrations in all mature samples. Sabinene and the intercellular or vascular tissue to GT on the leaf surface santoline triene were present in all leaves and leaf sections, would be unlikely. Previously, other diterpene compounds with the exception of new leaves. The amount of these have been isolated of GTs in different ethnomedicinal plant compounds decreased acropetally, but this was more pro- species, for example Salvonorin A, a neoclerodane diterpene nounced for sabinene, which showed lower concentrations was isolated from Salvia divinorum [29]; duvatrienediol and than santoline triene. Cis-geraniol was detected in the middle cis-abienol were obtained of GTs from Nicotiana tabacum and basal parts of more developed leaves. Whereas, elemol [30,31]; and of glandular trichomes from Helichrysum tenax was present in all zones of young, mature, and old leaves. (Asteracea) two diterpene compounds have been isolated [32]. The emission of the majority of the volatiles detected in It is not known whether KA and GA have a specific function this study did not seem to be restricted to a defined zone in in GTs. Has been speculated that kaurenoic acid could have a the leaves. Nevertheless, the developmental stage and the protective function in these glands [28]. In plants, the role of location of the GT on the leaves appeared to influence the type kaurenoic acid in the gibberellin biosynthetic pathway is well and concentration of volatiles that accumulated in the glands. Author's personal copy

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Table 1 Leaf surface glandular trichomes of Montanoa tomentosa

RI⁎ Compound Leaf developmental stage New Young Mature Old

Basal Middle Apical Basal Middle Apical Basal Middle Apical Basal Middle Apical Monoterpenes 888 α-Thujene 0.95 1.16 0.81 0.25 0.35 890 Santoline triene 1.75 3.06 4.89 3.87 3.66 1.13 3.25 2.49 6.27 1084 Sabinene 0.79 0.98 2.02 0.22 0.30 0.31 0.34 0.42 0.76 1494 α-Gurjunene 3.11 2.90 2.80 2.71 1.72 1.58 1.83 1.67 1.45 1.54

Oxygenated monoterpenes 1490 Z,β-Terpineol 0.97 0.73 1.93 1.89 2.21 1.64 1.52 1.61 1497 Linaool 0.19 0.31 0.24 0.30 0.14 1595 Nerol acetate 1.74 1.70 0.76 0.71 0.59 2.11 2.21 1.64 1.64 1.62 1.37 1599 β-Citral 0.26 0.35 2.96 2.65 2.44 2.46 2.05 1.99 1697 α-Citral 3.66 3.42 1.11 1.80 3.36 3.23 5.41 3.10 2.50 3.24 1699 Geranyl acetate 6.36 1.70 1.20 1.17 3.02 2.48 3.28 2.43 1.70 1798 Cis-Geraniol 0.83 0.70 1.33 1.45 0.96 0.97

Sesquiterpenes 1499 Caryophyllene 3.40 2.56 2.65 1.80 1.94 2.02 1.76 1.67 1.98 1695 Germacrene D 3.12 2.22 2.76 2.94 0.87 1.18 0.91 1.11 1.02 1696 α-Muurolene 0.55 0.36 0.42 0.33 0.38 1698 δ-Cadinene 1.39 1.67 2.72 1.80 2.32 1.85 2.15 1899 γ-Cadinene 0.46 0.58 1.07 1.15 0.89 1.11 0.88 1899 Caryophillene oxide 0.96 0.66 1.05 1.36 0.82 1.20 1.51 2198 Valencene 35.51 45.00 43.90 30.44 31.53 32.76 25.47 26.72 34.59 25.29 27.37 27.80

Oxygenated sesquiterpenes 2000 Elemol 3.49 3.40 3.50 3.40 2.87 9.68 3.13 3.28 2.07 2099 τ-Cadinol 1.78 2.36 2.20 2.50 2.86 2.65 1.18 2101 Agarospirol 4.67 4.15 4.50 4.04 4.14 2199 β-Eudesmol 34.58 49.89 56.10 26.77 27.20 32.70 27.90 31.40 40.75 27.45 29.87 33.71 2297 Carotol 3.46 3.57 3.00 2.02 1.53 2.06 2.17 1.80

Other 1491 1,5-Hexadiene-2,5-dimethyl-3-methylen 0.36 0.33 0.26 0.22 1999 1-Hydroxy-1,7-dimethyl-4-isopropyl-2, 3.86 7.24 6.36 6.04 2.22 2.86 2.12 2.12 7-cyclodecadiene 2100 5-Azulenemethanol-1,2,3,4,5,6,7,8-octahydro- 2.95 4.93 3.16 3.97 5.46 4.42 5.02 4.47 α,α,3,8-tetramethyl

Terpene content as percentual composition. Basal, middle and apical zones of different developmental stages leaves. ⁎RI: Kovats Index.

In M. x piperita, it was observed that there was a qualita- on the leaves. The microsampling and GC-MS analysis evi- tive and quantitative variability of the plant essential oil denced the accumulation of KA and GA and a variety of coincidental with the development of the plant. Presumably compounds, mainly terpenes. Considering the high number this is related to the age and distribution of the trichomes of glandular trichomes found in the leaves and the possible [19]. applications of the compounds found, the expectations for a Of the 26 volatiles identified, all of them in the basal sec- major study in the future, are opened. tion of old leaves, only two were found in the apical section of new leaves. These were β-eudesmol and valencene, which Acknowledgements were the most abundant volatiles in GTs. β-eudesmol has been reported to have cytotoxic activity [46], act as a cyano- To Consejo Nacional de Ciencia y Tecnología (CONACYT) bactericide [47], as an inhibitor of tumour growth [48] and for the scholarship 86454 to R.E.R.Z., and the financial support antiangiogenic activity [49]. There are a handful of reports for this project No 28029N. related to valencene, but none describe its possible biological function. Commercial interest in valencene might increase References since it is the precursor of nootkatone [50], a potent ter- miticidal, insecticidal and acaricidal [51,52] and a flavoring [1] Werker E. Adv Bot Res 2000;31:1. [2] Serrato-Valenti G, Bisio A, Cornara L, Ciarallo G. Ann Bot 1997;79:329. compound for soft drinks. Many of the volatile compounds [3] Turner GW, Gershenzon J, Croteau RB. Plant Physiol 2000;124:655. synthesized by the GT in M. tomentosa cells could have a [4] Gannon AJ, Bach CE. Environ Entomol 1996;25:1077. potential use in diverse commercial areas [53]. [5] Duke SO. Int J Plant Sci 1994;155:617. [6] Valkama E, Salminen JP, Koricheva J, Pihlaja K. Ann Bot 2004;94:233. In conclusion, M. tomentosa was shown to have glandular [7] X. Lozoya. Xiuhapatli, Herba officinalis. Ed. Secretaría de Salud-UNAM, trichomes; these trichomes were homogenously distributed México, D.F. 1999;33. Author's personal copy

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