Biopesticides from plants: Calceolaria integrifolia s.l

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Biopesticides from plants: Calceolaria integrifolia s.l ARTICLE in ENVIRONMENTAL RESEARCH · JULY 2014 Impact Factor: 4.37 · DOI: 10.1016/j.envres.2014.04.003

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Environmental Research 132 (2014) 391–406

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Environmental Research journal homepage: www.elsevier.com/locate/envres

Biopesticides from plants: Calceolaria integrifolia s.l. Carlos L. Céspedes a,n, Juan R. Salazar b, Armando Ariza-Castolo c, Lydia Yamaguchi d, José G. Ávila e, Pedro Aqueveque f, Isao Kubo g, Julio Alarcón a a

Basic Science Department, Faculty of Sciences, University of Bío Bío, Andres Bello Av, s/n, Chillán, P.O. Box 447, Ñuble 3780000, Chile Facultad de Ciencias Químicas, Universidad La Salle, México DF, México c Departamento de Química, CINVESTAV-IPN, México DF, México d Instituto de Química, Universidad de São Paulo, São Paulo, Brazil e Laboratorio de Fitoquímica, UBIPRO, FES-Iztacala, UNAM, México DF, México f Laboratorio de Microbiología y Micología Aplicada, Departamento de Agroindustrias, Facultad de Ingeniería Agrícola, Universidad de Concepción, Chillán, Chile g ESPM Department, University of California at Berkeley, USA b

art ic l e i nf o

a b s t r a c t

Article history: Received 30 January 2014 Received in revised form 31 March 2014 Accepted 2 April 2014

The effects of persistent organic pollutants (POPs) on humans and biodiversity are multiple and varied. Nowadays environmentally-friendly pesticides are strongly preferred to POPs. It is noteworthy that the crop protection role of pesticides and other techniques, i.e. biopesticides, plant extracts, prevention methods, organic methods, evaluation of plant resistance to certain pests under an integrated pest management (IPM), could improve the risks and beneﬁts which must be assessed on a sound scientiﬁc basis. For this directive it is crucial to bring about a signiﬁcant reduction in the use of chemical pesticides, not least through the promotion of sustainable alternative solutions such as organic farming and IPM. Biopesticides are derived from natural materials such as animals, plants, bacteria, and certain minerals. Most of them are biodegradable in relatively short periods of time. On this regard, substances from Calceolaria species emerge as a strong alternative to the use of POPs. The American genus Calceolaria species are regarded both as a notorious weeds and popular ornamental garden plants. Some have medicinal applications. Other taxa of Calceolaria are toxic to insects and resistant to microbial attack. These properties are probably associated with the presence of terpenes, iridoids, ﬂavonoids, naphthoquinones and phenylpropanoids previously demonstrated to have interesting biological activities. In this article a comprehensive evaluation of the potential utilization of Calceolaria species as a source of biopesticides is made. The chemical proﬁle of selected members of the Chilean Calceolaria integrifolia sensu lato complex represents a signiﬁcant addition to previous studies. New secondary metabolites were isolated, identiﬁed and tested for their antifeedant, insect growth regulation and insecticidal activities against Spodoptera frugiperda and Drosophila melanogaster. These species serve as a model of insect pests using conventional procedures. Additionally, bactericidal and fungicidal activity were determined. Dunnione mixed with gallic acid was the most active fungistatic and fungicidal combination encountered. Several compounds as isorhamnetin, combined with ferulic and gallic acid quickly reduced cell viability, but cell viability was recovered quickly and did not differ from that of the control. The effect of these mixtures on cultures of Aspergillus niger, Fusarium moniliforme, Fusarium sporotrichum, Rhizoctonia solani, and Trichophyton mentagrophytes, was sublethal. However, when fungistatic isorhamnetin and dunnione were combined with sublethal amounts of both ferulic and gallic acid, respectively, strong fungicidal activity against theses strains was observed. Thus, dunnione combined with gallic acid completely restricted the recovery of cell viability. This apparent synergistic effect was probably due to the blockade of the recovery process from induced-stress. The same series of phenolics (iridoids, ﬂavonoids, naphthoquinones and phenylpropanoids) were also tested against the Gram-negative bacteria Escherichia coli, Enterobacter agglomerans, and Salmonella typhi, and agaisnt the Gram-positive bacteria Bacillus subtilis, Sarcinia lutea, and Staphyllococcus aureus and their effects compared with those that of kanamycin. Mixtures of isorhamnetin/dunnione/kaempferol/ferulic/gallic acid in various combinations were found to have the most potent bactericidal and fungicidal activity with MFC between 10 and 50 μg/ml. Quercetin was found to be the most potent fungistatic single compound with an MIC of 15 mg/ml. A time-kill curve study showed that quercetin was fungicidal

Keywords: Insecticidal Antifungal activity Calceolariaceae Iridoids Flavonoids

n

Corresponding author. E-mail address: [email protected] (C.L. Céspedes).

http://dx.doi.org/10.1016/j.envres.2014.04.003 0013-9351/& 2014 Elsevier Inc. All rights reserved.

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against fungi assayed at any growth stage. This antifungal activity was slightly enhanced by combination with gallic acid. The primary antifungal action of the mixtures assayed likely comes from their ability to act as nonionic surfactants that disrupt the function of native membrane-associated proteins. Hence, the antifungal activity of isorhamnetin and other O-methyl ﬂavonols appears to be mediated by biophysical processes. Maximum activity is obtained when the balance between hydrophilic and hydrophobic portions of the molecules of the mixtures becomes the most appropriate. Diterpenes, ﬂavonoids, phenylpropanoids, iridoids and phenolic acids were identiﬁed by chromatographic procedures (HPLCDAD), ESI-MS, and NMR hyphenated techniques. & 2014 Elsevier Inc. All rights reserved.

1. Introduction The effects of persistent organic pollutants (POPs) on humans and biodiversity are multiple and varied. At present, environmentallyfriendly pesticides are strongly preferred to POPs. It is noteworthy that the crop protection role of pesticides, and other techniques, i.e. biopesticides, plant extracts, prevention methods, organic methods and plant resistance to certain pests under an integrated pest management (IPM). The risks and beneﬁts of these must be assessed on a sound scientiﬁc basis. It is crucial for this directive to bring about a signiﬁcant reduction in the use of chemical pesticides, not least through the promotion of sustainable alternative solutions such as organic farming and IPM (Rosner and Markowitz, 2013; Rhodes et al., 2013). Biopesticides are derived from natural materials such as animals, plants, bacteria, and certain minerals. They are usually biodegradable in short periods of time. Plants, the most common source of biopesticides, produce a great variety of secondary metabolites that lack apparent function in physiological or biochemical processes; these compounds (or allelochemicals) are important in mediating interactions between plants and their biotic environment (Berenbaum, 1989, 2002; Kessler and Baldwin, 2002). Some can be used as lead molecules for the development of protective agents against insects and fungi (Kubo et al., 1981, 1993, 2000, 2003a, 2003b), and enzyme inhibitors (Kubo, 1997; Keane and Ryan, 1999; Ortego et al., 1999; Céspedes et al., 2001a, 2001b; Kubo et al., 2000, 2003a, 2003b). As a result, there is increased interest for application of secondary metabolites in IPM, this has prompted the search for new sources of biologically active natural products, with new modes of action (Conner et al., 2000; Eisner et al., 2000; Meinwald, 2001), characteristics that which enhance their value as practical pesticides (Akhtar et al., 2008; González and Estevez-Braun, 1998; Isman, 2006; Valladares et al., 1997). Here, we review recent results on the bioactivity of extracts, fractions, mixtures and pure compounds from selected members of the Calceolaria integrifolia s.l. complex. These substances provide defense mechanisms against bacterial, fungal and herbivore predator attacks in these plants. Terpenes, phenolics and other compounds are accumulated in aerial parts, mainly in leaves and trichomes, resulting in unique biopesticides from these plants (Céspedes et al., 2013b, 2013c; Muñoz et al., 2013a, 2013b, 2013c). Plants from the genus Calceolaria (Calceolariaceae; formerly Scrophulariaceae) are distributed in temperate and tropical regions of New Zealand and Central and South America. (Di Fabio et al., 1995; Garbarino et al., 2000, 2004). Several species of Calceolaria are used as ornamental plants and in traditional medicine (Falcao et al., 2006). The aerial parts of these plants are used in Chile and South America for their analgesic, digestive and diuretic properties (Sacchetti et al., 1999), and as antimicrobials for stomach ailments (Sacchetti et al., 1999; Garbarino et al., 2004). Some species of this genus have substances with potential uses as insecticides (Khambay and Jewess, 2000), against tuberculosis (Woldemichael et al., 2003) and as growth inhibitors of TA3 tumor

cells and methotrexate resistant TA3 cells (Morello et al., 1995). Flavonoids, glucophenylpropanoids, and diterpenes have previously been identiﬁed in Calceolaria (Di Fabio et al., 1995; Nicoletti et al., 1986; Wollenweber et al., 1989; Garbarino et al., 2000; Muñoz et al., 2001). Approximately 86 species of this genus occur natively in Chile (Céspedes et al., 2013c); only 15% of them have been phytochemically characterized. The C. integrifolia sensu lato complex comprises nine species: C. andina, C. angustifolia, C. auriculata, C. georgiana, C. integrifolia s.str, C. rubiginosa, C. talcana, C. verbascifolia, and C. viscosissima. Each of these species has its own characteristic distribution pattern, which correlates with ecological and weather factors (Ehrhart, 2000, 2005). They are found in regions VII and VIII of Chile together with other species of Calceolaria. C. angustifolia, C. integrifolia, C. talcana and C. verbascifolia (Table 1), commonly known as “zapatito de doncella” or “capachito de hoja larga”, are strong erect shrubs, 150 cm tall or sometimes smaller with fragile ascending branches, internodes of 2–8 cm, and inﬂorescences and distal parts of stems that are glutinous or velutinous with erect hairs (Ehrhart, 2000, 2005). In previous reports on the antifeedant, insect growth regulatory (igr) and insecticidal activities induced by a series of phenolic and terpenes compounds from Calceolaria species, their maximum antifeedant and igr activity was shown to depend on the hydrophobic alkyl moieties and/or from the hydrophilic hydroxyl groups (Céspedes et al., 2013b, 2013c; Muñoz et al., 2013c). These compounds also possessed inhibitory effects on tyrosinase and acetylcholinesterase enzymes (Muñoz et al., 2013c; Céspedes et al., 2013b). On the past persistent organic pollutants mostly of synthetic origin (i.e. POPs) have been widely used, application of these substances has produced a strong impact on the environment, in many cases strains resistant to these compounds has resulted. Organic molecules of botanical origin may offer a safe and more efﬁcient source of compounds for pest management because most are environmentally friendlier resulting in an excellent alternative to POPs (Kubo, 1997). As many of them have low mammalian toxicity, limited persistence in the environment, and enhanced biodegradability the use of secondary metabolites for pest control has generated a growing interest in the search for new sources of biologically active natural products (Akhtar et al., 2008, 2012; González and Estevez-Braun, 1998; Isman and Akhtar, 2007; Isman, 2006). To date, the most widely used pesticides in global agricultural systems have been of synthetic origin such as carbamates, halogenated organic and organophosphorous (OP) compounds. Overuse has resulted in the generation of new strains of pests resistant to the original pesticides. The development of resistances is frequently related to modiﬁcation of receptors involved in the mechanisms and targets of action of certain molecules (Pang et al., 2012; Alout et al., 2012; Casida and Durkin, 2013) many of these pesticides target acetylcholinesterase. As a result of resistance, the scientiﬁc community has synthesized many new organic molecules with this target of action, resulting in dangerous health effects for animals. Acute or chronic

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Table 1 Antibacterial inhibitory activitiesa of compounds and mixtures from C. integrifolia s.l. on growth inhibition of bacteria (diameter in mm).a Sample

M1 quercetin M2 kaempferol M3 isorhamnetin M4 myricetin M5 dunnione Ferulic acid Dehydroabietinol Abietatrien-3β-ol chloramphenicol Kanamycin

Gram negative

Gram positive

E. coli

E. agglomerans

Salmonella sp.

B. subtilis

S. aureus

S. lutea

4.17 0.6a 3.9 7 0.4a 13.95 7 1.6b 9.3 7 0.7a 18.17 3.1c 12.5 7 2.6b 5.6 7 0.6a 4.2 7 0.6a 21.5 7 2.6c 16.5 7 2.1c 9.6 7 0.7a 12.9 7 1.6b 13.5 7 1.3b 20.0 7 2.6c 25.5 7 1.4c

3.9 7 0.4a 4.17 0.6b 10.5 7 1.6b 10.0 7 1.6b 40.0 7 3.6d 27.8 7 1.8c 4.5 7 0.7a 4.17 0.3a 42.0 7 3.7d 36.5 7 2.8d 12.3 7 1.1b 15.0 7 1.6b 15.8 7 1.2b 25.2 7 1.4c 56.4 7 0.6e

4.0 70.45a 4.17 0.5a 9.9 71.2b 8.5 70.9b 16.3 7 2.4b 16.0 7 1.6b 5.5 70.9b 0b 20.0 72.1c 14.4 7 2.2b 11.4 7 0.6b 14.5 7 1.6b 15.7 7 1.7b 25.0 71.6c 30.17 1.1c

4.5 70.45a 4.8 70.5a 0b,c 4.17 0.3a 12.3 70.9b 10.0 7 0.4b 3.9 70.2a 0b 12.3 71.2b 6.0 70.6a 9.9 70.9b 0b 0b 28.4 73.5c 22.4 70.9c

5.1 70.55a 4.9 70.46a 0b 4.9 70.5a 13.2 7 1.7b 11.0 7 1.1b 4.3 70.3a 0b 14.5 7 1.5b 8.17 0.7a 10.2 7 0.9b 0b 0b 22.2 73.5c 49.67 4.3e

4.9 7 03a 4.5 7 0.4a 0b 4.5 7 0.7a 14.5 71.4b 12.9 7 1.1b 4.17 0.2a 0b 11.0 7 0.9b 7.9 7 0.3a 15.0 71.3b 0b 0b 37.5 72.9c 21.7 7 1.4c

a Inhibitory effects at an equivalent concentration of 1600 μg per disc with M1 and M4, 800 μg per disc with M2 and 100 μg per disc with M3 and M5 is represented as mm of growth; mean value of diameter of inhibition zone: mm 7 standard error, of n ¼21 and its signiﬁcant difference from the control p o 0.01. b Activity not present. c Mean of three replicates. Means followed by the same letter within a column after7 standard error values are not signiﬁcantly different in a Student–Newman–Keuls (SNK) (treatments are compared by concentration to control), 95 % Conﬁdence limits. Negative control: N,N-Dimethylformamide (DMFA) 5 ml/disc. Positive controls: kanamycin and chloramphenicol 30 mg/disc. cActivity not present (in this table are shown only the more signiﬁcant inhibitory effects, above 4.0 mm).

poisoning caused by pesticides is a problem in many countries worldwide especially in developing countries (Francis, 2006; Fournier, 2005; Casida and Durkin, 2013; Green et al., 2013; Fournier and Mutero, 1994; Feyereisen, 1995). The search for new botanical pesticides (biopesticides) which have this target of action and remain harmless to animals and humans is relevant today. Our interest is centered on the study of shrubs belonging to the family Calceolariaceae, due to their notable resistance to pathogen attack observed in nature (Céspedes et al., 2013a) and their uses as medicinal plants. Limited availability of plant material of C. integrifolia s.l. has restricted our initial attempts to study the defense mechanism against pathogen attack on a molecular level. Therefore, based on previous results observed for the effects of extracts as insect growth inhibitors (Céspedes et al., 2013b; Muñoz et al., 2013a, 2013b), this study highlights bioactive phenolics isolated from selected plant species of the C. integrifolia s.l. complex as inhibitors of insects and fungi. Additionally, studies in Calceolaria species from Americas show the presence of many substances with agrichemical applications and pharmacological potential (Falcao et al., 2006; Woldemichael et al., 2003; Céspedes et al., 2013a, 2013b, 2013c; Muñoz et al., 2013a, 2013b, 2013c). To date, few studies of the phytochemical composition or biological activity of plants of the C. integrifolia sensu lato complex have been carried out. Céspedes et al. (2008, 2009, 2010a, 2010b)), Fraga et al. (1964), Harborne and Baxter (2001), Kubo and Himejima (1992), Montes (1987), Montes and Wilkomirsky (1978), Nicoletti et al. (1988a, 1988b). A few studies have investigated the sites and mechanisms of action of fungicidal, insecticidal and/or insect growth regulatory activity indicating that different secondary metabolites from these plants are enzymatic and metabolic inhibitors (Calderón et al., 2001; Céspedes et al., 2006; Feeny, 1976; Céspedes et al., 2013a, 2013b, 2013c; Muñoz et al., 2013a, 2013b, 2013c) and have insecticidal, IGR and antifeedant effects on phytophagous insects (Rhoades and Cates, 1976; Swain, 1979; Simmonds et al., 1996; Xie et al., 1993; Ortego et al., 1995; Mullin et al., 1997; Muñoz et al., 2013a, 2013b, 2013c). We have previously demonstrated that diverse secondary metabolites have different sites of action and different molecular targets when they interact with enzymes and metamorphosis processes (Céspedes et al., 2006, 2013a, 2013b, 2013c).

The general objective of this study was to establish the fungicidal and bactericidal activity of fractions, mixtures and pure compounds isolated from n-hexane and ethyl acetate extracts and also to measure the effect of these compounds on the metamorphosis of insect pest models. Several secondary metabolites from other Calceolaria species have shown biocidal activities Falcao et al., 2006; Woldemichael et al., 2003; Céspedes et al., 2013c; Muñoz et al., 2013c and their occurrence in Chilean Calceolariaceae has been reported (Céspedes et al., 2013c; Garbarino et al., 2000, 2004). The long-term goal is to examine the role of the phytochemicals of this complex of species in the fungistatic/ fungicidal effects and in the inhibitory behavior on growth and development of insects, namely Drosophila melanogaster (Diptera: Drosophilidae) and Spodoptera frugiperda (J.E. Smith, Lepidoptera: Noctuidae) as model systems of pest insects. In brief, we are in search of botanicals for potential use as biopesticides. In continuation with our investigation this is a report on the bactericidal, fungicidal and insect growth regulatory effects of selected members of Calceolaria complex (Muñoz et al., 2013b, 2013c; Céspedes et al., 2013b). The bioactive isolates from the most polar of the ethyl acetate extract F-7, a mixture M4 of naphthoquinones 6, 7 and iridoids and ﬂavonoids were assayed against D. melanogaster (fruit ﬂy) and S. frugiperda (fall armyworm) and aspects such as mortality, rate of development, time of pupation, adult emergence and deformities were measured. These data were compared with those of gedunin, Yucca and Cedrela MeOH extracts, known growth inhibitors of S. frugiperda (Céspedes et al., 2000, 2006; Isman, 2006; Torres et al., 2003). Because previously gathered information about this genus and our own ﬁeld observations indicated that these plant species appear to possess a strong resistance to pathogen attack in the ﬁeld, we undertook examination of Chilean members of this C. integrifolia s.l. complex. 2. Materials and methods 2.1. Plant material Samples of aerial parts of C. talcana Grau & C. Ehrhart, was collected along the roadside 4.7 km NW of Conﬂuencia on the road to Trehuaco on the north shore of the Itata River (361370 21″S, 721280 16″W, elev. 172 m), Ñuble province, VIII Región,

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Chile, in November, 2010. C. integrifolia was collected on the rural freeway M-80-M from Cobquecura to Buchupureo (361040 51″S, 721480 15″W, elev. 82 m) Ñuble province, VIII Region, Chile, in November 2010. C. talcana x integrifolia was collected on the rural freeway 126 from Quirihue to Cauquenes (361070 16″S, 721270 01″W, elev. 216 m), Ñuble-Linares Province, VII–VIII Region, Chile, in November 2011 and 2012. C. angustifolia was collected along the freeway L-391 from Linares to National Reserve “Los Bellotos del Melado” shore to Rio Ancoa (361050 48″S, 711220 38″W, elev. 451 m), Linares Province, VII Region, Chile, in November 2012. Voucher specimens have been deposited in the Herbarium of the Basic Science Department, University of Bío-Bío (Voucher DS-2010/05-16243/44) and in the Herbarium of the University of Illinois, at Urbana–Champaign, IL, USA (ILL, Voucher DS-16243/44). The samples were identiﬁed by Prof. David S. Seigler, Ph.D. (Emeritus Professor of Plant Biology and Curator of the Herbarium of the University of Illinois at Urbana– Champaign).

2.2. Extracts of aerial parts Samples of aerial parts were air dried at room temperature, milled and extracted with methanol overnight; the process was repeated ﬁve times. The resulting methanol extract was concentrated at reduced pressure in a rotatory evaporator at 40 1C and 200 mb to yield a syrupy methanol extract (645 g). A portion of the methanol extract (410 g) was dissolved in distilled water, diluted with methanol to a ratio of 60/40 methanol/water, placed in a separatory funnel, and washed with n-hexane (150 mL, 20 times). The n-hexane phases were combined and concentrated under reduced pressure. Identical procedures were carried out with CH2Cl2 and ethyl acetate extracts.

For hyphenated analyses the following were used: 1H NMR and 13C NMR spectra were recorded at 400 MHz, and 125 MHz, respectively on Avance 400 and 600 MHz on NMR Bruker spectrometers, chemical shifts (ppm) are related to (CH3)4Si as internal reference (δ 0), CDCl3, MeOD and acetone-d6 from Aldrich Chemical Co were used as solvents; coupling constants are quoted in Hz. GC/MS HP5989A, LC/MSD-TOF Agilent. Additionally, EIMS and TOF data were determined on a Q-TOF Waters and JEOL JMSAX505HA mass spectrometer at 70 eV. FABMS were obtained on a JEOL JMS-SX102A mass spectrometer operated with an acceleration voltage of 10 kV. Samples were desorbed from a nitrobenzyl alcohol matrix using 6 keV Xenon atoms. In addition to LC/MSD-TOF (6500 Series Agilent), and GC/MS with GC/MSD 5977A Agilent systems a HPLC system consisted of a Hewlett Packard Series 1100 HPLC instrument with DAD and UV detectors set at 320 nm. The column was obtained from Supelco Technologies C18 (150 mm 4.6 mm, 5 mm). The eluent was a mixture of 4% tetrahydrofuran in acetonitrile and water (35:65, v/v) and contained 0.04% phosphoric acid. The ﬂow rates were 0.85, 1.0 and 1.7 mL/min and the column temperature and pressure were 30 1C and 149 bar, respectively, and the injection volume was 20 mL. Verbascoside, linarin and syringin served as standards. 2.4. Chemicals and solvents All reagents used were either A.R. grade or chromatographic grade, methanol, CH2Cl2, CHCl3, NaCl, KCl, NaOH, KOH, acetonitrile, water, butanol, silica gel GF254 analytical chromatoplates, silica gel grade 60 (70–230, 60 Å) for column chromatography; n-hexane, and ethyl acetate were purchased from Merck-Chile, Santiago de Chile.

2.3. Apparatus

2.5. Extraction, isolation and puriﬁcation of diterpenes, iridoids, ﬂavonoids, naphthoquinones and phenylpropanoids

1 H NMR spectra were recorded at 300 and 500 MHz, 13C NMR at 75 and 125 MHz respectively, on Bruker DPX 300 MHz and DRX500 MHz spectrometers, chemical shifts (ppm) are relative to (CH3)4Si as internal reference. CDCl3 and acetone-d6 from Aldrich Chemical Co. were used as solvents, and coupling constants are reported in Hz. IR spectra were obtained as KBr pellets on Perkin Elmer 283-B and FT-IR Nicolet Magna 750 spectrophotometers. UV spectra of pure compounds were determined on a Shimadzu UV-160 and Spectronic model Genesys 5 spectrophotometers; CHCl3 was used as solvent. Optical rotations were measured on a JASCO DIP-360 spectropolarimeter; CHCl3 was used as solvent. Melting points were obtained on a Fisher-Johns apparatus and remain uncorrected. Nunc 24-well polystyrene multidishes were purchased from Cole-Parmer. LAB-LINE Chamber model CX14601A, with adjustable Hi–Lo protection thermostats safeguard samples. A Spectronic model Genesys 5 and a microplate reader Epoch-Biotek UV–vis (200–999 nm) spectrophotometers were used to carry out the spectrophotometric measurements of the cholinesterase activity. EIMS and TOF data were determined on a Q-TOF Waters and JEOL JMSAX505HA mass spectrometer at 70 eV. FABMS were obtained on a JEOL JMS-SX102A mass spectrometer operated with an acceleration voltage of 10 kV. Samples were desorbed from a nitrobenzyl alcohol matrix using 6 keV Xenon atoms. Column chromatography was carried out on Kiesel-gel G (Merck, Darmstadt, Germany); TLC was performed on Si gel 60 F254.

The concentrated n-hexane extract was subjected to a silica gel column chromatography (column diameter 2.5 cm, height 55 cm, 200–425 mesh) to yield 5 fractions (F-1–F-5). A similar procedure was applied to ethyl acetate extract (EtOAc); two fractions were obtained (F-6 and F-7) (see Scheme 1). From F-1 and F-2, waxes, fatty acids and carotenes, respectively, were encountered. From F-3, the solvent mixture of n-hexane/EtOAc (8:2) yielded 157 mg of a terpenoid mixture that was analyzed by NMR, LC/MS and TLC analysis. The compounds were isolated by preparative TLC and then puriﬁed by HPLC (Céspedes et al., 2013c). In continuation of the phytochemical analyses with hyphenated techniques, in F-3, it was possible to identify the diterpenes 1,10-α-cyclopropyl-4,13-dimethyl-19α-hydroxy-9-epi-ent-7,15-isopimaradiene (1,10-cyclopropyl-9-epi-ent-isopimarol) 5 (Chamy et al., 1991; Muñoz et al., 2013b), 19α-hydroxy-8,11,13-abietatriene (dehydroabietinol) 6 (Chamy et al., 1987; Woldemichael et al., 2003; Muñoz et al., 2013b), 2α,19-dihydroxydehydroabietane 4 (Chamy et al., 1995a, 1995b), 17-hydroxy-9-epi-ent-isopimara-7,15-diene 1 (Chamy et al. 1998a, 1998b) and 18-hydroxy-9-epi-ent-isopimara-7,15-diene 2 (Chamy et al., 1998a, 1998b). These compounds had identical chromatographic and spectral data as those from literature. Their presence is hereby reported. Fraction F-4 contains a mixture of α-lupeol, β-sitosterol, ursolic acid and a complex mixture of sterols, their properties were identical to those literature values and were not further studied.

Aerial parts of sample Calceolaria spp. Dried Methanol Extract (A) MeOH/H2O

n-hexane

Ethyl acetate Partition (C)

MeOH/H2O Residue (D)

vacuum chromatography On silica-gel

F-1

waxes

F-2

carotenes

F-3

diterpenes

F-4

triterpenes

F-5

naphthoquinones

F-6

flavonoids

F-7

phenylpropanoids

Scheme 1. Method of obtaining extracts, partitions, fractions, and compounds (Céspedes et al., 2013c).

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395

Fig. 2. Chemical structures of phenolic acids and iridoids.

Fig. 1. Chemical structures of diterpenes. Fraction F-5 contained to a mixture of hydroxylnaphthoquinones 43, αdunnione 41, and 2-acetoxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone 42 (Chamy et al., 1995a; Morello et al., 1995). These compounds were also isolated by fast extraction air-dried leaves with a CH2Cl2/n-hexane mixture (1:1) following the procedure described by Mercado et al., 2010, with modiﬁcations. Whole air-dried aerial stems and leaves (253 g) (without ﬂowers), were soaked in a solvent mixture (n-hexane/CH2Cl2, 6:4). The solution was then ﬁltered (Whatman ﬁlter paper # 1) and evaporated in a rotatory evaporator to yield 3.79 g of intensely colored crude residue. This mixture was dissolved in MeOH/CH2Cl2 (7:3) samples and were worked by TLC and HPLC-DAD–MS (Figs. 5 and 8). Verbascoside 31, martynoside 32 and phenylethanoid mixtures 33–37 (Fig. 4) were isolated from fraction F-7, obtained from the ethyl acetate extract, that was fractionated by open cc using silica gel 60 F254 (0.063–0.200-mm particle size, 70–230 mesh ASTM, 60 Å pore diameter) and silica gel 60 F254 precoated aluminum sheets (0.2 mm layer thickness) purchased from Merck (Darmstadt, Germany). Elution was carried out with hexane-ethyl acetate in different ratios and methanol was added to increase the polarity of the gradient until 100% methanol was reached. All fractions were analyzed by TLC using ceric sulfate as the visualization system. Fractions obtained with CH2Cl2–MeOH (8:2) system contained verbascoside 31 as the major compound (4.345 g, 7.23% from the ethyl acetate extract). Verbascoside, further was puriﬁed by silica gel TLC using CH2Cl2–MeOH (7:3) as the eluting system together martynoside which has a similar Rf value. Chemical structures were determined by comparison with spectroscopic data from authentic samples and previously reported data (Domínguez et al., 2007; Céspedes et al., 2013c). These two compounds were further puriﬁed with Sephadex LH-20 column chromatography yielded the two puriﬁed phenylpropanoid compounds (Muñoz et al., 2013c). The most polar part of the methanol extractable fraction of the EtOAc extract F-7 consists of verbascoside 31 and martynoside 32, verbenalin (cornin) 17, hastatoside 18, the iridoids 19, 20 (Fig. 2), and a mixture of phenylpropanoid glycosides including calceolariosides A 33, B 36, C 34, D 37, E 35 (Fig. 4). These last compounds have been reported previously (Di Fabio et al., 1995; Garbarino et al., 2000, 2004). These glycosides were identiﬁed by HPLC-DAD–ESI-MS and NMR hyphenated techniques. Column chromatography of F-6 from the same ethyl acetate extract over Sephadex LH-20, when eluted with MeOH removed ﬂavonoids from similar amounts of terpenoid material. HPLC-DAD–ESI-MS and NMR hyphenated techniques were used to identify rutin 30, kaempferol 25, its 3-O-glucoside 26, quercetin 23, its 3-O-glucoside 24, isorhamnetin 27, its 3-O-glucoside 28,

myricetin 21, its 3-O-glucoside 22, together with monoterpenoid phenolic acids 7– 12 (Figs. 2–5). The chemical structures of iridoids, ﬂavonoids and phenylpropanoid glycosides were determined by spectroscopic analyses, comparing data with those reported in literature and direct comparison with authentic samples using TLC, HPLC-DAD–ESI-MS and NMR hyphenated techniques. 2.6. Estimation of total phenolic content by Folin–Ciocalteau Method The total phenolic content of extracts was determined using the Folin– Ciocalteau reagent: 10 mL sample or standard (10–100 mM catechin) plus 150 mL diluted Folin–Ciocalteau reagent (1:4 reagent: water) was placed in each well of a 96-well plate, and incubated at RT for 3 min. Following addition of 50 mL sodium carbonate (2:3 saturated sodium carbonate: water) and a further incubation of 2 h at room temperature, the absorbance was read at 725 nm. Results are expressed as mmol Cat E per gram. All tests were conducted in triplicate (Domínguez et al., 2005). 2.7. Evaluation of antimicrobial activity (microorganisms and growth medium) The antibacterial and antifungal activities of the M1–M5 [M1 (quercetinþ ferulic acid), M2 (kaempferolþ ferulic acid), M3 (isorhamnetinþferulic acid), M4 (myricetinþ ferulic acid) and M5 (dunnioneþ gallic acid)], ethyl acetate extract, diterpenes, iridoids, phenylpropanoids and ﬂavonoids were determined. Because of the small amount of 13–18, these compounds were not examined. For antibacterial activity, paper disks (6 mm, Whatman #1 ﬁlter paper) were impregnated with 10 mL of solution containing 100 mg of each compound to perform the test against the Gramnegative bacteria, Escherichia coli (ATCC25922), Enterobacter agglomerans (ATCC27155), Salmonella typhi (ATCC19430), and the Gram-positive bacteria, Bacillus subtilis (ATCC6633), Sarcina lutea (wild-type 1), and Staphylococcus aureus (ATCC12398). For the antifungal activity the fungi strains used were Aspergillus niger (ATCC64958), Fusarium moniliforme (ATCC96574), F. sporotrichum (wild-type 2), Rhizoctonia solani (wild-type 2a), and Trichophyton mentagrophytes (ATCC9972). Wild-type 1: the strain was cultured and donated by Laboratorio de Microbiología of FES-Cuautitlan (UNAM). Wild-Type 2: the strain was cultured and donated by Laboratorio de Análisis Clínicos of FES-Iztacala (UNAM). Wild-Type 2a: The strain was isolated from infected bean cultures by Prof. Dr. Rodolfo de la Torre, Laboratorio de Microbiología, FES-Iztacala (UNAM). Wild-Types: Strains Cultivation were maintained under freezing and, before the bioassays were done, were cultured in sterile Erlenmeyer ﬂasks with 10 mL of YEB liquid medium (ﬂasks were maintained in incubation for 72 h at 37 1C). The bioassay was made by paper

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C.L. Céspedes et al. / Environmental Research 132 (2014) 391–406 10 mg/disk ketoconazole. The cultures were incubated at 28 1C for 14 days and checked every 24 h. Inhibition of radial mycelial growth diameters was measured daily and recorded as mean percentages (%) of growth (53). 2.8. Antifungal assays (FC50) and minimum fungicidal concentration (MFC). These tests were carried out to analyze the fungicidal activity exhibited by each compound and extract. For quantitative assays of the extracts, three doses were added to Czapek-Dox agar (4 mL) at 45 1C, mixed rapidly, and poured into three separate 6 cm Petri dishes. After the agar had cooled at room temperature, a small amount of mycelium (1 mm 1 mm) was inoculated, and the same amount was added to each plate. DMFA was only employed as a negative control. After incubation at 23 1C for 72 h, the area of the mycelial colony was measured and the inhibition of fungal growth and hence the IC50 were determined. Fourteen days after the beginning of the assay, a circle of agar around the central hole was obtained. Ketoconazole (0.9 mg/mL) was used as a positive control. At the end of this period, the MFC values were recorded (53). 2.9. Bioassays with fall armyworm

Fig. 3. Chemical structures of ﬂavonoids. disk method (AOAC, 1070); disks of 6 mm in diameter were soaked with samples, respectively. These disks were impregnated with 10 mL of a 40 mg/mL solution of sample and with 10 mL of a 400 mg/mL solution of sample and placed in Petri dishes containing YEB medium previously inoculated with bacteria (100 mL). Cultures were incubated at 37 1C for 24 h. The diameter of the growth inhibition zone was determined (mm, included the paper disk). The mean value of at least three different experiments was used for statistical analysis, and each experiment was made in triplicate. Bioassays with ATCC, Wild-Types 1 and 2 Strains. Bacteria that were grown in brain heart infusion broth (Bioxon, Mexico City, Mexico, 112-1) for stock cultures and Mueller–Hinton broth (Bioxon, 260-1) were used as test media due to their low interaction with the assayed compounds. The antibacterial activity of the extract and pure compounds was assessed with the disc-diffusion method using Mueller–Hinton agar (52), and the determination of inhibition zones at different dilutions of compounds and extracts was evaluated. In this case, ﬁlter paper disks (6 mm diameter, Whatman #1) with pure compounds were impregnated with 10 mL of a 10 mg/mL solution of each sample, and the ﬁlter paper disks of the crude total extract were impregnated with 10 mL of a 40 mg/mL solution of sample and 10 mL of a 400 mg/mL solution of sample, respectively, and placed in Petri dishes containing the test organisms. Cultures were incubated at 37 1C, and after 72 h, the diameter of inhibition zone was determined (mm). The mean value of at least three different experiments was used for statistical analysis, and each experiment was made in triplicate. The treatments were evaluated with a completely randomized design and were subjected to a one-way analysis of variance (ANOVA). Means were compared with the Student–Newman–Keuls (SNK) test (Po 0.05) under MicroCal Origin 8.0 Microsoft statistical program. Kanamycin and chloramphenicol were used as positive controls. The antifungal property of the extract and compounds was tested by the agarwell diffusion method using Sabouraud dextrose agar. Standard reference antibiotics were used in order to control the sensitivity of the tested microorganisms, which were inoculated in Czapek-Dox broth medium. Plates containing only the culture medium, with the addition or not of the solvents (methanol or water 10 mL/disk), were used as viability controls for each fungus studied. The fungal inocula (10 mL of 3 106 spores/mL) were placed in a hole (0.4 mm2) made in the center of each Petri dish after solidiﬁcation of the agar. Extract doses were up to 4 mg/disk (0.4 mg of M2 and 4.0 mg of M1, respectively); positive control,

S. frugiperda J.E. Smith (Lepidoptera: Noctuidae) larvae used for experiments were obtained from our cultures at the laboratory of Phyotchemical Ecology of the Basic Science Department, Faculty of Science, University of Bío-Bío, Chillán, Chile, and at the Laboratorio de Fitoquímica y Síntesis Orgánica, Facultad de Ciencias Quimicas, Universidad La Salle, Mexico DF, Mexico and were maintained under previously described conditions (Muñoz et al., 2013a). An artiﬁcial diet containing 800 mL of sterile water, 10.0 g of agar, 50.0 g of soy meal, 96.0 g of corn meal, 40.0 g of yeast extract, 4.0 g of wheat germ, 2.0 g of sorbic acid, 2.0 g of choline chloride, 4.0 g of ascorbic acid, 2.5 g of p-hydroxybenzoic acid methyl ester, 7.0 mL of Wesson salt mixture, 15.0 mL of Vanderzant vitamin mixture for insects, 2.5 mL of formaldehyde, 0.1 unit of streptomycin, 5.0 g of aureomycin, and 20.0 g of milled ear of corn grain (for 1 kg of diet) were used for the bioassay, which was prepared by the procedure described earlier (Céspedes et al., 2000, 2001a, 2001b, 2004, 2005; Céspedes and Alarcón, 2011; Mihm, 1987). Polystyrene multidishes with 24-wells were ﬁlled with the liquid diet, and then left for 20 min at room temperature under sterile conditions. The 3.4 mL wells measure 17 mm in depth 15 mm in diameter with a 1.9 cm2 culture area. All test compounds were dissolved in 95% ethanol and layered on top of each well with the artiﬁcial diet using up to six concentrations (see Table 1) and a control (1 mL 95% ethanol) allowing evaporation of solvent. Hexane and MeOH extracts (1.0 and 3.5 ppm) were used, because these extracts showed the greatest inhibitory activity in preliminary trials (data not shown). For each concentration used and control, a single S. frugiperda neonate ﬁrst instar larva was placed on the diet mixture in each well for 7 days. Thus each experiment contains 72 total larvae (each plate of 24 wells with three replicates). After 7 days, surviving larvae were measured, weighed and then transferred to separate vials containing fresh stock diet. Larval weight gains and mortality were recorded after 21 days of incubation, as the pupation average is 23 71 days (Table 1). Other lifecycle measurements, such as time to pupation, mortality of larvae and adult emergence and deformities (data not shown) were recorded. All treatments were carried out in a controlled environmental chamber with an 18 L: 6 D photoperiod, at 25 1C day and 19 1C night temperature regime, and a relative humidity of 8075%. There were three replications for each assay. Control assays (24-wells) contained the same number of larvae, volume of diet, and ethanol as the test solutions (Céspedes et al., 2000; Torres et al., 2003). 2.10. Acute toxicity on S. frugiperda Acute toxicity was determined by topical application to S. frugiperda of last stage larvae. Larvae of S. frugiperda were iced to stop their movement and treated on their abdomens with each of test compounds, at concentrations of 5.0, 10.0, 20.0, 35.0, 50.0 and 75.0 mg/mL. Additional concentrations (25.0 and 2.0 ppm) were used for MeOH-Cedrela and MeOH-Yucca extracts, respectively (Table 2). Solvent used was 10.5 mL of acetone injected with 50 mL microsyringe, and control was only treated with 10.5 mL of acetone. After 24 h survivals were recorded. Five larvae were used for each concentration, respectively. LD50 is the lethal dose producing 50% survival (Céspedes et al., 2000; Torres et al., 2003). 2.11. Not choice test (insecticidal bioassay) against D. melanogaster The bioassay for insecticidal activity against larvae of D. melanogaster was carried out as follows (Muñoz et al., 2001): ﬁve concentrations (5.0, 10.0, 20.0, 50.0, and 100.0 ppm of sample) were used for determining LC50 values (Table 3). Test samples were dissolved in 50 mL of EtOH and mixed in 1 mL of artiﬁcial diet [brewers' yeast (60 g), glucose (80 g), agar (12 g), and propionic acid (8 mL) in water (1000 mL)]. A control diet was treated with 50 mL of EtOH only. About 100 adults from the colonies of D. melanogaster were introduced into a new culture

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397

Fig. 4. Chemical structures of phenylpropanoids. bottle, into which artiﬁcial diet had been poured into a Petri dish, and allowed to oviposit at 25 1C and relative humidity 460% for 3 h. The diet was taken out of the bottle, and 10 new eggs were collected and transplanted onto each diet (1 mL) in glass tubes and reared at 25 1C and relative humidity 490% for 8 days. One day after transplantation, the larvae were hatched and fed each test extracts with the artiﬁcial diet. At 25 1C, larvae generally change to pupae after 7 days. In each instance, the developmental stage was observed and the numbers of pupae were recorded and compared with those of a control. Ten new eggs were used in each of three replicates. The LC50, the concentration that produces 50% mortality, was determined using Origin 8.0 statistical and graphs PC program. 2.12. Statistical analysis. Data shown in tables are the average of ﬁve replicates and independent experiments and are presented as averages7standard errors of the mean. Data were evaluated by variance analysis (ANOVA). Signiﬁcant differences between means were identiﬁed by GLM procedures. Results are given in the text as probability values, with p o0.05 adopted as the criterion of signiﬁcance; differences between treatments means were established with a SNK test. The I50 (CF50) values for each activity were calculated based on percentage of inhibition obtained at each concentration of the samples compared with control. I50 is the concentration producing 50% inhibition. The complete statistical analysis was performed by means of the OriginLab, Origin 8.0, statistical and graphs PC program.

3. Results and discussion Persistent organic pollutants (POPs) are certain chemicals that are persistent in the environment for long period of time,

migrating in air, water, soil and sediments and accumulating to levels that can harm wildlife and human health. These pollutants are organic compounds of natural or synthetic origin that possess a particular combination of physical and chemical properties such that, once released into the environment, they remain intact because they resist photolytic, chemical means and biological degradation (Wong et al., 2005; Rosner and Markowitz, 2013). The more common POPs include chemicals such as the chlorinated organic compounds hexachlorobenzene (HCB), hexachlorocyclohexane (HCH), dichlorodiphenyltrichloroethane (DDT), polychlorinated biphenyls (PCBs) and multiple organochlorine (OCPs) and organophosphate (OPs) pesticides that were among the most widely used during last century. At present they are banned or restricted in the majority of industrialized countries, but are still used in Africa, South Asia, Central and South America (Kumar et al., 2005; Selin and Eckley, 2003; Villa et al., 2003; Rhodes et al., 2013). POPs have become a matter of concern because of their toxicity and tendency to accumulate in food chains (Mason and Barak, 1990). In the environment they are transported at low concentrations by water and, as they are semi-volatile, are transported over long distances in the atmosphere. The result is widespread distribution of POPs across the globe, including humans, animals, fruits, vegetables and foods (El-Shahawi et al., 2010). In our screening program for biopesticides of plant origin from Chile and South America, it was found that selected specimens

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Fig. 5. Chemical structures of catechins and naphthoquinones.

Table 2 Results of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of mixtures M3 and M5 (μg/mL). Bacteria

E. coli E. agglomerans B. subtilis S. aureus

M3

M5

MIC

MBC

MIC

MBC

250 180 300 400

300 180 180 400

450 200 180 450

↑450 250 300 ↑450

Negative control: 70 μl/ml N,N-dimethylformamide (DMFA). Positive control 30 μg/ml chloramphenicol. The arrows mean that MBC can be greater than showed values.

of C. integrifolia s.l. complex showed antifeedant, antifungal, insecticidal and antibacterial activities (Muñoz et al., 2013a, 2013b, 2013c; Céspedes et al., 2013a, 2013b). Based on this information and the strong resistance to insect and pathogen attack by this plant, we carried out a biodirected phytochemical study of the aerial parts of C. angustifolia, C. integrifolia, C. talcana and a C. talcana x integrifolia hybrid. 3.1. Phytochemical analysis. Previously we reported the isolation and identiﬁcation of two diterpenes from C. talcana (Céspedes et al., 2013c; Muñoz et al.,

2013b) whose 1H and 13C NMR signals were in accord with those previously reported (Wenkert and Buckwalter, 1972; Buckwalter et al., 1975; Piovano et al., 1988; Ulubelen and Topcu, 1992; Chamy et al., 1992, 1995a, 1998a; Shimbo et al., 2000; Woldemichael et al., 2003) (Fig. 1). Upon discovery that the n-hexane extract of C. talcana exhibited a good antifeedant index activity against S. frugiperda and D. melanogaster with values of 55.5% and 87.5% at 50.0 mg/mL, respectively (Muñoz et al., 2013b), bioassay-guided fractionation of the n-hexane extract was undertaken as described in the Experimental Section. Extensive chromatographic puriﬁcation of components of the most bioactive fractions (F-3, F-4 and F-5) afforded two new diterpenes for this plant species, as well as several known triterpenes and naphthoquinone compounds whose structural identities were published previously (Muñoz et al., 2013b). Compounds 1–6 were isolated previously from C. talcana as a mixture of diterpenes, together with triterpenes and sterols that remain unidentiﬁed (Fig. 1) (Céspedes et al., 2013c). F-5 corresponds to a mixture of naphthoquinones that were identiﬁed as 2-hydroxy-3-(1,1-dimethylallyl-1,4-naphthoquinone) 43, α-dunnione 41 and acetoxynaphthoquinone 42 (Fig. 5) previously isolated from Calceolaria sessilis (Chamy et al., 1993) and C. integrifolia (Rüedi and Eugster, 1977), together with other compounds in minute amounts that remain unidentiﬁed (Céspedes et al., 2013a, 2013b, 2013c). The structures of these compounds were determined by 1H and 13C NMR data and reported previously (Muñoz et al., 2013a, 2013b, 2013c). Similarly, the polar part of the

C.L. Céspedes et al. / Environmental Research 132 (2014) 391–406

Table 3 The effect of samples from Calceolaria s.l. as antifungal growth inhibition activity on fungi preparation inoculaa (diameter in mm)c.

Table 5 Insecticidal activity on Spodoptera frugiperda and Drosophila melanogaster.a Samples

Ethyl acetate extract M3 Isorhamnetin M5 Dunnione Ferulic acid Dehydroabietinol Abietatrien-3β-ol 3 Gallocatechingallate Gallic acid Verbascoside Ketoconazole

R. solani

F. sporotrichum F. moniliforme A. niger

16.4 7 1.7b 18.17 0.9b 8.17 0.5a 35.87 0.8c 35.17 0.7c 16.5 7 0.5b 107 0.9a 127 0.4a 157 0.3a 31.2 7 0.9c 8.0 7 0.2a 45.9 7 0.3d

35.17 4.1c 25.17 1.9c 17.9 7 2.2b 38.2 7 0.5c 27.9 7 0.5c 14.3 7 0.6a 11.0 7 0.8a 12.0 7 0.4a 13.0 7 0.5a 29.8 7 0.6c 5.6 7 0.5a 40.9 7 0.6d

40.4 74.5d 36.1 73.1c 23.05 72.4c 44.5 70.5d 41.0 70.3d 18.9 70.2b 12.0 70.9a 8.0 70.5a 15 70.5a 23 70.9b 6.3 70.7a 47.8 70.5d

25.3 7 2.3b 33.97 2.7c 15.5 7 1.9a 39.8 7 1.5c 42.0 7 1.5d 39.0 7 0.5c 12.0 7 1.5a 9.2 7 1.1a 10.0 7 1.2a 28.9 7 0.5c 5.5 7 1.8a 52.3 7 0.9d

a Inhibitory effects at an equivalent concentration of 2000 μg per disc with M3, 200 μg per disc with M5 and 100 μg per disc with compounds is represented as mm of growth; mean value of diameter of inhibition zone: mm 7 standard error, of N ¼21 and its signiﬁcant difference from the control p o 0.01. c Mean of three replicates. Means followed by the same letter within a column after7standard error values are not signiﬁcantly different in a Student–Newman– Keuls (SNK) test (treatments are compared by concentration to control), 95 % Conﬁdence limits.

399

5 6 α-Lupeol β-Sitosterol Ursolic acid 43 41 31 32 M10 g M20 g M30 g M40 g F-3h F-4h F-5h F-7h Me-Yuc Gedunin

Doses (mg/ml)

35.0 100.0 n.d.f n.d. 100.0 50.0 n.d. 50.0 n.d. 35.0 35.0 35.0 20.0 50.0 50.0 50.0 35.0 50.0 50.0

D. melanogasterb Mortality %d

MC50e

100 82.9 – – 100 100 – 100 – 100 100 100 100 100 100 100 100 90.5 100

9.2 16.1 – – 41.3 15.8 – 8.08 – 9.48 16.9 7.51 6.99 15.9 38.9 15.6 13.2 17.04 11.93

Doses (mg/ml)

10.0 50.0 n.d. n.d. 35.0 20.0 n.d. 20.0 n.d. 20.0 35.0 20.0 20.0 35.0 50.0 50.0 35.0 25.0 50.0

S. frugiperdac Mortality %d

MC50e

96.5 96.1 – – 92.1 100 – 100 – 89.3 89.5 90.6 100 100 100 100 100 95.0 100

3.02 17.5 – – 10.2 3.64 7.78 3.31 9.22 4.99 5.01 25.3 22.5 19.9 12.0 8.0 10.78

a

Several values were taken from Muñoz et al., 2013a, 2013b. Mortality percentage of D. melanogaster larvae, after 72 h of the application of the samples at concentration with maximum effect, IC50 in mg/ml. c Mortality percentage of S. frugiperda larvae of last stage in acute test, values recorded at 24 h of the application of the samples at concentration with maximum effect. d Each value corresponds to the average of the three experiments. e Values of IC50 are in mg/ml. f n.d. ¼not determined because their concentrations were greater than 1000 mg/ml. g M10 ; M20 ; M30 ; M40 (see Muñoz et al., 2013b). h For F-3; F-4; F-5 and F-7, please see Materials and Methods. F-7 ¼ Phenolic fraction from ethyl acetate extract (verbascoside, martynoside, ﬂavonoids, iridoids) (Céspedes et al., 2013b; Muñoz et al., 2013b). b

Table 4 Antifungal bioassay of the compounds 6 and 7. Evaluation of FC50 and minimum fungicidal concentration (MFC).a Samples

M3 (mg/mL)

M5 (mg/mL)

Fungi

FC50

MFC FC50 MFC FC50

MFC FC50

MFC

Rhyzoctonia solani Fusarium sporotrichum Fusarium monoliforme Aspergillus niger Trichophyton mentagrophytes

62.5 125 125 125 62.5

125 250 250 250 125

250 250 250 250 125

25 15 25 25 10

18.5 25.0 18.5 15.0 12.5

32 35 32 25 15

Gallic acid (mg/mL)

125 125 125 125 62.5

ketoconazole (mg/mL)

12.5 7.5 12.5 12.5 5.0

a

MFC is deﬁned as the lowest concentration providing complete inhibition of mycelial growth. The average of three replicates was measured for 14 days after incubation. mg/disc, the negative control (C-) was N,N-dimethylformamide (DMFA), with a maximum dilution of (20 ml/disc).

methanol extractable fraction of the ethyl acetate extract F-7 consists almost exclusively of verbascoside 31 and martynoside 32 accompanied by a complex mixture of other compounds such as iridoids and ﬂavonoids, whose chemical structures are reported here (Figs. 2–4). In addition, when the fractionation was also guided by the artiﬁcial diet feeding assay against the fruit ﬂy, the same compounds were characterized as active principles (Muñoz et al., 2013a, 2013b, 2013c) (Table 5). In order to obtain more details about sites and mechanisms of action of the insecticidal activity of n-hexane, ethyl acetate, and MeOH-residue extracts, the mixtures M10 –M40 [M10 ¼5 þ6; M20 ¼mix of triterpenes (Céspedes et al., 2013b); M30 ¼41 þ43; M40 ¼31 þ32], fractions F-3, F-5, F-7, and several isolated compounds were assayed as cholinesterase inhibitors. Forsythoside B, leucosceptoside B, quercetin, and galanthamine were used as controls in the inhibitory activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) bioassays that were carried out (Céspedes et al., 2013b). Interestingly, verbascoside 31 and martynoside 32 were characterized from an early CC fraction obtained from the ethyl acetate extract. However, these compounds could not be detected in the

HPLC analytical determination of the n-hexane extract nor in the MeOH/H2O residue from sample material plants in this study. On the other hand, diterpenes, triterpenes and 2-hydroxy-3-(1, 1-dimethylallyl)-1,4-naphthoquinone 43 and α-dunnione 41 are abundant and easily detectable in the n-hexane/CH2Cl2 extract part of leaves and stems (Scheme 1). In this work, we report the identiﬁcation of eight diterpenes, two new iridoids, three naphthoquinones, eleven ﬂavonoids, six phenylpropanoids and diverse monoterpene phenolic acids that were identiﬁed from all samples obtained from the aerial part of these selected Calceolaria species used in this work (Figs. 1–8). The structures of these compounds were established by combined chromatographic procedures with standards and by HPLC-DAD– ESI-MS, IR, UV and NMR hyphenated techniques. 17-Hydroxy-9epi-ent-isopimara-7,15-diene 1, 18-hydroxy-9-epi-ent-isopimara-7, 15-diene 2, 3β-hydroxydehydroabietane 3, 2α,19-dihydroxy-dehydroabietane 4, 1,10-α-cyclopropyl-4,13-dimethyl-19-α-hydroxy-9epi-ent-7,15-isopimaradiene (1,10-cyclopropyl-9-epi-ent-isopimarol) 5, 19α-hydroxy-8,11,13-abietatriene (dehydroabietinol) 6, verbascoside 31, martynoside 32, calceolariosides A 33, B 36, C 34, D 37, E 35, verbenalin 17, hastatoside 18, the iridoids 19, 20 as well as the aglycone ﬂavonoids myricetin 21, kaempferol 25, quercetin 23, isorhamnetin 27, catechin 40, catechin gallate 38, gallocatechin gallate 39, rutin 30 and as glycosides myricetin-3-O-glycoside 22, iso-rhamnetin-3-O-glycoside 28, kaempferol-3-O-glycoside 26 and quercetin-3-O-glycoside 24, together with monoterpenoid phenolic acids including cinnamic acid 7, caffeic acid 9, ferulic acid 10, 5-hydroxyferulic acid 12, gallic acid 11, p-coumaric acid (hydroxycinnamic acid) 8, diboa 15, dimboa 16, HBOA 13, and TRIBOA 14

400

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Abundance

26000 24000 22000 20000 18000 16000 mAU

14000 12000 10000 8000 6000 4000 2000 5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

Minutes Fig. 6. HPLC-DAD of phenolic acids and ﬂavonoids at 280 nm.

3000

mAU

2000

1000

0

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

Minutes Fig. 7. HPLC-DAD of phenylpropanoids glycosides: iridoid 1, verbenalin 2, calceolarioside D 3, verbascoside 4, martynoside 5, calceolarioside E 6, calceolarioside C 7, calceolarioside A 8, calceolarioside B 9, at 280 nm.

were found. Control compounds were purchased from Sigma-Aldrich and Merck. 3.2. Previous enzyme inhibitory effects by naphthoquinones, phenylethanoids, and other secondary metabolites The ethyl acetate extract and fraction F-7 were more active against acetylcholinesterase (AChE) than the ﬂavonoid quercetin, phenylethanoids (forsythoside B and leucosceptoside B) and M40 , above 25.0 mg/ml. The inhibitory activity of AChE showed by

fractions F-5, F-7, mixtures M30 , M40 , and ethyl acetate extract occurred in a dose-dependent manner (Céspedes et al., 2013b). Thus, it is possible to infer that phenolic compounds in these samples could be the active inhibitors of acetylcholinesterase in C. angustifolia, C. integrifolia, C. talcana and in C. talcana x integrifolia hybrid. Additionally, the presence or absence of a methyl group in phenylethanoids respectively increases or decreases the strength of these compounds on inhibition of AChE as in the case of forsythoside B compared with leucosceptoside B and verbascoside

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401

800

mAU

600

400

200

0

5.0

10.0

15.0

20.0

25.0

Minutes Fig. 8. HPLC-DAD of naphthoquinones: dunnione 1, acetoxynaphthoquinone 2, hydroxynaphthoquinone 3, at 350 nm.

31 compared with martynoside 32 (Georgiev et al., 2011; Céspedes et al., 2013b). Thus, we suggest that insect growth inhibitory and insecticidal activity of the ethyl acetate extracts, F-5, and F-7 are not caused by one strong inhibitor, but rather by a synergistic effect of the components in the mixtures. The inhibition of AChE activity by phenolics has been reported for related insecticidal effects (Pang et al., 2012; Pinho et al., 2013). Therefore, plant phenolics may be considered AChE antagonists (Pinho et al., 2013). Similarly, naphthoquinones (Changwong et al., 2012) and phenylethanoids (Georgiev et al., 2011) have been shown to act as AChE inhibitors. Verbascoside 31 and its methyl ether martynoside 32 showed cholinesterase inhibitory activity; in contrast, naphthoquinones (Fig. 8) appeared to be most active at minor concentrations (Table 6), these effects were similar to observed in the insecticidal inhibitory activity (Table 5). It is obvious that the nature of the ether substituent on the phenyl moiety of cinnamic and caffeic acids, respectively, plays an important role for the insecticidal and cholinesterase activities of the phenylethanoids, F-5, F-7 and mixture M40 . In anticholinesterase assays, the most active compounds (hydroxynaphthoquinone 43 and verbascoside 31) contained a small and relatively hydrophilic hydroxyl group, whereas dunnione 41 and martynoside 32, with a bulky and more lipophilic ether group, exhibited minor activity level (Céspedes et al., 2013b). These results conﬁrm previous ﬁndings on the quantitative structure–activity relationship of phenolic derivatives, namely that the antifeedant activity of the respective natural product depends on the polarity and on the size of the ether substituents (Uriarte-Pueyo and Calvo, 2011; Pang et al., 2012; Pinho et al., 2013; Georgiev et al., 2011; Changwong et al., 2012; Park, 2010) (Fig. 9). With respect to the phenylethanoids, our ﬁndings indicate that the presence of a methoxy substituent in the aromatic rings seems to be the cause of increasing or decreasing inhibitory activities of BChE as shown by compounds 31 and 32 with 70.0% and 29.5% of inhibition at 200.0 lg/ml, respectively; and 45.9% and 19.9% of inhibition at 100.0 lg/ml, respectively (Table 6) in contrast to

Table 6 AChE and BChE inhibitory activity of compounds extracts and reference compounds IC50 [μg/mL].a Samples tested

AChE

BChE

Inhibition type

n-Hexane F-3 F-4 F-5 Ethyl acetate F-7 5 6 Ursolic acid 43 41 31 32 MeOH-residue M10 M20 M30 M40 Quercetin Forsythoside B Leucosceptoside B Galanthamine

n.d. n.d. n.d. 90.7 20.1 15.7 n.d. n.d. n.d. n.d. n.d. 189.8 n.d. n.d. n.d. n.d. 102.5 19.7 53.7 n.d. 20.1 13.2

20.4 n.d. n.d. 59.6 18.2 14.7 n.d. n.d. 168.1 142.4 n.d. 105.9 n.d. 50.7 n.d. n.d. 95.1 24.5 79.8 27.6 n.d. 7.3

Mixed – – Competitive Mixed Competitive – – Competitive Competitive – Competitive – Mixed – – Mixed Mixed Competitive Mixed Competitive Mixed/non-competitive

a Values correspond to average of three experiments; units of concentrations are expressed as [μg/mL]. n.d. – Not determined (Céspedes et al., 2013b).

methoxylated leucosceptoside B from V. xanthophoeniceum, where the methyl ether in the aromatic rings of the phenylethanoids structure decreased the antiBChE activity, being more active against AChE (Georgiev et al., 2011). Our results were notably different from those of the study of Georgiev's group: they obtained 9.22% and 39.19% of inhibition of BChE at 100 and 200 mg/ml and we obtained 45.9% and 70.0% of inhibition at 100 and 200 mg/ml, respectively. This difference may be attributed to the origin of cholinesterase sources (Legay, 2000).

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0.50 0.45 0.40

Normalized Intensity

0.35 0.30 0.25 0.20 0.15 0.10 0.05 0 8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

Chemical Shift (ppm) Fig. 9. Representative H-NMR of ethyl acetate extracts.

The high activity against AChE and BChE from mixture M40 (31 þ32) and F-7 could be due to synergistic effects of the respective components. It is important to mention that the innocuous effect of verbascoside 31 was reported showing that it was not genotoxic for Drosophila wing spot test crosses (SantosCruz et al., 2012). Those effects are in accordance with other results in D. melanogaster assays (Heres-Pulido et al., 2005) and show that is possible to use verbascoside 31 on live organisms without secondary effects. In relation to naphthoquinones, our results are moderate, but are somewhat consistent with those reported by Changwong et al. (2012). The strong activity against AChE and BChE observed with mixture M30 and F-5 also could be attributed to synergistic effects by the components. Additionally, F-7, hydroxynaphthoquinone and verbascoside proved be the most active samples against tyrosinase. Fraction F-7 had the highest activity level; this fraction inhibited tyrosinase as well as two digestive proteinases used as a protease enzyme model (Muñoz et al., 2013b). In this fraction, we have determined the presence of ﬂavonoids and iridoids. The inhibitory effect of ﬂavonoids on cholinesterases and tyrosinase has been reported by many authors (Masuoka et al., 2012; Cao et al., 2013; Orhan et al., 2007, 2013; Orhan, 2013; Sheng et al., 2009; Zhao et al., 2013). It may be logical to assume that phenolics such as verbascoside and hydroxynaphthoquinone are synthesized during preliminary attacks on C. integrifolia s.l. by an unidentiﬁed insect observed on these plants, which after several hours searches for another plant of different species (Muñoz et al., 2013b). 3.3. Antibacterial activity Gram-positive and Gram-negative bacteria were used in the antibacterial bioassays. At different levels, ﬂavonoids, ferulic acid and diterpenes 5 and 6 were active against almost all Gram-positive and -negative bacteria assayed. The mixture M3 (isorhamnetinþferulic acid) was the most active sample. Tables 1 and 3 show the inhibitory zone diameter (mm) of the

M1 (quercetinþferulic acid), M2 (kaempferolþferulic acid), M3 (isorhamnetinþ ferulic acid), M4 (myricetinþferulic acid) and M5 (dunnioneþ gallic acid) against enterobacteria and fungi, respectively. M3 showed an inhibitory effect against E. coli, E. agglomerans, Salmonella sp., B. subtilis, S. aureus and S. lutea; this effect was noticeably greater in E. coli, E. agglomerans and Salmonella sp than the other bacteria assayed; the inhibition diameter was 18 mm (40% at 72 h) against E. coli, 40.0 mm against E. agglomerans, and 16.3 mm against Salmonella sp., and (712 mm) against B. subtilis, S. aureus and S. lutea, (Table 1). These results show that the M3 extract and their components had signiﬁcant inhibitory activity against Gram-negative bacteria and have moderate activity against Gram-positive bacteria (there was no zone of inhibition at a concentration of o400 mg/disk) (data not shown). The mixtures M3, M5 and diterpenes were also assayed against R. solani, F. sporotrichum, F. moniliforme, and A. niger (Table 3). The activity level shown by dunnione and M5 against these fungi was relatively high as compared with the positive control (ketoconazole) (data not shown); M5 was the most active of all samples assayed in a manner similar to that reported by Khambay et al., 2003 (Table 3). In addition, compound 13 and M2 exhibited total inhibition (100%) against these fungi at concentrations above 400 mg/disk (data not shown). A similar effect was shown by M3, which completely inhibited the mycelial growth of these fungi above 4000 mg/disk. The growth of A. niger, F. monoliforme, F. sporotrichum, and R. solani was completely inhibited by M5 in a range of 1500–5000 mg/disk, and partial inhibition was observed between 500–1500 (480%) and between 100–1000 (450%) (data not shown). In view of the strong activity of M3 against bacteria, and M5 against fungi, these samples and several compounds were assayed against different fungal and bacterial strains and their minimum inhibitory concentration (MIC), minimum bactericide concentration (MBC), FC50, and MFC values were obtained (Tables 2 and 4). Nonetheless, ferulic acid and isorhamnetin, a phenolic acid and an O-methylﬂavonoid, respectively, showed a good activity against

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bacteria. These facts suggest that the vicinal methoxy moiety to hydroxyl positions could be important for antibacterial and antifungal activity. In this case, it is probable that the methoxy group, together with the vicinal hydroxyl groups, electronically activate the aromatic ring and increase the lipophilicity of the compounds; consequently, these structural features may be responsible for inhibiting the growth of bacteria strains tested by these compounds. Additionally, the fungicidal evaluation showed that the mixture of dunnione with gallic acid M5, was most active followed by M3 in a signiﬁcant minor potency (Table 3). For this reason, the FC50 and MFC values were determined; this result shows that M3 and M5 are the most potent fungicides assayed in this study. Their FC50 and MFC values against R. solani, F. sporotrichum, F. monoliforme, A. niger, and T. mentagrophytes are presented in Table 4. The values for these compounds are close to those of the positive control; this renders them quite important due to the possible use of these substances as nutraceuticals. In the Calceolaria isolates F-7 showed a composition mainly of naphthoquinones, phenolic acids and ﬂavonoids, gallic acid, kaempferol and quercetin (435.0%) (the most abundant), followed by naphthoquinones (420.0%), diterpenes (410.0%), triterpenes (5.0%), and other secondary metabolites in a very minor amount (1.5%). Many of these compounds have also been previously reported for other species of genus Calceolaria (Di Fabio et al., 1995; Céspedes et al., 2013c). Interestingly, the total MeOH extract of C. integrifolia x C. talcana and C. integrifolia have the largest percentage of phenolic compounds. The hybrid species biosynthesized larger amounts of phenolics compounds. This could explain the strong resistance of these species to the attack of pathogenic organisms as they exhibited a strong inhibition to bacterial and fungal growth, and in the insect model used in this study, showed the greatest antifeedant activities. These results suggest a possible synergistic effect of secondary metabolites present in these species. The activity showed by these phenolics compounds could be due to the lipophilic and antioxidant properties of the components, based on data reported by Schulz and Nicholas (2000, 2002). The compounds assayed had an inhibitory effect against some human and phytopathogenic fungi. The presence of phenolic compounds has already been described in many botanical families, and it is well-established that these compounds have different biological properties. Nonetheless, to the best of our knowledge, until now, the fungitoxic and bactericidal activities of extracts from this plant have not been described completely in the literature. The results show that the activity of Calceolaria isolates may be explained by their lipophilic properties as they have an activity comparable with positive control. In summary, when the antifungal activity was assayed with methoxylated compounds, the FC50 values were ﬁve and 10 times higher than those observed with the positive control ketoconazole. This result suggests a synergistic effect of the gallic acid/ ferulic acid and/or naphthoquinones/ﬂavonoid composition of the extract, which has not been observed until now. Synergistic effects are one of the most important characteristics exhibited by natural extracts. This increases the efﬁcacy of the components in contrast to that which could be obtained with an equivalent amount of the active constituents alone. Additional synergistic effects of these compounds are under study. These results indicate that extract of Calceolaria species act on bacteria and phytopathogenic fungi. This could indicate that this plant can play an important role in food preservation and food preparation and as an excellent source of biopesticides. 3.4. Synergistic effects of gallic acid on ﬂavonoids Each antimicrobial phytochemical plays important role in the defense against pathogen attacks in living plants, but their individual

403

activities are usually not potent enough to be considered for practical use. This is an important question when the biological activities of phytochemicals, especially their antimicrobial activity, is considered. Hence, studies to enhance their biological activities are needed. Combining two or more phytochemicals in order to enhance total biological activities is a promising strategy. Combining more than two compounds may be even better than a single antimicrobial compound to prevent the development of resistance mechanisms in microorganisms, in addition to enhancing and/or broadening the total biological activity. However, a rational basis for this approach is still in an embryonic stage. As a test, the antimicrobial activities of anethole and eugenol were ﬁrst tested against selected microorganisms by a two-fold serial broth dilution method. Based on the MIC values, both phenylpropanoids have broad spectra and nearly comparable activity. Neither anethole nor eugenol alone may qualify as an effective antimicrobial agent. Studies to enhance the observed activity are needed. However, the fungicidal activity of anethole against S. cerevisiae can be enhanced by inclusion of a sublethal amount of polygodial (Kubo and Himejima, 1991). Explanation of this synergism on a molecular basis remains unclear (Trombetta et al., 2002). Against C. albicans, a sublethal concentration of anethole was reported to enhance the fungicidal activity of polygodial 128-fold (Kubo and Himejima, 1991). In our experiments, mixtures of quercetin, kaempferol, isorhamnetin, ferulic and gallic acid acts in similar manner to other synergic mixtures of antifungal agents. For example, the fungicidal activity of quercetin, kaempferol, and isorhamnetin against A. niger, F. moniliforme, F. sporotrichum, R. solani and T. mentagrophytes was enhanced 8fold in combination with a sublethal amount (equivalent to ½ MFC) of gallic acid. Thus, the MFC of ﬂavonoids was lowered from 2000 to 250 mg/mL in combination with 100 mg/mL of gallic acid. As mentioned previously ﬂavonoids alone did not exhibit any fungicidal activity up to 1600 mg/mL, but the combination with 50 mg/mL of gallic acid was fungicidal. On the other hand, half of the MIC of gallic acid alone did not show any growth inhibitory effects. Gallic acid seemed to limit recovery from the ﬂavonoid-induced damage. In addition, the ﬂavonoids alone decreased the number of colonyforming unit (CFU) for the ﬁrst 24 h but recovered thereafter. Because adaptation to ﬂavonoid stress was not observed under the treated conditions, protein synthesis may be necessary for this process. Further investigations are in progress.

3.5. Insecticidal activity of Calceolaria species The n-hexane extract of aerial parts of Calceolaria talcana afforded diterpenes 1–6 together with other known triterpenes and naphthoquinones. In addition to n-hexane extract, diterpenes, triterpenes and several fractions showed insecticidal and insect growth regulatory activity against fall armyworm (S. frugiperda, Lepidoptera: Noctuidae), and fruit ﬂy (Drosophila melanogaster, Diptera: Drosophilidae) insect pests of corn and fruits, respectively. The most active samples, a mixture (M10 ) of the diterpenes 5 þ6 (6:4), showed insecticidal activity between 10 and 20 ppm. Additionally, a mixture of triterpenes (M20 ) (see Muñoz et al., 2013b), the fraction 5 (F-5), a mixture of two naphthoquinones 41–43 (M30 ), and the n-hexane extract, all these samples separately had signiﬁcant effects between 20.0 and 35.0 ppm in diets. Almost all samples were insecticidal with lethal doses between 35 and 50 ppm. Our results indicate that these compounds at low concentrations appear to have selective effects on the preemergence metabolism of the insect. The results were fully comparable to known natural insect growth inhibitors such as gedunin, MeOH-Cedrela and MeOH-Yucca extracts and have a possible role as natural insecticidal agents (Muñoz et al., 2013b).

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It is important to note that similar insect growth regulatory activity on S. frugiperda (fall armyworm) was shown by other secondary metabolites (Céspedes et al., 2006). It has been reported that certain diterpenes have insect antifeedant activity. Diterpenes, triterpenes and naphthoquinones have been detected in signiﬁcant amounts in Ajuga (Coll and Tandron, 2007), Salvia spp. (Simmonds et al., 1996), and Gutierrezia microcephala (Calderón et al., 2001) with signiﬁcant antifeedant and insecticidal activity, similarly as was observed with our compounds. On the other hand, there are no insecticidal reports of pimaradienes and abietatrienes, but only for antibacterial activity of some of them (Falcao et al., 2006; Woldemichael et al., 2003). However, antifeedant activities have been reported for clerodane-type diterpenoids against Spodoptera littoralis (Simmonds et al., 1996). Thus, the presence of hydroxyl (hydroxypimaradiene and hydroxynaphthoquinone) or vinyl functionalities, in a manner similar to compounds previously reported seems to be necessary for insecticidal activity (Simmonds et al., 1996; Mullin et al., 1997; Céspedes et al., 2006). Former experimental observations suggest that our compounds may bind to and inhibit the proteinases, ETH and polyphenol oxidases (PPO), as was showed by the acute toxicity and growth inhibitory activity. These targets have been demonstrated for other compounds of natural origin (Kessler and Baldwin, 2002). We are presently studying elucidation of the activities, sites and mechanisms of action. These activities probably correspond to a combination of antifeedant action, which is known at the molecular level to involve the following targets: midgut phenol oxidase, proteinase, ETH, tyrosinase or other PPOs and cuticle synthesis inhibition. The active compounds also have moulting sclerotization toxicity, as has been found for other natural compounds (Kubo et al., 2003a, 2003b, Kubo, 1997, Céspedes et al., 2000, 2006; Torres et al., 2003) and extracts (Feng et al., 1995). In summary, the insecticidal activity of extracts from aerial parts of C. integrifolia s.l. may involve a synergistic effect shown by the ecdysone-like activity of the extracts in the test system used in this investigation. Based on these results, we suggest that insect growth inhibitory activity of mixtures M10 , M20 and M30 could be caused not only by a strong inhibitor, but by a synergistic effect of the mixtures involved. Signiﬁcant inhibition of insect growth activity by these compounds suggests that these samples may serve as efﬁcient IGR agents. Compounds 5, 6, ursolic acid, 43, as well as M10 , M20 and M30 proved to have potent insecticidal and growth inhibitory activities. The nature of the substituent at the acidic group plays an important role for the insecticidal activity and IGR of these compounds. The most active diterpenoids compound contained a small and relatively lipophilic group. Additionally, naphthoquinone compound with one hydrophilic hydroxyl group and two carbonyls exhibited one of the largest activity levels (Akhtar et al., 2012). These plant isolates are efﬁcient insect growth regulators (IGR), with activity similar to that of phytoecdysteroids, as was evidenced by their signiﬁcant inhibition of molting processes.

4. Concluding remarks Our ﬁndings show that acute toxicity and insect growth inhibition observed may be due to the inhibition of tyrosinase, acetylcholinesterase or butyrylcholinesterase enzymes. These targets have also been demonstrated for similar compounds (Céspedes et al., 2013b; Muñoz et al., 2013c; Alout et al., 2012; Cao et al., 2013; Changwong et al., 2012; Georgiev et al., 2011; Orhan et al., 2007, 2013; Orhan, 2013). In summary, the insecticidal activity of n-hexane, ethyl acetate extracts, F-5, and F-7 fractions from aerial parts of selected members of the C. integrifolia s.l. complex may be due to synergistic effects shown by the components of the mixtures in the test

system used in this investigation. Comparing insecticidal activities of compounds from Calceolaria species, these samples exhibited potent antifeedant activity against S. frugiperda and D. melanogaster (Muñoz et al., 2013a, 2013b). These results are supportive of the potency of the n-hexane and ethyl acetate extracts from Calceolaria complex as inhibitors of insect cholinesterases (Table 6). The sites, mechanism and mode of action of these compounds are presently under investigation. This activity may be due to enzyme inhibition or a combination of antifeedant action as midgut esterase inhibition, postdigestive toxicity and other PPO binding with the isolates, since these targets were also found for other secondary metabolites (Céspedes et al., 2006, 2013a, 2013b; Casida, 2009; Casida and Durkin, 2013; Green et al., 2013; Muñoz et al., 2013c; Orhan, 2013). In addition, the presence of methoxy group seems to be important for increasing or decreasing activity as was shown for the most potent compounds in this study: isorhamnetin 27, verbascoside 31 and dunnione 41. Furthermore, the great inanition (extreme weakness from lack of nourishment) observed in insect growth regulatory and insecticidal assays may be due to inhibition of cholinesterases as well. The activity of this plant and its metabolites, fractions and n-hexane and ethyl acetate extracts is comparable to that of the insect growth regulator gedunin, which suggests potential for further development as biopesticides that prevent the emergence of resistance in insect pest control and avoid the use of POPs. The activity of this plant, their metabolites and mixtures could to help to explain its resistance to pathogen attack.

Conﬂict of interest The authors declare that there are no conﬂicts of interest and they have no actual or potential competing ﬁnancial interests.

Ethics committee approval The study was reviewed and approved, prior to its conduct, by The Committee of Bioethics of University of Bío Bío, Chile and UNAM, Mexico.

Acknowledgments The authors are indebted to Drs. J.G. Avila, P. Aqueveque and J.R. Salazar for performing the antimicrobial and insecticidal assay in part. CLC is grateful to CONICYT Chile through FONDECYT Program Grants # 1101003 and # 1130242 for ﬁnancial support in part. The authors wish to thank to Ana Ma. Bores for technical assistance, FES-Iztacala, UNAM, Mexico. Technicians from Dept. of Chemistry at CINVESTAV-IPN, Mexico, by HPLC–MS–NMR hyphenated techniques and to Miss Jessica Cote for language revision of the text. LY acknowledge to Projeto Temático Fapesp 09/51850-9 and to technicians of Instituto de Quimica, University of São Paulo, Brasil, by NMR resonances. IK and CLC acknowledge the support in part by UCBerkeley/CONICYT-Chile Seed Grant. References Akhtar, Y., Yeoung, Y.R., Isman, M.B., 2008. Comparative bioactivity of selected extracts from Meliaceae and some commercial botanical insecticides against two noctuid caterpillars, Trichoplusia ni and Pseudaletia unipuncta. Phytochem. Rev. 7, 77–88. Akhtar, Y., Isman, M.B., Lee, Ch-H., Lee, S.-G., Lee, H.-S., 2012. Toxicity of quinones against two-spotted spider mite and three species of aphids in laboratory and green house conditions. Ind. Crop Prod. 37 (1), 536–541.

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Biopesticides from plants: Calceolaria integrifolia s.l

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