Biochemical Systematics and Ecology 43 (2012) 101–107

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Biochemical Systematics and Ecology

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Chemical characterization of the aphid antifeedant extracts fromDittrichia viscosa and Ferula communis

Erjon Mamoci a, Ivana Cavoski b, Ma Fe Andres c, Carmen Elisa Díaz d,Azucena Gonzalez-Coloma c,*

aDipartimento di Science per l’Ambiente, Università degli Studi di Napoli “Parthenope” Centro Direzionale, Isola C4, 80143 Napoli, Italyb Istituto Agronomico Mediterraneo di Bari, Via Ceglie 9, 70010 Valenzano, Italyc Instituto de Ciencias Agrarias-CSIC, Serrano 115 Dpdo, 28006 Madrid, Spaind Instituto de Productos Naturales y Agrobiología-CSIC, Avda. Astrofísico F. Sánchez 3, 38206 La Laguna, Tenerife, Spain

a r t i c l e i n f o

Article history:Received 26 September 2011Accepted 19 February 2012Available online 30 March 2012

Keywords:Dittrichia viscosaFerula communisTomentosinInuviscolideFerulenolAntifeedantNematicidal

* Corresponding author.E-mail address: azu@ccma.csic.es (A. Gonzalez-C

0305-1978/$ – see front matter � 2012 Elsevier Ltddoi:10.1016/j.bse.2012.02.012

a b s t r a c t

The plant defensive activities of the hexane and ethanolic extracts from Dittrichia viscosaand Ferula communis against generalist herbivorous insects (Spodoptera littoralis andMyzuspersicae), are described here. Additionally we tested for their effects on nematodes(Meloidogyne javanica) and plants (Lactuca sativa and Lolium perenne). These plant extractsshowed variable biological effects, their aphid antifeedant action being the most impor-tant. The bioassay-guided search for aphid antifeedant compounds resulted in the isolationof tomentosin (1), inuviscolide (2) and ferulenol (3). Tomentosin (1) rich extracts ofD. viscosa were more active than ferulenol (3) rich extracts from F. communis.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Dittrichia viscosa (L.) Greuter (syn Inula viscosa (L.) (Asteraceae) is a perennial weed, native to the Mediterranean Basin(Pignatti, 1982). In traditional pharmacopoeia, D. viscosa has been used for different therapeutic purposes where it functionsas a diuretic, topical anti-inflammatory, hemostatic agent (Ali-Shtayeh et al., 1998; Lev and Amar, 2000) and it is used in thetreatment of diabetes (Yaniv et al., 1987) and gastroduodenal disorders (Lastra et al., 1983). Moreover, its extracts haveexhibited antimicrobial effects (Blanc et al., 2006; Cafarchia et al., 2002; Maoz and Neeman, 2000; Silva et al., 2005).Numerous compounds have been isolated and identified from D. viscosa such as flavonoids (Grande et al., 1985; Martin et al.,1988; Wollenweber et al., 1991; Máñez et al., 1999; Hernández et al., 2007), monoterpenes (Pérez-Alonso et al., 1996) tri-terpenoids, sesquiterpene lactones and sesquiterpene acids all structurally related to ilicic acid (Ulubelen et al., 1987; Sanzet al., 1991; Máñez et al., 1999; Grande and Bellido, 1992; Maoz et al., 1999; Zarga et al., 1998, 2003; Camacho et al., 2000;Fontana et al., 2007) and polyphenols (Danino et al., 2009). Extracts from this plant have also exhibited phytotoxic (Stephanouand Manetas, 1995; Levizou et al., 2002, 2004; Dor and Hershenhorn, 2004), nematicidal (Oka et al., 2001, 2006), antifungal(Abou-Jawdah et al., 2004;Wang et al., 2004; Cohen et al., 2002, 2006) and acaricidal activity (Mansour et al., 2004). D. viscosaplays an important role as the host plant for the aphid predator Macrolophus melanotoma (Perdikis et al., 2007), however itsdefensive chemistry against herbivore insects remains unknown.

oloma).

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E. Mamoci et al. / Biochemical Systematics and Ecology 43 (2012) 101–107102

The Genus Ferula belongs to the Apiaceae family and consist of approximately 130 species distributed throughout theMediterranean to Central Asia. In traditional pharmacopoeia, several species of this genus were used as tranquilizers and forthe treatment of digestive disorders, rheumatism, headache, arthritis, dizziness and toothache (Gonzalez and Barrera, 1995).Several phytochemical studies of Ferula communis L. have reported the presence of coumarino-sesquiterpenes (Appendinoet al., 1988), daucane esters (Miski and Mabry, 1985; Valle et al., 1987) and phenylpropanoid compounds (Miski andJakupovic, 1990) in fruits and roots. Compounds isolated from this plant were reported to have displayed different proper-ties such as anticoagulant (Monti et al., 2007), cytotoxic activity against various human tumor cell lines (ferulenol) (Boccaet al., 2002), apoptotic effects in the human Jurkat T-cell line (Macho et al., 2004), mitochondrial disfunction (Lahouelet al., 2007) and antimicrobial effects (Al-Yahya et al., 1998; Mossa et al., 2004; Appendino et al., 2004), however itsdefensive chemistry against insects and plant parasitic nematodes remains unknown.

The aim of this work was to screen D. viscosa and F. communis plant extracts for their plant defensive effects against insectherbivores (such as Spodoptera littoralis andMyzus persicae) and to identify and quantify the compounds responsible for theseactivities. Additionally we tested the nematicidal (Meloidogyne javanica) and phytotoxic (Lactuca sativa and Lolium perenne)activity of these extracts and compounds.

2. Materials and methods

2.1. General experimental procedures

1H and 13C NMR spectra weremeasured on a Bruker Advance-400 and AMX-500 spectrometers with pulsed-field gradient(400 and 500 MHz for 1H; 100 and 125 MHz for 13C), using CDCl3 as internal standard. Multiplicities of 13C signals weredetermined by distortionless enhancement of polarization transfer (DEPT) experiments. The programs used in two-dimensional (2D) NMR experiments (COSY, NOESY, HSQC and HMBC) were those furnished with the Bruker software.Exact mass measurements and electronic impact-mass spectroscopy (EI-MS) results were recorded on a Micromass Autospecinstrument at 70 eV, temp 220 �C. Preparative and semipreparative flash chromatography was carried out in an automatedgradient flash system (FlashMaster Personal, Jones Chromatography) with prepacked silica column (Isolute flash silica,20 g/70 mL, International Sorbent Technology Ltd. Tucson, USA). Silica gel 0.025–0.04 and 0.040–0.015 mm (Macherey–NagelGmbH&Co.KG, Düren, Germany) was used for vacuum column chromatography (VLC) and column chromatography (CC).Compounds were visualized on TLC with H2SO4 (5%) and vanillin (5%) in EtOH.

2.2. Plant material

F. communis L. was collected during flowering (April 2009) in Sammichele, Bari, Italy and D. viscosa (L.) Greuter wascollected during leaf-rosette (May 2009) and flowering (October 2009) Modugno, Bari, Italy. A voucher specimen(F. communis L. 35678, 35681 and D. viscosa (L.) Greuter 35679) was deposited at the herbarium of the Botany Department,University of Bari.

2.3. Extraction

Air-dried plants were grounded with a blade mill and extracted with n-hexane (Hex) and ethanol (EtOH) successivelywith a Soxhlet apparatus. Four plant extracts were obtained from air-dried plant parts of D. viscosa young shoots (DvAHexwith n-hexane, DvAEtOH with ethanol) and F. communis roots (FcRHex with n-hexane, FcREtOH with ethanol). Extracts werestored in a refrigerator at 4 �C.

2.4. Compound isolation

The D. viscosa hexane extract (30.2895 g, 6.12%) active against M. persicae (%SI ¼ 87 � 5) was fractionated by VLC andeluted with a Hex:EtOAc gradient to give 24 fractions. The most active fractions (Fr9, 1.8201 g, 0.43%, %SI ¼ 99 � 1 and Fr17,0.8729 g, 0.2%, %SI ¼ 77 � 7) were further chromatographed to give compound 1 (75:25 Hex:EtOAc, 290.1 mg, 58.7 � 10�3%)and 2 (40:50 Hex:EtOAc, 101.4 mg, 20.5 � 10�3%).

The F. communis hexane extract (24.2976 g, 2.5%) active against M. persicae (%SI ¼ 94 � 2) was fractionated by VLC andeluted with a Hex:EtOAc gradient to give 13 fractions. The most active fraction (Fr5, 90:10 Hex:EtOAc, 2.7134 g, 0.29%, %SI ¼ 90 � 5) was further chromatographed to give 3 (85:15 Hex:EtOAc, 437.2 mg, 44.3 � 10�3%).

1H NMR, MS and 13C NMR spectra of 1 coincided with those reported by Bohlmann et al. (1978) and Willuhn et al. (1998)and was identified as tomentosin.

Spectroscopic data (1H NMR, MS and 13C NMR) of 2 coincided with those reported by Zdero et al. (1987) and Sanz et al.(1991) and was identified as inuviscolide.

Spectroscopic data (1H NMR, MS and 13C NMR) of 3 coincided with those reported by Lamnaouer et al. (1987) and wasidentified as ferulenol.

E. Mamoci et al. / Biochemical Systematics and Ecology 43 (2012) 101–107 103

2.5. Quantification of isolated compounds

The HPLC system Ultimate 3000 (Dionex, Germering, Germany) was equipped with a photodiode array detector, fluo-rescence detector (FR 2000), low pressure pump Ultimate 3000, 20 ml Rheodyne injector loop (Rheodyne, USA), the C18reverse Acclaim column (150 � 4.6 mm; 3 mm) and C18 reverse Acclaim precolumn (10 � 4.6 mm; 5 mm) and column oven.The HPLC was controlled and data were elaborated using Chromeleon Software vs 6.8 (Dionex, Germering, Germany).Compounds were eluted with an acetonitrile/water gradient (45–85%) for 5 min maintained for 40 min at 1 ml/min flow rateand a column temperature of 30 �C. The compounds were detected at 254 (1 and 2), 283 and 307 (3) nm. The calibrationcurves were built with known concentrations (0.01–5 mg/ml) of tomentosin (1), inuviscolide (2), and ferulenol (3) forquantification purposes with a correlation coefficient of 0.9999 (Fig. 1).

2.6. Insect bioassays

S. littoralis was reared on artificial diet and M. persicae and R. padi were reared on their host plants(Capsicum annuum Var.Californiaworder s/calibra, Ramiro Arnedo S.A, Calahorra, España and Hordeum vulgare, La Poveda-ICA/CSIC, Arganda Del Rey)andmaintained at 22�1 �C,>70% relative humiditywith aphotoperiod of 16:8h (L:D) in a growth chamber. The bioassayswereconductedwithnewlyemerged S. littoralis L6 larvaeor tenM.persicae/R. padi adults as described inBurgueño-Tapia et al. (2008).

2.7. Phytotoxic activity

The experiments were conducted with L. sativa var. Carrascoy (Ramiro Arnedo S.A, Calahorra, España) and L. perenne var.Perenne Nui (InterSemillas, Valencia, España) seeds. Juglonewas included as a positive control. 2.5 cm diam. filter paper diskswith 20 ml of the test compound (10 mg/ml for extracts and 5 mg/ml for pure compounds) were placed on 12-well plates (Falcon).500 ml H2O/well and 10/5 seeds (L. sativa/L. perenne pre-soaked in distilled water for 12 h) were added and the covered platesplaced in a plant growth chamber (25 �C, 70% RH,16:8 L:D). Germinationwasmonitored for 6 days and rootlet/leaf length wasmeasured at the end of the experiment (25 plantlets randomly selected for each experiment and digitalized with theapplication ImageJ 1.43, http://http//rsb.info.nih.gov./ij/).

Tomentosin 1

Inuviscolide 2

Ferulenol 3

Fig. 1. Chemical structure of tomentosin (1), inuviscolide (2) and ferulenol (3).

Table 1Nematicital and antifeedant effects of hexane and ethanolic extracts of F. communis root (FcRHex, FcREtOH) and D. viscosa aerial (DvAHex and DvAEtOH)extracts (concentrations of 1 mg/ml for M. javanica and 100 mg/cm2 for insects).

Plant extracts M. javanica S. littoralis R. padi M. persicae

%INa %FR %C %T %SI %C %T %SI

FcRHex 0c 42 � 12 77*�3 23 � 3 66 � 5 95*�2 5 � 2 94 � 3FcREtOH 8b � 1 69*�8 91*�3 9 � 3 86 � 6 74*�6 26 � 6 60 � 10DvAHex 16b � 1 65*�8 83*�3 17 � 3 75 � 6 91*�3 9 � 3 87 � 6DvAEtOH 74a � 3 63*�5 77*�3 23 � 5 63 � 10 85*�5 15 � 5 76 � 9

%IN, Percentage of immobile nematodes.%FR, Percent feeding reduction.*P � 0.05, Wilcoxon Signed Rank Paired test. %SI (Settling inhibition index) ¼ 1-(%T/%C) � 100, where %T and %C are the percentages of aphids on treatmentand control surface respectively.

a Values within column followed by different letters are significantly different according to Tukeys test (P � 0.05).

E. Mamoci et al. / Biochemical Systematics and Ecology 43 (2012) 101–107104

2.8. Nematode bioassays

M. javanica population was maintained on Lycopersicon esculentum plants (var. Marmande) in pot cultures at 25 � 1 �C,>70% relative humidity. Second stage juveniles (J2) hatched within a 24-h period from egg masses handpicked from infectedtomato roots were used. The experiments were carried out in 96-well microplates (Becton, Dickinson) as described(Hernández-Carlos et al., 2011). Percent J2 immobilized was recorded after 72 h. All treatments were replicated four times.The data is presented as percent paralyzed corrected according to Scheider-Orelli’s formula.

3. Results and discussion

Table 1 shows the nematicidal and insect antifeedant effects of the extracts tested. The ethanolic extract of D. viscosa(DvAEtOH) reduced nematode mobility. Leaf powder and formulated leaf extracts of D. viscosawere previously described asnematicidal against M. javanica J2 in pot and field trial experiments (Oka et al., 2001, 2006). This nematicidial activity ofD. viscosawas attributed to sesquiterpenic acids (costic acid and isocostic acid) found in the acetone extract (Oka et al., 2001).

F. communis root ethanolic (FcREtOH) and both D. viscosa extracts (DvAHex and DvAEtOH) exhibited a significant butmoderate reduction in S. littoralis larvae feeding. All the extracts significantly affected the feeding behavior of the two aphidswithM. persicae beingmore sensitive to these extracts. The hexane extracts (FcRHex and DvAHex) displayed themost activityagainstM. persicae. FcREtOH exhibited themost activity against R. padi followed by DvAHex.We therefore proceededwith thebioassay-guided isolation of the active compounds in FcRHex and DvAHex on M. persicae.

Previous reports have shown that D. viscosa extracts had slight effects on the antennae and proboscis of Oncopeltus fas-ciatus (Alexenizer and Dorn, 2006) and an acaricidal effect on Tetranychus cinnabarinus (Mansour et al., 2004). However this isthe first report on the aphid antifeedant effects of D. viscosa and F. communis.

Table 2 shows the phytotoxic activity of the extracts on L. sativa and L. perenne. Overall, L. perenne was more sensitive tothese extracts than L. sativa. After 24 h of incubation, all extracts reduced the seed germination rate of L. sativa. However, after48 h of incubation there were no significant effects. Root length was moderately affected by DvAHex and DvAEtOH extracts.L. perenne seed germination was moderately reduced by DvAEtOH after 168 h (similar to juglone). The root and leaf lengthswere reduced by all the treatments except for DvAEtOH. DvAHex and FcREtOH were the most active extracts.

Leaf epicuticular water-soluble exudates of D. viscosa composed of sesquiterpene acids and flavonoid aglycones stronglyinhibited the seed germination of lettuce (Levizou et al., 2002). However, the flavonoid aglycones had no effects on seedlingradicle growth (Levizou et al., 2004). Furthermore, the phytotoxic effects of D. viscosa have been attributed to the sesqui-terpene lactone tayunin (Dor and Hershenhorn, 2004).

The bioassay-guided isolation of the hexane extract of D. viscosa shoots and F. communis root resulted in the isolation ofcompounds 1–3. Table 3 shows the antifeedant effects of 1–3 on M. persicae. Tomentosin (1) was a strong aphid repellent

Table 2Phytotoxic activity of hexane and ethanolic extracts of F. communis root (FcRHex, FcREtOH) and D. viscosa aerial (DvAHex and DvAEtOH) extracts at100 mg/cm2 and juglone at 50 mg/cm2 against L. sativa and L. perenne expressed as percentage of control.

Extracts L. sativa L. perenne

Germination Root length Germination Root length Primary leaf

24 h 48 h 72 h 96 h 120 h 144 h 168 h

Juglone 0c� 10b � 7 19d � 4 0b 10c � 5 41c � 9 58c � 11 77b � 9 30d � 4 50a � 9FcRHex 65b � 14 98a � 3 103a � 8 79a � 10 93ab � 6 91ab � 5 94ab � 3 94ab � 3 63b � 4 77b � 7FcREtOH 97a � 5 100a 105a � 4 104a � 6 107a � 6 106a � 6 103a � 6 103a � 6 53bc�5 68ab � 5DvAHex 56b � 8 93a � 5 64c � 5 68a � 12 70b � 11 84ab � 8 82abc�8 82ab � 8 36cd � 4 70ab � 9DvAEtOH 68ab � 8 100a 84b � 7 64a � 10 67b � 8 69bc�9 67bc�9 77b � 9 80a � 8 71ab � 9

Values within column followed by different letters are significantly different according Duncan’s multiple range test (P � 0.05).

Table 3Effect of tomentosin (1), inuviscolide (2) and ferulenol (3) on settling inhibition of M. persicae.

Compound Concentration (mg/cm2) %C %T %SIa ED50 (mg/cm2)

1 10 39 � 6 61 � 6 19c � 750 65 � 6 35 � 6 48b � 9 31.9

100 98*�1 2 � 1 98a � 1 (R2 ¼ 0.8722)2 10 57 � 7 43 � 7 38 � 10

50 61 � 8 39 � 8 50 � 10 –

100 69*�7 31 � 7 56 � 93 10 72 � 11 28 � 11 63 � 14

50 80*�6 20 � 6 71 � 9 –

100 74*�8 26 � 8 62 � 11

%C and %T are percent aphids settled on control and treated leaf disks, respectively. *P � 0.05, Wilcoxon Signed Rank Paired test. %SI (Settling inhibitionindex) ¼ 1-(%T/%C) � 100.ED50 linear regression analysis (%SI on log dose). – not determined.

a Values within column followed by different letters are significantly different according Duncan’s multiple range test (P � 0.05).

Table 4Content of isolated compounds in hexane and ethanolic extracts of F. communis root (FcRHex, FcREtOH) and D. viscosa aerial (DvAHex and DvAEtOH) extracts.

Extracts Compounds (mg/g extract)

1 2 3 Others (%)

FcRHex – – 104.70 89.53FcREtOH – – 56.60 94.34DvAHex 235.41 43.00 – 72.16DvAEtOH 39.35 6.90 – 95.37

E. Mamoci et al. / Biochemical Systematics and Ecology 43 (2012) 101–107 105

followed by ferulenol (3), however the settling inhibition index was not significantly different among the three testedconcentrations.

Tomentosin (1) and inuviscolide (2) are cytotoxic (Topçu et al., 1993; Rozenblat et al., 2008) and anti-inflammatory(Abrham et al., 2010; Hernández et al., 2001; Mañez et al., 1999, 2007) sesquiterpene lactones. Some inuviscolide (2)isomers have been described as potent antifungals (Momen-Roknabadi et al., 2008) and cytotoxic agents (Lee et al., 2002) andtomentosin (1) is a fungistatic agent (Cafarchia et al., 2001). Ferulenol (3) was first isolated from F. communis as an antico-agulant (Carboni et al., 1964) and its anticoagulant and cytotoxic effects have been reviewed (Nazari and Iranshahi, 2011). Ithas antimycobacterial effects (Appendino et al., 2004) and it interferes with the mitochondrial respiratory chain (Nadia et al.,2009; Lahouel et al., 2007). The presence of 3 in F. communis is an indication of its toxicity (Aragno et al., 1988). However this isthe first report on the insect antifeedant effects of 1 and 3.

The quantity of 1 and 2 in D. viscosa extracts varied according to the solvent used for extraction (Table 4), with the hexaneextract containing the highest concentrations (3 and 6 times more of 1 and 2 respectively than the EtOH extract). Tomentosin(1) was the major compound present in DvAHex and DvAEtOH extracts. The highest content of ferulenol (3) was found in theFcRHex extract. Therefore the aphid antifeedant effect of D. viscosa hexane extracts could be attributed to tomentosin (1).However, the fractions from which inuviscolide (2) and ferulenol (3) were isolated were more active than the isolatedcompounds, suggesting a possible synergistic activity of themixture. It is interesting to note thatD. viscosa plays an importantrole as the host plant for the aphid predator M. melanotoma (Perdikis et al., 2007) while being defended against aphids.

In summary, this study showed that the plant extracts tested had variable biological effects, their aphid antifeedant actionbeing the most important. The bioassay-guided search for the active compounds resulted in the isolation of tomentosin (1),inuviscolide (2) and ferulenol (3). Tomentosin (1) rich extracts of D. viscosawere more active than ferulenol (3) rich extractsfrom F. communis.

Acknowledgments

This work has been supported by a MICINN (CTQ2009-14629-C01) grant and E.M. by a Parthenope University of Naplespredoctoral fellowship. S. Carlin is acknowledged for language revision.

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