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Biopesticides from plants: Calceolaria integrifolia s.l

Biopesticides from plants: Calceolaria integrifolia s.l
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  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 b 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 a r t i c l e i n f o  Article history: Received 30 January 2014Received in revised form31 March 2014Accepted 2 April 2014 Keywords: InsecticidalAntifungal activityCalceolariaceaeIridoidsFlavonoids a b s t r a c t 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 thecrop protection role of pesticides and other techniques, i.e. biopesticides, plant extracts, preventionmethods, organic methods, evaluation of plant resistance to certain pests under an integrated pestmanagement (IPM), could improve the risks and bene fi ts which must be assessed on a sound scienti fi cbasis. For this directive it is crucial to bring about a signi fi cant reduction in the use of chemicalpesticides, not least through the promotion of sustainable alternative solutions such as organic farmingand IPM. Biopesticides are derived from natural materials such as animals, plants, bacteria, and certainminerals. Most of them are biodegradable in relatively short periods of time. On this regard, substancesfrom  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. Somehave medicinal applications. Other taxa of   Calceolaria  are toxic to insects and resistant to microbialattack. These properties are probably associated with the presence of terpenes, iridoids,  fl avonoids,naphthoquinones and phenylpropanoids previously demonstrated to have interesting biological activ-ities. In this article a comprehensive evaluation of the potential utilization of   Calceolaria  species as asource of biopesticides is made. The chemical pro fi le of selected members of the Chilean  Calceolariaintegrifolia  sensu lato complex represents a signi fi cant addition to previous studies. New secondarymetabolites were isolated, identi fi ed and tested for their antifeedant, insect growth regulation andinsecticidal activities against  Spodoptera frugiperda  and  Drosophila melanogaster  . These species serve as amodel of insect pests using conventional procedures. Additionally, bactericidal and fungicidal activitywere determined. Dunnione mixed with gallic acid was the most active fungistatic and fungicidalcombination encountered. Several compounds as isorhamnetin, combined with ferulic and gallic acidquickly reduced cell viability, but cell viability was recovered quickly and did not differ from that of thecontrol. The effect of these mixtures on cultures of   Aspergillus niger, Fusarium moniliforme, Fusariumsporotrichum ,  Rhizoctonia solani , and  Trichophyton mentagrophytes,  was sublethal. However, whenfungistatic isorhamnetin and dunnione were combined with sublethal amounts of both ferulic andgallic acid, respectively, strong fungicidal activity against theses strains was observed. Thus, dunnionecombined with gallic acid completely restricted the recovery of cell viability. This apparent synergisticeffect was probably due to the blockade of the recovery process from induced-stress. The same series of phenolics (iridoids,  fl avonoids, naphthoquinones and phenylpropanoids) were also tested against theGram-negative bacteria  Escherichia coli, Enterobacter agglomerans,  and  Salmonella typhi,  and agaisnt theGram-positive bacteria  Bacillus subtilis, Sarcinia lutea,  and  Staphyllococcus aureus  and their effectscompared with those that of kanamycin. Mixtures of isorhamnetin/dunnione/kaempferol/ferulic/gallicacid in various combinations were found to have the most potent bactericidal and fungicidal activitywith MFC between 10 and 50  μ g/ml. Quercetin was found to be the most potent fungistatic singlecompound with an MIC of 15  m g/ml. A time-kill curve study showed that quercetin was fungicidal Contents lists available at ScienceDirect journal homepage: Environmental Research &  2014 Elsevier Inc. All rights reserved. n Corresponding author. E-mail address: (C.L. Céspedes).Environmental Research 132 (2014) 391 – 406  against fungi assayed at any growth stage. This antifungal activity was slightly enhanced by combinationwith gallic acid. The primary antifungal action of the mixtures assayed likely comes from their ability toact as nonionic surfactants that disrupt the function of native membrane-associated proteins. Hence, theantifungal activity of isorhamnetin and other  O -methyl  fl avonols appears to be mediated by biophysicalprocesses. Maximum activity is obtained when the balance between hydrophilic and hydrophobicportions of the molecules of the mixtures becomes the most appropriate. Diterpenes,  fl avonoids,phenylpropanoids, iridoids and phenolic acids were identi fi ed by chromatographic procedures (HPLC-DAD), ESI-MS, and NMR hyphenated techniques. &  2014 Elsevier Inc. All rights reserved. 1. Introduction The effects of persistent organic pollutants (POPs) on humans andbiodiversity are multiple and varied. At present, environmentally-friendly pesticides are strongly preferred to POPs. It is noteworthythat the crop protection role of pesticides, and other techniques,i.e. biopesticides, plant extracts, prevention methods, organicmethods and plant resistance to certain pests under an integratedpest management (IPM). The risks and bene fi ts of these must beassessed on a sound scienti fi c basis. It is crucial for this directive tobring about a signi fi cant reduction in the use of chemical pesti-cides, not least through the promotion of sustainable alternativesolutions such as organic farming and IPM (Rosner and Markowitz,2013; Rhodes et al., 2013).Biopesticides are derived from natural materials such as ani-mals, plants, bacteria, and certain minerals. They are usuallybiodegradable in short periods of time. Plants, the most commonsource of biopesticides, produce a great variety of secondarymetabolites that lack apparent function in physiological or bio-chemical processes; these compounds (or allelochemicals) areimportant in mediating interactions between plants and theirbiotic environment (Berenbaum, 1989, 2002; Kessler and Baldwin, 2002). Some can be used as lead molecules forthe development of protective agents against insects and fungi(Kubo et al., 1981, 1993, 2000, 2003a, 2003b), and enzymeinhibitors (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 fornew sources of biologically active natural products, with newmodes of action (Conner et al., 2000; Eisner et al., 2000; Meinwald,2001), characteristics that which enhance their value as practicalpesticides (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 membersof the  Calceolaria integrifolia  s.l. complex. These substances providedefense mechanisms against bacterial, fungal and herbivore pre-dator attacks in these plants. Terpenes, phenolics and othercompounds are accumulated in aerial parts, mainly in leaves andtrichomes, resulting in unique biopesticides from these plants(Céspedes et al., 2013b, 2013c; Muñoz et al., 2013a, 2013b, 2013c).Plants from the genus  Calceolaria  (Calceolariaceae; formerlyScrophulariaceae) are distributed in temperate and tropicalregions of New Zealand and Central and South America. (DiFabio et al., 1995; Garbarino et al., 2000, 2004). Several speciesof   Calceolaria  are used as ornamental plants and in traditionalmedicine (Falcao et al., 2006). The aerial parts of these plants areused in Chile and South America for their analgesic, digestive anddiuretic properties (Sacchetti et al.,1999), and as antimicrobials forstomach ailments (Sacchetti et al., 1999; Garbarino et al., 2004).Some species of this genus have substances with potential uses asinsecticides (Khambay and Jewess, 2000), against tuberculosis(Woldemichael et al., 2003) and as growth inhibitors of TA3 tumorcells and methotrexate resistant TA3 cells (Morello et al., 1995).Flavonoids, glucophenylpropanoids, and diterpenes have pre-viously been identi fi ed 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 genusoccur 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. integrifolias.str  ,  C. rubiginosa ,  C. talcana ,  C. verbascifolia , and  C. viscosissima .Each of these species has its own characteristic distributionpattern, 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), commonlyknown as  “ zapatito de doncella ”  or  “ capachito de hoja larga ” , arestrong erect shrubs, 150 cm tall or sometimes smaller with fragileascending branches, internodes of 2 – 8 cm, and in fl orescences anddistal parts of stems that are glutinous or velutinous with erecthairs (Ehrhart, 2000, 2005).In previous reports on the antifeedant, insect growth regulatory(igr) and insecticidal activities induced by a series of phenolicand terpenes compounds from  Calceolaria  species, their maximumantifeedant and igr activity was shown to depend on thehydrophobic alkyl moieties and/or from the hydrophilic hydroxylgroups (Céspedes et al., 2013b, 2013c; Muñoz et al., 2013c).These compounds also possessed inhibitory effects on tyrosinaseand acetylcholinesterase enzymes (Muñoz et al., 2013c; Céspedeset al., 2013b).On the past persistent organic pollutants mostly of syntheticorigin (i.e. POPs) have been widely used, application of thesesubstances has produced a strong impact on the environment, inmany cases strains resistant to these compounds has resulted.Organic molecules of botanical srcin may offer a safe and moreef  fi cient source of compounds for pest management because mostare environmentally friendlier resulting in an excellent alternativeto POPs (Kubo, 1997). As many of them have low mammaliantoxicity, limited persistence in the environment, and enhancedbiodegradability the use of secondary metabolites for pest controlhas generated a growing interest in the search for new sourcesof 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 globalagricultural systems have been of synthetic srcin such as carba-mates, halogenated organic and organophosphorous (OP) com-pounds. Overuse has resulted in the generation of new strainsof pests resistant to the srcinal pesticides. The development of resistances is frequently related to modi fi cation of receptorsinvolved in the mechanisms and targets of action of certainmolecules (Pang et al., 2012; Alout et al., 2012; Casida andDurkin, 2013) many of these pesticides target acetylcholinesterase.As a result of resistance, the scienti fi c community has synthe-sized many new organic molecules with this target of action,resulting in dangerous health effects for animals. Acute or chronic C.L. Céspedes et al. / Environmental Research 132 (2014) 391 – 406 392  poisoning caused by pesticides is a problem in many countriesworldwide especially in developing countries (Francis, 2006;Fournier, 2005; Casida and Durkin, 2013; Green et al., 2013;Fournier and Mutero, 1994; Feyereisen, 1995). The search fornew botanical pesticides (biopesticides) which have this target of action and remain harmless to animals and humans is relevanttoday. Our interest is centered on the study of shrubs belonging tothe family Calceolariaceae, due to their notable resistance topathogen attack observed in nature (Céspedes et al., 2013a) andtheir uses as medicinal plants.Limited availability of plant material of   C. integrifolia  s.l. hasrestricted our initial attempts to study the defense mechanismagainst pathogen attack on a molecular level. Therefore, based onprevious results observed for the effects of extracts as insectgrowth inhibitors (Céspedes et al., 2013b; Muñoz et al., 2013a,2013b), this study highlights bioactive phenolics isolated fromselected plant species of the  C. integrifolia  s.l. complex as inhibitorsof insects and fungi. Additionally, studies in  Calceolaria  speciesfrom Americas show the presence of many substances withagrichemical applications and pharmacological potential (Falcaoet 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 orbiological activity of plants of the  C. integrifolia  sensu lato complexhave 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 investi- gated the sites and mechanisms of action of fungicidal, insecticidaland/or insect growth regulatory activity indicating that differentsecondary metabolites from these plants are enzymatic andmetabolic 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 antifeedanteffects 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 havepreviously demonstrated that diverse secondary metabolites havedifferent sites of action and different molecular targets when theyinteract with enzymes and metamorphosis processes (Céspedes etal., 2006, 2013a, 2013b, 2013c). The general objective of this study was to establish thefungicidal and bactericidal activity of fractions, mixtures and purecompounds isolated from  n -hexane and ethyl acetate extracts andalso to measure the effect of these compounds on the metamor-phosis of insect pest models. Several secondary metabolites fromother  Calceolaria  species have shown biocidal activities Falcaoet al., 2006; Woldemichael et al., 2003; Céspedes et al., 2013c; Muñoz et al., 2013c and their occurrence in Chilean Calceolaria-ceae has been reported (Céspedes et al., 2013c; Garbarino et al.,2000, 2004). The long-term goal is to examine the role of thephytochemicals of this complex of species in the fungistatic/fungicidal effects and in the inhibitory behavior on growth anddevelopment 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 insearch of botanicals for potential use as biopesticides.In continuation with our investigation this is a report on thebactericidal, 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 themost polar of the ethyl acetate extract F-7, a mixture M4 of naphthoquinones 6, 7 and iridoids and  fl avonoids were assayedagainst  D. melanogaster   (fruit fl y) and  S. frugiperda  (fall armyworm)and aspects such as mortality, rate of development, time of pupation, adult emergence and deformities were measured. Thesedata were compared with those of gedunin,  Yucca  and  Cedrela MeOH extracts, known growth inhibitors of   S. frugiperda  (Céspedeset al., 2000, 2006; Isman, 2006; Torres et al., 2003). Becausepreviously gathered information about this genus and our own fi eld observations indicated that these plant species appear topossess a strong resistance to pathogen attack in the  fi eld, weundertook 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 theroadside 4.7 km NW of Con fl uencia on the road to Trehuaco on the north shore of the Itata River (36 1 37 0 21 ″ S, 72 1 28 0 16 ″ W, elev. 172 m), Ñuble province, VIII Región,  Table 1 Antibacterial inhibitory activities a of compounds and mixtures from  C. integrifolia s.l.  on growth inhibition of bacteria (diameter in mm). a Sample Gram negative Gram positive E. coli E. agglomerans Salmonella  sp.  B. subtilis S. aureus S. lutea M1  4.1 7 0.6a 3.9 7 0.4a 4.0 7 0.45a 4.5 7 0.45a 5.1 7 0.55a 4.9 7 03aquercetin 3.9 7 0.4a 4.1 7 0.6b 4.1 7 0.5a 4.8 7 0.5a 4.9 7 0.46a 4.5 7 0.4a M2  13.95 7 1.6b 10.5 7 1.6b 9.9 7 1.2b 0 b,c 0 b 0 b kaempferol 9.3 7 0.7a 10.0 7 1.6b 8.5 7 0.9b 4.1 7 0.3a 4.9 7 0.5a 4.5 7 0.7a M3  18.1 7 3.1c 40.0 7 3.6d 16.3 7 2.4b 12.3 7 0.9b 13.2 7 1.7b 14.5 7 1.4bisorhamnetin 12.5 7 2.6b 27.8 7 1.8c 16.0 7 1.6b 10.0 7 0.4b 11.0 7 1.1b 12.9 7 1.1b M4  5.6 7 0.6a 4.5 7 0.7a 5.5 7 0.9b 3.9 7 0.2a 4.3 7 0.3a 4.1 7 0.2amyricetin 4.2 7 0.6a 4.1 7 0.3a 0 b 0 b 0 b 0 b M5  21.5 7 2.6c 42.0 7 3.7d 20.0 7 2.1c 12.3 7 1.2b 14.5 7 1.5b 11.0 7 0.9bdunnione 16.5 7 2.1c 36.5 7 2.8d 14.4 7 2.2b 6.0 7 0.6a 8.1 7 0.7a 7.9 7 0.3aFerulic acid 9.6 7 0.7a 12.3 7 1.1b 11.4 7 0.6b 9.9 7 0.9b 10.2 7 0.9b 15.0 7 1.3bDehydroabietinol 12.9 7 1.6b 15.0 7 1.6b 14.5 7 1.6b 0 b 0 b 0 b Abietatrien-3 β -ol 13.5 7 1.3b 15.8 7 1.2b 15.7 7 1.7b 0 b 0 b 0 b chloramphenicol 20.0 7 2.6c 25.2 7 1.4c 25.0 7 1.6c 28.4 7 3.5c 22.2 7 3.5c 37.5 7 2.9cKanamycin 25.5 7 1.4c 56.4 7 0.6e 30.1 7 1.1c 22.4 7 0.9c 49.6 7 4.3e 21.7 7 1.4c a Inhibitory effects at an equivalent concentration of 1600  μ g per disc with  M1  and  M4 , 800  μ g per disc with  M2  and 100  μ g per disc with  M3  and  M5  is represented asmm of growth; mean value of diameter of inhibition zone: mm 7 standard error, of   n ¼ 21 and its signi fi cant 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 after 7 standard error values are not signi fi cantly different in a Student – Newman – Keuls(SNK) (treatments are compared by concentration to control), 95 % Con fi dence limits. Negative control:  N,N  -Dimethylformamide (DMFA) 5  m l/disc. Positive controls:kanamycin and chloramphenicol 30  m g/disc.  c Activity not present (in this table are shown only the more signi fi cant inhibitory effects, above 4.0 mm). C.L. Céspedes et al. / Environmental Research 132 (2014) 391 – 406  393  Chile, in November, 2010.  C. integrifolia  was collected on the rural freeway M-80-Mfrom Cobquecura to Buchupureo (36 1 04 0 51 ″ S, 72 1 48 0 15 ″ W, elev. 82 m) Ñubleprovince, VIII Region, Chile, in November 2010.  C. talcana x integrifolia  was collectedon the rural freeway 126 from Quirihue to Cauquenes (36 1 07 0 16 ″ S, 72 1 27 0 01 ″ W,elev. 216 m), Ñuble-Linares Province, VII – VIII Region, Chile, in November 2011 and2012.  C. angustifolia  was collected along the freeway L-391 from Linares to NationalReserve  “ Los Bellotos del Melado ”  shore to Rio Ancoa (36 1 05 0 48 ″ S, 71 1 22 0 38 ″ W,elev. 451 m), Linares Province, VII Region, Chile, in November 2012. Voucherspecimens 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 theUniversity of Illinois, at Urbana – Champaign, IL, USA (ILL, Voucher DS-16243/44).The samples were identi fi ed 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 andextracted with methanol overnight; the process was repeated  fi ve times. Theresulting methanol extract was concentrated at reduced pressure in a rotatoryevaporator at 40  1 C and 200 mb to yield a syrupy methanol extract (645 g). Aportion of the methanol extract (410 g) was dissolved in distilled water, dilutedwith 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 werecombined and concentrated under reduced pressure. Identical procedures werecarried out with CH 2 Cl 2  and ethyl acetate extracts.  2.3. Apparatus 1 H NMR spectra were recorded at 300 and 500 MHz,  13 C NMR at 75 and125 MHz respectively, on Bruker DPX 300 MHz and DRX500 MHz spectrometers,chemical shifts (ppm) are relative to (CH 3 ) 4 Si as internal reference. CDCl 3  andacetone- d 6   from Aldrich Chemical Co. were used as solvents, and couplingconstants are reported in Hz. IR spectra were obtained as KBr pellets on PerkinElmer 283-B and FT-IR Nicolet Magna 750 spectrophotometers. UV spectra of pure compounds were determined on a Shimadzu UV-160 and Spectronic modelGenesys 5 spectrophotometers; CHCl 3  was used as solvent. Optical rotations weremeasured on a JASCO DIP-360 spectropolarimeter; CHCl 3  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-LINEChamber model CX14601A, with adjustable Hi – Lo protection thermostats safe-guard samples.A Spectronic model Genesys 5 and a microplate reader Epoch-Biotek UV  – vis(200 – 999 nm) spectrophotometers were used to carry out the spectrophotometricmeasurements of the cholinesterase activity. EIMS and TOF data were determinedon a Q-TOF Waters and JEOL JMSAX505HA mass spectrometer at 70 eV. FABMSwere obtained on a JEOL JMS-SX102A mass spectrometer operated with anacceleration voltage of 10 kV. Samples were desorbed from a nitrobenzyl alcoholmatrix using 6 keV Xenon atoms. Column chromatography was carried out onKiesel-gel G (Merck, Darmstadt, Germany); TLC was performed on Si gel 60 F 254 .For hyphenated analyses the following were used:  1 H NMR and  13 C NMR spectra were recorded at 400 MHz, and 125 MHz, respectively on Avance 400 and600 MHz on NMR Bruker spectrometers, chemical shifts (ppm) are related to(CH 3 ) 4 Si as internal reference ( δ   0), CDCl 3 , MeOD and acetone- d 6   from AldrichChemical Co were used as solvents; coupling constants are quoted in Hz. GC/MSHP5989A, LC/MSD-TOF Agilent. Additionally, EIMS and TOF data were determinedon a Q-TOF Waters and JEOL JMSAX505HA mass spectrometer at 70 eV. FABMSwere obtained on a JEOL JMS-SX102A mass spectrometer operated with anacceleration voltage of 10 kV. Samples were desorbed from a nitrobenzyl alcoholmatrix 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 aHewlett Packard Series 1100 HPLC instrument with DAD and UV detectors set at320 nm. The column was obtained from Supelco Technologies C18 (150 mm  4.6 mm, 5  m m). The eluent was a mixture of 4% tetrahydrofuran in acetonitrile andwater (35:65, v/v) and contained 0.04% phosphoric acid. The  fl ow rates were 0.85,1.0 and 1.7 mL/min and the column temperature and pressure were 30  1 C and149 bar, respectively, and the injection volume was 20  m L. Verbascoside, linarin andsyringin served as standards.  2.4. Chemicals and solvents All reagents used were either A.R. grade or chromatographic grade, methanol,CH 2 Cl 2 , CHCl 3 , NaCl, KCl, NaOH, KOH, acetonitrile, water, butanol, silica gel GF 254 analytical chromatoplates, silica gel grade 60 (70 – 230, 60 Å) for column chromato-graphy;  n -hexane, and ethyl acetate were purchased from Merck-Chile, Santiagode Chile.  2.5. Extraction, isolation and puri  fi cation of diterpenes, iridoids,  fl avonoids,naphthoquinones and phenylpropanoids The concentrated  n -hexane extract was subjected to a silica gel columnchromatography (column diameter 2.5 cm, height 55 cm, 200 – 425 mesh) to yield5 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 , thesolvent mixture of   n -hexane/EtOAc (8:2) yielded 157 mg of a terpenoid mixturethat was analyzed by NMR, LC/MS and TLC analysis. The compounds were isolatedby preparative TLC and then puri fi ed 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-19- α -hydroxy-9-epi- ent  -7,15-isopimaradiene (1,10-cyclopropyl-9-epi- ent  -isopimarol) 5  (Chamy et al., 1991; Muñoz et al., 2013b), 19 α -hydroxy-8,11,13-abietatriene(dehydroabietinol)  6  (Chamy et al., 1987; Woldemichael et al., 2003; Muñozet al., 2013b), 2 α ,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 fromliterature. Their presence is hereby reported.Fraction  F-4  contains a mixture of   α -lupeol,  β -sitosterol, ursolic acid and acomplex mixture of sterols, their properties were identical to those literaturevalues and were not further studied. Aerial parts of sample Calceolaria spp. Dried Methanol Extract ( A ) n- hexaneMeOH/H 2 O Residue ( D )MeOH/H 2 Ovacuum chromatographyOn silica-gel F-1 F-3 F-4 Ethyl acetate Partition ( C )waxescarotenesditerpenes triterpenes naphthoquinonesphenylpropanoidsflavonoids F-2 F-5 F-6 F-7 Scheme 1.  Method of obtaining extracts, partitions, fractions, and compounds (Céspedes et al., 2013c). C.L. Céspedes et al. / Environmental Research 132 (2014) 391 – 406 394  Fraction  F-5  contained to a mixture of hydroxylnaphthoquinones  43 ,  α -dunnione  41 , and 2-acetoxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone  42  (Chamyet al., 1995a; Morello et al., 1995). These compounds were also isolated by fastextraction air-dried leaves with a CH 2 Cl 2 / n -hexane mixture (1:1) following theprocedure described by Mercado et al., 2010, with modi fi cations. Whole air-driedaerial stems and leaves (253 g) (without  fl owers), were soaked in a solvent mixture( n -hexane/CH 2 Cl 2 , 6:4). The solution was then  fi ltered (Whatman  fi lter paper # 1)and evaporated in a rotatory evaporator to yield 3.79 g of intensely colored cruderesidue. This mixture was dissolved in MeOH/CH 2 Cl 2  (7:3) samples and wereworked 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 wasfractionated by open cc using silica gel 60 F 254  (0.063 – 0.200-mm particle size,70 – 230 mesh ASTM, 60 Å pore diameter) and silica gel 60 F 254  precoated aluminumsheets (0.2 mm layer thickness) purchased from Merck (Darmstadt, Germany).Elution was carried out with hexane-ethyl acetate in different ratios and methanolwas added to increase the polarity of the gradient until 100% methanol wasreached. All fractions were analyzed by TLC using ceric sulfate as the visualizationsystem. Fractions obtained with CH 2 Cl 2 – MeOH (8:2) system contained verbasco-side  31  as the major compound (4.345 g, 7.23% from the ethyl acetate extract).Verbascoside, further was puri fi ed by silica gel TLC using CH 2 Cl 2 – MeOH (7:3) as theeluting system together martynoside which has a similar  R f   value.Chemical structures were determined by comparison with spectroscopic datafrom authentic samples and previously reported data (Domínguez et al., 2007;Céspedes et al., 2013c). These two compounds were further puri fi ed with SephadexLH-20 column chromatography yielded the two puri fi ed phenylpropanoid com-pounds (Muñoz et al., 2013c). The most polar part of the methanol extractablefraction 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 identi fi ed by HPLC-DAD – ESI-MSand NMR hyphenated techniques.Column chromatography of   F-6  from the same ethyl acetate extract overSephadex LH-20, when eluted with MeOH removed  fl avonoids from similaramounts of terpenoid material. HPLC-DAD – ESI-MS and NMR hyphenatedtechniques 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,  fl avonoids and phenylpropanoid glycosideswere determined by spectroscopic analyses, comparing data with those reported inliterature and directcomparisonwith authentic samplesusingTLC, HPLC-DAD – ESI-MSand 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  m L sample or standard (10 – 100  m M catechin) plus 150  m L diluted Folin – Ciocalteau reagent (1:4 reagent: water) was placed in each well of a96-well plate, and incubated at RT for 3 min. Following addition of 50  m L sodiumcarbonate (2:3 saturated sodium carbonate: water) and a further incubation of 2 hat room temperature, the absorbance was read at 725 nm. Results are expressedas  m mol 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  (quercetin þ ferulic acid),  M2  (kaempferol þ ferulic acid),  M3  (isorhamnetin þ ferulic acid),  M4 (myricetin þ ferulic acid) and  M5  (dunnione þ gallic acid)], ethyl acetate extract,diterpenes, iridoids, phenylpropanoids and  fl avonoids were determined. Because of the small amount of   13 – 18 , these compounds were not examined. For antibacterialactivity,paperdisks(6 mm,Whatman#1 fi lterpaper)wereimpregnatedwith10  m L of solution containing 100  m g of each compound to perform the test against the Gram-negative bacteria,  Escherichia coli (ATCC25922), Enterobacteragglomerans  (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 Micro-biología of FES-Cuautitlan (UNAM). Wild-Type 2: the strain was cultured anddonated 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 Cultivationwere maintained under freezing and, before the bioassays were done, werecultured in sterile Erlenmeyer  fl asks with 10 mL of YEB liquid medium ( fl askswere maintained in incubation for 72 h at 37  1 C). The bioassay was made by paper Fig. 1.  Chemical structures of diterpenes. Fig. 2.  Chemical structures of phenolic acids and iridoids. C.L. Céspedes et al. / Environmental Research 132 (2014) 391 – 406  395
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