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Identification of an insecticidal polyacetylene derivative from Chrysanthemum macrotum leaves
Industrial Crops and Products 34 (2011) 1128–1134
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Identification of an insecticidal polyacetylene derivative from Chrysanthemum macrotum leaves Dalila Haouas a,∗ , Flamini Guido b,2 , Ben Halima-Kamel Monia a,1 , Ben Hamouda Mohamed Habib a,1 a Unité de Recherche: invertébrés, micro-organismes et malherbes nuisibles: méthodes alternatives de lutte, Institut Supérieur Agronomique de Chott Mariem, University of Sousse, 4042 Sousse, Tunisia b Dipartimento di Scienze Farmaceutiche, Sede Chimica Bioorganica e Biofarmacia, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy
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Article history: Received 10 November 2010 Received in revised form 25 March 2011 Accepted 29 March 2011 Available online 30 April 2011 Keywords: C. macrotum Asteraceae Insecticidal activity Spodoptera littoralis Polyacetylene Spiroketal enol ether
a b s t r a c t Compounds responsible for insecticidal properties of Chrysanthemum macrotum (D.R.) Ball. leaves against Spodoptera littoralis Boiduval caterpillars have been investigated. The screening of the insecticidal activity was performed by incorporating methanol, buthanol or ethyl acetate extracts, or some chromatographic fractions to the caterpillars’ artificial diet. It was noted that extracts and fractions ameliorated or disturbed nutritional indexes, being not always toxic for caterpillars. Among the tested fractions, one pure compound with a high insecticidal activity (percentage of mortality 66.7%) was purified. The nuclear magnetic resonance study allowed its identification as a polyacetylene derivative, in particular a spiroketal enol ether one. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Insect pests have mainly been controlled with synthetic insecticides in the last fifty years. Most insecticidal compounds fall within four main classes, the organochlorines, organophosphates, the carbamates and pyrethroids. Out of these the major classes in use today are organophosphates and carbamates. There are problems of pesticide resistance and negative effects on nontarget organisms including man and the environment (Kabaru and Gichia, 2001). Since few decades, many investigations on plant insecticide potential were carried in order to substitute chemical molecules. More than 2000 plant species are a rich source of novel insecticides (Klocke, 1989), where Meliaceae, Rutaceae, Asteraceae, Annonaceae, Labiatae and Canellaceae are the promising families (Wheeler and Isman, 2001), the two famous compounds are azadirachtin extract from the Indian neem tree (Azadirachta indica) (Jilani and Saxena, 1990) and pyrethrum from Chrysanthemum cinerareafolium (Prakash and Rao, 1997). Recently, over than 2000 polyacetylenes are known, with more than 1100 in the plant family Asteraceae (Minto and Blacklock, 2008). These compounds are fatty
∗ Corresponding author. Tel.: +216 73 327 546; fax: +216 73 327 591. E-mail addresses: dalila [email protected], [email protected] (D. Haouas). 1 Tel.: +216 73 327 546; fax: +216 73 327 591. 2 Tel.: +39 0502219686; fax: +39 0502219660. 0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.03.032
acid derivatives characterized with long hydrocarbon chains (Baek et al., 1995) they are generally stored in secretory plant cells, called laticifers (Ellis, 1997), secreting latex. Most of them have an insecticidal, ovicidal, larvicidal, fungicidal, nematocidal and phytotoxicity proprieties (Stevens et al., 1990). Some polyacetylenes, such as the relatively stable ␣-terthienyl and phenylheptatriyne are phototoxic to insects and fungi at very low concentrations when introduced into insect diets or tested against cultured fungi by using the disk diffusion method (Towers and Champagne, 1987; Downum, 1992). Early study realized by Wrang and Lam (1975) identified fifteen polyacetylenes from Chrysanthemum leucanthemum, from which five are new in this herb. Bohlmann and Zdero (1975) have also described four new polyacetylenes from Chrysanthemum macrotum: 8-(2-thienyl)-3t,5t-ocradien7-in-1-ol-acetat, 8-(2-thienyl)-3t,5t-uctadien-7-in-1-ol, 2t,4tundecadien-8,10-diinsaure-4,6-heptadiinylester and 1-(2,3dihydro-2-furyl)-4-(2-thienyl)-1t-buten-3-in. Nevertheless, no biological activity investigation of these compounds has been ˜ made. Some years later, Bowers and Aregullin (1987), Cunat et al. (1990), Sanz et al. (1990) and Song et al. (2005) identified nine polyacetylenes in Chrysanthemum coronarium, from which four compounds have an insect antijuvenile hormone activity against Oncopeltus fasciatus. The biological properties of these compounds make them of a great interest to plant pathologists and pharmacologists. Many of the polyacetylenes and related compounds require UV-light (300–400 nm) for being toxic and having other biological activities (Christensen and Lam, 1990).
D. Haouas et al. / Industrial Crops and Products 34 (2011) 1128–1134
As part of our search within the Chrysanthemum genus and after a preliminary study on eight Chrysanthemum species (Haouas et al., 2003, 2005, 2008, 2009) we focus to identify a polyacetylene(s) derivative from C. macrotum leaves endowed with insecticidal activity against Spodoptera littoralis caterpillars.
2. Materials and methods 2.1. Plant material C. macrotum (D.R.) Ball. leaves were collected in 2007 during its flowering stage in April from the region of Zaghouan (Tunisia). The sampling was made on the rocky mountain slopes (36◦ 23 13.15 N, 10◦ 07 51.49 E) at about 305 m above the sea level which belong to the sub-humid bioclimatic stage. Voucher specimens were deposited in the National Gene Bank of Tunisia. Leaves were dried in open air in the shade.
2.2. Chemical study One hundred gramme of the dried powdered leaves of C. macrotum were extracted at room temperature with MeOH (1 l × three times during 3 days). After filtering, extracts were combined and dried at reduced pressure. 45 g of residues were re-dissolved in methanol–water (3:1) and partitioned in a separatory funnel with EtOAc and BuOH, in the order. After solvents removal at reduced pressure, respective EtOAc (8.7 g), BuOH (3.6 g) and MeOH–H2 O (32.4 g) extracts were obtained. After bio-insecticidal activity evaluation of these three crude extracts, it was established that the most effective was the EtOAc one. Thus, a portion of this latter extract (7 g) was re-dissolved in MeOH and submitted to size-exclusion chromatography on a Sephadex LH-20 column, eluting with 100% MeOH. According to TLC (Merck Kieselgel 60 F254) analysis, chromatograms were visualized under UV light at 254 and 366 nm and/or sprayed with cerium sulphate or Naturstoffereagenz A-PEG reagents. Collected fractions were combined in nine homogeneous fractions (ACI –ACIX ). All of them were tested for their insecticidal activity. Only ACI and ACII (0.98 g) fractions resulted effective and were further fractionated on a silica gel 60 column, eluting with CHCl3 : MeOH mixtures (9:1, 8.5:1.5, 7:3, 5:5, to 100% MeOH). After TLC analysis, 21 homogeneous fractions (ACS1 –ACS24 ) were obtained. Finally, a pure secondary metabolite (1, 3.5 mg), endowed with a high insecticidal activity was isolated (Flamini et al., 1997, 2004; Song et al., 2005). Structural determination of (1) was performed by spectroscopic analysis. Melting points (uncorrected) were determined with Kofler apparatus; 1H and 13C NMR spectra were obtained with a Bruker Avance II 250 spectrometer in CDCl3 , using TMS as internal standard. All experiments were performed using the standard Bruker library of microprograms. Known compounds were identified by comparison of their spectral data to those of the literature (Flamini et al., 1997, 2004).
2.3. Insects Insects were obtained from a culture of S. littoralis and maintained under standard conditions of temperature (27 ± 1 ◦ C), photoperiod (L16:D8) and relative humidity (60–70%). caterpillars were reared in Petri dishes with small artificial diet cubes based on wheat germ (Poitout and Bues, 1974), while adults with a 15% honey water solution. The culture was continuously supplemented with wild moths captured with a light trap in High Institute of Agronomy, Tunisia.
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2.4. Insect assay 2.4.1. Extracts and fractions effects on insect survival Thirty 3rd instar caterpillars were individually placed in glass Petri dishes (1 cm high and 9 cm in diameter) and provided with appropriate artificial diet added with 0 (control), 0.1, 1 and 10 mg/g of C. macrotum leaves methanol crude extract, or 1 mg/g of semipurified BuOH, EtOAc or MeOH–H2 O extracts. Fractions obtained from the Sephadex column were tested at 1 mg/g, while those from silica gel at 0.1 mg/g. All fractions are dried and well mixed with the artificial diet after preparation and cooling according to the procedure of Martinez and Van Emden (2001). Insect mortality was recorded in the end of experiment and adjusted for control using Abbott’s correction (Abbott, 1925). Mc =
Mo − Me × 100 100 − Me
Mo = mortality rate of treated insects (%); Me = mortality rate of control (%); Mc = corrected mortality rate (%). 2.4.2. Extracts and fractions effects on food consumption and utilization The effect of extracts and fractions on food consumption and utilization by third instar caterpillars was investigated using reared caterpillars on control diet after the second molt (<24 h). They were weighed and individually placed in Petri dishes. They were fed with known weights of diets containing 0, 0.1, 1 and 10 mg/g of extract (n = 30 and five replications for each concentration) and left to feed for 2 days, a period slightly shorter than instar duration. At the end of the experiment, caterpillars and faeces were weighed and food consumption was determined. Nutritional indices, namely relative consumption rate (RCR), relative growth rate (RGR), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD) and approximate digestibility (AD) were calculated as follows: RCR = I/Ba T RGR = B/Ba T ECI = (B/I) × 100 AD = [(I − F)/I] × 100 ECD = [B/(I − F)] × 100 where: I = weight of consumed food; Ba = arithmetic mean of insect weight during the experiment = [(PF − PI)/log (PF/PI)]; PF = caterpillars final weight (mg); PI = caterpillars starting weight (mg); T = feeding period in hours; B = change in body weight; F = weight of faeces produced during the feeding period (Waldbauer, 1968; Farrar et al., 1989). 2.5. Statistical analyses Nutritional indexes obtained after treatments with methanol and ethyl acetate extracts and fractions from sephadex column, as well as mortality, for all different treatments were compared using analysis of variance (ANOVA) followed by Duncan test for multiplecomparison when significant differences were observed at P = 0.01. Nutritional indexes resulting on caterpillars treated on fractions obtained from silica gel column were analyzed using the principal compound analysis (Tanagra version 1.4.38). 3. Results 3.1. Effect of methanol extract After preliminary study related to the insecticidal activity of flowers and leaves Chrysanthemum powders and their methanolic extracts against S. littoralis caterpillars (Haouas et al., 2008; Haouas,
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Table 1 Effect of C. macrotum methanolic extract on nutritional index and toxicity of the 3rd instar S. littoralis caterpillars at concentration of 0.1; 1 and 10 mg/g.a
Control 0.1 mg/g 1 mg/g 10 mg/g
RCR (mg/mg/day)
AD (%)
± ± ± ±
94.87 95.14 96.41 97.70
0.1064 0.1262 0.1357 0.1047
0.03b 0.02a 0.04a 0.006b
ECD (%) ± ± ± ±
1.47c 1.32c 0.94b 1.21a
8.12 4.39 2.40 2.66
± ± ± ±
ECI (%) 2.93a 1.19b 0.80c 0.91c
7.69 4.18 2.31 2.59
± ± ± ±
RGR (mg/mg/day) 2.76a 1.18b 0.76c 0.89c
0.0077 0.0051 0.0029 0.0027
± ± ± ±
0.0019a 0.0007b 0.0007c 0.0008c
Mtotb (%) 22.9 ± 1.2b 97.0 ± 3.2a 100.0 ± 0.0a
RCR, relative consumption rate; AD, approximate digestibility; ECD, efficiency of conversion of digested food; ECI, efficiency of conversion of ingested food; RGR, relative growth rate; Mtot, total mortality. a Means in the same column followed by the same letters are not significantly (P < 0.01) according to Duncan test. b Values were adjusted for control mortality using Abbott’s correction (Abbott, 1925).
2010), obtained results showed that C. macrotum leaves methanol extract was the most active one. For this raison it was selected to continue its chemical study. The incorporation of the MeOH extract of C. macrotum leaves in artificial diet of S. littoralis caterpillars at concentrations of 0.1, 1 and 10 mg/g affected positively or negatively all nutritional indexes. In fact, these doses of extract stimulate: (i) the relative consumption rate for caterpillars at concentrations of 0.1 and 1 mg/g and (ii) the approximate digestibility when employed at 10 mg/g. The comparison of RGR for each concentration showed that this parameter was affected at low concentration (0.1 mg/g). We noted a significant difference (P < 0.01) between caterpillars treated at 0.1 mg/g and control (respectively 0.0051 ± 0.0007 mg/mg/day and 0.0077 ± 0.0019 mg/mg/day). Concerning the RCR we noted an increasing effect of crude extract at the concentration of 1 and 0.1 mg/g. The mortality analysis showed a dose–effect correlation. In fact, at 0.1 mg/g, mortality was 22.9% and it increased to 100% at 10 mg/g (Table 1).
ACVIII fractions is noted, whereas fraction ACIII increases significantly the same parameter. In addition, approximate digestibility decreases significantly when ACII fraction is added to the artificial diet of Spodoptera caterpillars. The same result is also obtained for the ECD after adding ACIII and ACVI fractions to the caterpillars’ diet. Moreover, statistical analysis of the relative growth rate of treated caterpillars shows that ACIX and ACV fractions slow significantly the insect growth. In contrast the ACIV fraction is responsible for the growth perfection rate. These results highlight the specific effect of each fraction on nutritional indexes (Table 3). However, the effect of fractions is notably important on insects’ survival. Indeed, the addition of ACI and ACII fractions in artificial diet contribute to the mortality of 88% of treated caterpillars (Table 3). Knowing that ACI and ACII are collected successively from the sephadex column, have the highest rate of mortality, the same effect on nutritional indexes and practically the same TLC chromatograms, we suggest combine and simplified them using Silica gel column. 3.4. Activity of the fractions from Silica gel column chromatography
3.2. Effects of EtOAc, BuOH and MeOH–H2 O extracts
ACI and ACII fractions were combined and loaded on a silica gel column, obtaining 21 homogeneous fractions that were screened for their insecticidal activity. Results of principal compound analysis shows, on one side, an opposition between the effect of fractions on ECD, ECI and RGR (high positive correlation with the first axis) and their effect on RCR and AD (significant negative correlation with the first axis) on another side. Axis one is an axis of high fractions effect on ECD, ECI and RGR but it is low on RCR and AD. Axis two is an axis of high fractions effect on AD (Fig. 1). Statistical analysis of total mortality evidenced those fractions ACS9 , ACS10 , ACS11 , ACS12 , ACS16 , ACS1789 and ACS21 cause more than 50% death rate (Fig. 2). We remark that most died insects present molt difficulties and lethal malformation of new chrysalides.
These three extracts were tested for their insecticidal activity against S. littoralis caterpillars. Statistical analysis of relative consumption rate demonstrates that there are no significant differences between treatments and control and within different extracts. However, the analysis of the ECD, ECI and AD showed that the BuOH extract do not significantly affect the performance of the caterpillars. The most significant parameter that differenced the three extracts was the mortality rate. In fact, the EtOAc extract caused death to 84.4% of treated caterpillars. We remark also that many insects are died during molting and chrysalides formation. All combined results oriented us to choose the EtOAc extract for further bio-guided insecticidal assays (Table 2). 3.3. Effect of ethyl acetate fractions
3.5. Compound identification The ethyl acetate extract was chromatographed over Sephadex LH-20 column and the obtained fractions were added to the artificial diet of S. littoralis caterpillars (1 mg/g). By calculating nutritional indexes, a disturbing action, either as improvement or degradation, was evidenced for most fractions. Indeed, a significant reducing in the relative consumption rate caused by ACVI and
After insects assays the remaining amount of tested fractions are very low (ACS9 = 6.9 mg, ACS10 = 3.5 mg, ACS11 = 13.2 mg, ACS12 = 13.9 mg, ACS16 = 7.0 mg, ACS1789 = 23.5 and ACS21 = 1.6 mg). The TLC results of the fractions ACS9 , ACS11 , ACS12 , ACS16 and ACS1789 and ACS21 show a mixture of compounds, but taking in
Table 2 Action of ethyl acetate, buthanol and methanol extracts of C. macrotum on nutritional index and toxicity of the 3rd instar S. littoralis caterpillars at concentration of 1 mg/g.a
Control EtOAc BuOH MeOH–H2 O
RCR (mg/mg/day)
AD (%)
± ± ± ±
95.07 95.10 94.49 95.99
0.1160 0.1248 0.1334 0.1336
0.05a 0.02a 0.04a 0.024a
ECD (%) ± ± ± ±
1.63b 1.19b 1.33b 0.89a
7.92 6.13 7.85 5.50
± ± ± ±
ECI (%) 3.22a 1.60b 2.82a 1.46b
7.51 5.83 7.41 5.27
± ± ± ±
RGR (mg/mg/day) 3.03a 1.51b 2.67a 1.39b
0.0077 0.0070 0.0091 0.0068
± ± ± ±
0.0019b 0.0012b 0.0019a 0.0012b
Mtotb (%) 84.7 ± 2.0 a 18.8 ± 1.4c 42.0 ± 2.1b
RCR, relative consumption rate; AD, approximate digestibility; ECD, efficiency of conversion of digested food; ECI, efficiency of conversion of ingested food; RGR, relative growth rate; Mtot, total mortality. a Means in the same column followed by the same letters are not significantly (P < 0.01) according to Duncan test. b Values were adjusted for control mortality using Abbott’s correction (Abbott, 1925).
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Table 3 Results obtained on nutritional index and toxicity after treatment of the 3rd instar S. littoralis caterpillars with ethyl acetate fraction at 1 mg/g.a
Control ACI ACII ACIII ACIV ACV ACVI ACVII ACVIII ACIX
RCR (mg/mg/day)
AD (%)
± ± ± ± ± ± ± ± ± ±
95.07 93.79 93.25 94.95 94.05 94.55 93.55 94.40 93.78 95.08
0.1160 0.1245 0.1219 0.1545 0.1269 0.1137 0.0934 0.1048 0.0947 0.1067
0.05bcd 0.03bc 0.03bcd 0.02a 0.02b 0.03bcd 0.02e 0.02de 0.02e 0.02cde
ECD (%) ± ± ± ± ± ± ± ± ± ±
1.63a 2.35ab 3.71b 1.42a 1.35ab 1.87ab 3.81ab 1.44ab 2.27ab 1.71a
7.92 6.66 6.99 4.77 6.96 4.75 7.74 6.58 8.24 5.89
± ± ± ± ± ± ± ± ± ±
ECI (%) 3.22a 2.56ab 3.29ab 1.85c 2.48ab 2.85c 3.65ab 1.87abc 2.92a 2.54bc
7.51 6.20 6.43 4.51 6.52 4.47 7.19 6.20 7.67 5.58
± ± ± ± ± ± ± ± ± ±
Mtotb (%)
RGR (mg/mg/day) 3.03a 2.20ab 2.82ab 1.68c 2.26ab 2.64c 2.84ab 1.71ab 2.57a 2.36bc
0.0077 0.0073 0.0071 0.0067 0.0079 0.0046 0.0065 0.0063 0.0070 0.0057
± ± ± ± ± ± ± ± ± ±
0.0019ab 0.0012abc 0.0017abc 0.0020abcd 0.0020a 0.0021e 0.0012bcd 0.0013 cd 0.0017abc 0.0023d
88.9 88.9 22.2 22.2 33.2 66.7 44.3 44.4 55.6
± ± ± ± ± ± ± ± ±
2.6a 3.1a 3.2f 2.4f 3.1e 2.0b 2.4d 2.2d 2.6c
RCR, relative consumption rate; AD, approximate digestibility; ECD, efficiency of conversion of digested food; ECI, efficiency of conversion of ingested food; RGR, relative growth rate; Mtot, total mortality. a Means in the same column followed by the same letters are not significantly (P < 0.01) according to Duncan test. b Values were adjusted for control mortality using Abbott’s correction (Abbott, 1925).
Fig. 1. Principal compound analysis of the fractions effect on nutritional indexes of S. littoralis.
account the remaining amount, only fractions ACS11 , ACS12 and ACS1789 are loaded on a silica gel column. The quantities of obtained fractions are very small but not pure. So these fractions are not sufficient to continue insects’ tests not to identify the compound structure (Fig. 3). However, ACS10 TLC relieved a simple chromatogram. Nuclear magnetic resonance study of ACS10 led to the structure determination of the active compound (Table 4). The results were in agreement with those of literature for a C13 H10 O3 spiroketal enol ether derivative, in particular it was identified as (E)-7-(2,4-hexadiynylidene)-1,6-dioxaspiro[4.4]nona-2,8-dien4-ol (Fig. 4) (Zeisberg and Bohlmann, 1974).
4. Discussion The reduction in growth rate of caterpillars accurately reflects a reduction in the efficiency of conversion into biomass of the ingested and digested food. Similar results were reported by Pavela and Chermenskaya (2004) and Pavela (2004), when S. littoralis caterpillars were fed on Achillea ptarmica L., Ambrosia artemisiifolia L., Artemisia vulgaris L., Taraxacum officinal Weber and Cnicus benedictus L. methanol extracts. The sesquiterpene lactones isolated from Parthenium species showed a growth inhibition activity on Heliothis zea and Spodoptera exigua (Isman and Rodriguez, 1983). But the combination between toxicity and growth reduction could
Table 4 Chemical shift of hydrogen and carbon (CDCl3 , 250 MHz (1 H) and 62.5 MHz (13 C)). 1
Fig. 2. Mortality rate of S. littoralis caterpillars treated with fractions eluted from silica gel column.
1 2 3 4 5 6 7 8 9 10 11 12 13
H
1.92, 3H, d, J = 1.1 Hz – – – – 5.03, 1H, brs – 6.7, 1H, d, J = 5.7 Hz 6.28, 1H, dd, J = 5.7 and 1.9 Hz – 5.66, 1H, dd, 2.0 and 2.0 Hz 5.13, 1H, dd, 3.0 and 3.0 Hz 6.53, 1H, dd, 3.0 and 2.0 Hz
Coupling
13
H6 – – – – H1, H8, H9 – H6, H9 H6, H8 – H12, H13 H11, H13 H12, H11
4.6 80.3 65.3 77.4 70.1 83.0 168.6 133.7 125.8 117.2 76.0 106.6 147.0
C
DEPT CH3 C C C C CH C CH CH C CH CH CH
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Fig. 3. Chromatographic steps led to the isolation of compound (1).
1
CH3
2
C
4
3
C
C (1)
8
5
C H
9
6
C
E
10
7
O
Fig. 4. Structure of the spiroketal enol ether derivative.
O OH 11
13
12
D. Haouas et al. / Industrial Crops and Products 34 (2011) 1128–1134
be due to another compound, such as the chromene encecalin, isolated from Encelia genus responsible for mortality (77%) and growth inhibition of Peridroma saucia (Noctuidae) caterpillars (Isman and Proksch, 1985). The ingestion of C. macrotum EtOAc, BuOH and the MeOH–H2 O extracts affected the nutritional indexes. The follow-up of insects mortality treated with these same extracts showed that the EtOAc one causes an important toxicity (84.7%) in treated caterpillars. Similar results were obtained by Faini et al. (1997) which showed that Flourensia thurifera leaves and stems extracts were endowed with insecticidal activity against S. littoralis caterpillars because of their content in coumarins and chromenes. Fractions collected from the Sephadex column showed different activities against S. littoralis caterpillars, suggesting a different distribution of the active compounds. Most toxic fractions (ACI and ACII ) were further simplified and tested against S. littoralis caterpillars again. In this way, four of the nine fractions maintained a high mortality (more than 50%), that can be due to the perturbation of nutritional indexes. These observations allow affirming that nutritional imbalance can be one factor contributing the caterpillars mortality. A compound responsible for this toxicity is identified as a spiroketal enol polyacetylene, in particular as (E)-7-(2,4hexadiynylidene)-1,6-dioxaspiro[4.4]nona-2,8-dien-4-ol. In the Chrysanthemum genus, this compound was isolated for the first time by Wrang and Lam (1975) from C. leucanthemum L., roots, but without any further study on its biological activity. This compound is characterized by the presence of oxygen atoms, both as alcoholic groups and heterocyclic, chiral centers and an apolar part, typical characteristics of a potential insecticidal natural product, as reported by Ujváry (2002). Natural polyacetyenes are found as C10 –C17 derivatives. Compounds with 13 carbon atoms, such as this active spiroketal, are widely distributed in the Asteraceae family. Bohlmann and Zdero (1975) identified from C. macrotum polyacetylenes with 12 and 13 carbons without reported any activity for them. Later, Bohlmann and Fritz (1979) isolated a new sulfurcontaining polyacetylene in C. coronarium. Other studies performed by Gao et al. (1996) highlight the presence of spiroketal enol ether from C. segetum and C. coronarium endowed with an anti-feeding activity against Pieris brassicae (Gao et al., 1998; Yin et al., 2003, 2004; Chen et al., 2004, 2005). Phytochemical studies on some Asteraceae species (A. vulgaris L., C. leucanthemum Centautia spp. and Dahlia spp.) contribute to the isolation of 14 polyacetylenes compounds and some thiophene derivatives (Wat et al., 1981). Most of these compounds were characterized by a high toxic activity against Aedes aegypti, Simulium vitatum larvae and Caenorhabditis elegans nematodes. The toxicity of these polyacetylenics compounds can be attributed to different mode of actions. In absence of light the polyacetylenes are antifeedant to insects (Champagne et al., 1986). This can explain our results concerning the perturbation of nutritional indexes on S. littoralis caterpillars. In presence of light, there are two hypotheses explaining the insect toxicity. The first one can owe photocatalic cycle of single oxygen generation and other excited state molecule that leads to rapid lipid peroxidation and cell death. The second one can contribute to a photogenotoxicity, in which compounds intercalate into DNA, and upon irradiation with near UV light form mono- and bi-functional adducts with pyrimidine bases (Arnason and Bernards, 2010). Phototoxic compounds can lead to redding and thinning of cuticle in lepidopteran caterpillars exposed to dietary furanocoumarins (Berenbaum, 1987). They can also lead to the appearance of black cuticular lesions and prevent a normal molt and died before metamorphosing in the case of Manduca sexta (tobacco hornworm) when it was exposed to oral or topical of thiophene, poyine or hypericin (Downum et al., 1984; Champagne et al., 1986; Samuels and Knox, 1989).
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In this study the structure of a compound endowed with toxic properties against the S. littoralis caterpillars was determined in discussing with its possible phototoxicity. Further studies will be considered to highlight the mechanism of action of this compound under different light regimes. Acknowledgments We are grateful to the International Foundation for Science (IFS grant No. F/3968-1) and to the Organization for the Prohibition of Chemical Weapons (OPCW) for financial support. We thank also, Dr. Fethia Skhiri-Harzallah for her help in Chrysanthemum identification species. References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267, in Tapondjou, A.L., Adler, C., Fontem, D.A., Bouda, H., Reichmuth, C., 2005. Bioactivities of cymol and essential oils of Cupressus sempervirens and Eucalyptus saligna against Sitophilus zeamais Motschulsky and Tribolium confusum du Val. J. Stored Prod. Res. 41, 91–102. Arnason, J.T., Bernards, M.A., 2010. Impact of constitutive plant natural products on herbivores and pathogens. Can. J. Zool. 88, 615–627. Baek, N., Park, J.D., Lee, Y.H., Jeong, S.Y., Kim, S.I., 1995. A novel polyacetylene from Cirsium spp. Yakhak Hoeji 39 (3), 268–275. Berenbaum, M.R., 1987. Charge of the light brigade; photoxicity as a defense against insects. In: Heitz, J.R., Downum, K.R. (Eds.), Light-Activated Pesticides. ACS Symp Ser, vol. 339. American Chemical Society, Washington, pp. 206–216. Bohlmann, F., Fritz, U., 1979. Neue lyratolester aus Chrysanthemum coronarium. Phytochemistry 18, 1888–1889. Bohlmann, F., Zdero, C., 1975. Polyacetylenverbindungen, 2331) über die Inhaltsstoffe von Chrysanthemum macrotum (Dur.) Ball. Chem. Ber. 108, 739–742. Bowers, W.S., Aregullin, M., 1987. Discovery and identification of an antijuvenile hormone from Chrysanthemum coronarium. Mem. Inst. Oswaldo Cruz, Rio de Janeiro 82 (Suppl. III), 51–54. Champagne, D.E., Arnason, J.T., Philogene, B.J.R., Morand, P., Lam, J., 1986. Lightmediated allelochemical effects of naturally occurring polyacetylenes and thiophenes from Asteraceae on herbivorous insects. J. Chem. Ecol. 12, 835–858. Chen, L., Xu, H.H., Hu, T.S., Wu, Y.L., 2005. Synthesis of spiroketal enol ethers related to tonghaosu and their insecticidal activities. Pest Manag. Sci. 61, 477–482. Chen, L., Yin, B.L., Xu, H.H., Chiu, M.H., Wu, Y.L., 2004. Study on tonghaosu and its analogs: isolation, structure identification and synthesis of antifeedant B-ringhomo-tonghaosu. Chin. J. Chem. 22, 92–99. Christensen, L.P., Lam, J., 1990. Acetylenes and related compounds in Cynareae. Phytochemistry 29, 2753–2785. ˜ P., Primo, E., Sanz, I., March, M.C., Bowers, W.S., et Martinez-Pardo, R., 1990. Cunat, Biocidal activity of some Spanish Mediterranean plants. J. Agric. Food Chem. 38 (2), 497–500. Downum, K.R., Rosenthal, G.A., Towers, G.H.N., 1984. Phototoxicity of the allelocheical, ␣-terthienyl, to larvae of Mascuda sexta (L.) (Sphingidae). Pest Biochem. Physiol. 22, 104–109. Downum, K.R., 1992. Light-activated plant defence. New Phytol. 122 (3), 401–420. Ellis, B.E., 1997. Metabolism of defence and communication. In: Dennis, D.T., Turpin, D.H., Lefebvre, D.D., Layzell, D.B. (Eds.), Plant Metabolism. , 2nd edition. Longman, Singapore, pp. 148–160. Faini, F., Labbe, C., Salgado, I., Coll, J., 1997. Chemistry, toxicity and antifeedant activity of the resin of Flourensia thurifera. Biochem. Syst. Ecol. 25 (3), 189–193. Farrar, R.R., Barbour, J.D., Kenedy, G.G., 1989. Quantifying food consumption and growth in insects. Ann. Entomol. Soc. Am. 82, 593–598. Flamini, G., Braca, A., Cioni, P.L., Morelli, I., 1997. Three new flavonoids and other constituents from Lonicera implexa. J. Nat. Prod. 60, 449–452. Flamini, G., Stoppelli, G., Morelli, I., Ertugrul, K., Dural, H., Tugay, O., Demirelma, H., 2004. Secondary metabolites from Centaurea isaurica from Turkey and their chemotaxonomical significance. Biochem. Syst. Ecol. 32, 553–557. Gao, Y., Wu, W.L., Wu, Y.L., Ye, B., Zhou, R., 1998. A straightforward synthetic approach to the spiroketal-enol ethers synthesis of natural antifeeding compound tonghaosu and its analogs. Tetrahedron 54, 12523–12538. Gao, Y., Wu, W.L., Ye, B., Zhou, R., Wu, Y.L., 1996. Convenient syntheses of tonghaosu and two thiophene substituted spiroketal enol ether natural products. Tetrahedron Lett. 37 (6), 893–989. Haouas, D., Ben Halima Kamel, M., Skhiri Harzallah, F., Ben Hamouda, M.H., 2005. Bioactivities of seven Chrysanthemum species flowers powder on Spodoptera littoralis (Boiduval) caterpillars. Commun. Appl. Sci. Ghent Univ. 70 (4), 799–807. Haouas, D., Ben Halima-Kamel, M., Ben Hamouda, M.H., 2009. Impact des espèces de Chrysanthemum sur la consommation et la survie des chenilles de Spodoptera littoralis (Boisduval) (Lepidoptera; noctuidae). Actes du congrès Internationale sur la biodiversité des invertébrés en milieux agricoles et forestiers. INA El Harrach. Alger 128–138.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Identification of an insecticidal polyacetylene derivative from Chrysanthemum macrotum leaves Dalila Haouas a,∗ , Flamini Guido b,2 , Ben Halima-Kamel Monia a,1 , Ben Hamouda Mohamed Habib a,1 a Unité de Recherche: invertébrés, micro-organismes et malherbes nuisibles: méthodes alternatives de lutte, Institut Supérieur Agronomique de Chott Mariem, University of Sousse, 4042 Sousse, Tunisia b Dipartimento di Scienze Farmaceutiche, Sede Chimica Bioorganica e Biofarmacia, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy
a r t i c l e
i n f o
Article history: Received 10 November 2010 Received in revised form 25 March 2011 Accepted 29 March 2011 Available online 30 April 2011 Keywords: C. macrotum Asteraceae Insecticidal activity Spodoptera littoralis Polyacetylene Spiroketal enol ether
a b s t r a c t Compounds responsible for insecticidal properties of Chrysanthemum macrotum (D.R.) Ball. leaves against Spodoptera littoralis Boiduval caterpillars have been investigated. The screening of the insecticidal activity was performed by incorporating methanol, buthanol or ethyl acetate extracts, or some chromatographic fractions to the caterpillars’ artificial diet. It was noted that extracts and fractions ameliorated or disturbed nutritional indexes, being not always toxic for caterpillars. Among the tested fractions, one pure compound with a high insecticidal activity (percentage of mortality 66.7%) was purified. The nuclear magnetic resonance study allowed its identification as a polyacetylene derivative, in particular a spiroketal enol ether one. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Insect pests have mainly been controlled with synthetic insecticides in the last fifty years. Most insecticidal compounds fall within four main classes, the organochlorines, organophosphates, the carbamates and pyrethroids. Out of these the major classes in use today are organophosphates and carbamates. There are problems of pesticide resistance and negative effects on nontarget organisms including man and the environment (Kabaru and Gichia, 2001). Since few decades, many investigations on plant insecticide potential were carried in order to substitute chemical molecules. More than 2000 plant species are a rich source of novel insecticides (Klocke, 1989), where Meliaceae, Rutaceae, Asteraceae, Annonaceae, Labiatae and Canellaceae are the promising families (Wheeler and Isman, 2001), the two famous compounds are azadirachtin extract from the Indian neem tree (Azadirachta indica) (Jilani and Saxena, 1990) and pyrethrum from Chrysanthemum cinerareafolium (Prakash and Rao, 1997). Recently, over than 2000 polyacetylenes are known, with more than 1100 in the plant family Asteraceae (Minto and Blacklock, 2008). These compounds are fatty
∗ Corresponding author. Tel.: +216 73 327 546; fax: +216 73 327 591. E-mail addresses: dalila [email protected], [email protected] (D. Haouas). 1 Tel.: +216 73 327 546; fax: +216 73 327 591. 2 Tel.: +39 0502219686; fax: +39 0502219660. 0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.03.032
acid derivatives characterized with long hydrocarbon chains (Baek et al., 1995) they are generally stored in secretory plant cells, called laticifers (Ellis, 1997), secreting latex. Most of them have an insecticidal, ovicidal, larvicidal, fungicidal, nematocidal and phytotoxicity proprieties (Stevens et al., 1990). Some polyacetylenes, such as the relatively stable ␣-terthienyl and phenylheptatriyne are phototoxic to insects and fungi at very low concentrations when introduced into insect diets or tested against cultured fungi by using the disk diffusion method (Towers and Champagne, 1987; Downum, 1992). Early study realized by Wrang and Lam (1975) identified fifteen polyacetylenes from Chrysanthemum leucanthemum, from which five are new in this herb. Bohlmann and Zdero (1975) have also described four new polyacetylenes from Chrysanthemum macrotum: 8-(2-thienyl)-3t,5t-ocradien7-in-1-ol-acetat, 8-(2-thienyl)-3t,5t-uctadien-7-in-1-ol, 2t,4tundecadien-8,10-diinsaure-4,6-heptadiinylester and 1-(2,3dihydro-2-furyl)-4-(2-thienyl)-1t-buten-3-in. Nevertheless, no biological activity investigation of these compounds has been ˜ made. Some years later, Bowers and Aregullin (1987), Cunat et al. (1990), Sanz et al. (1990) and Song et al. (2005) identified nine polyacetylenes in Chrysanthemum coronarium, from which four compounds have an insect antijuvenile hormone activity against Oncopeltus fasciatus. The biological properties of these compounds make them of a great interest to plant pathologists and pharmacologists. Many of the polyacetylenes and related compounds require UV-light (300–400 nm) for being toxic and having other biological activities (Christensen and Lam, 1990).
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As part of our search within the Chrysanthemum genus and after a preliminary study on eight Chrysanthemum species (Haouas et al., 2003, 2005, 2008, 2009) we focus to identify a polyacetylene(s) derivative from C. macrotum leaves endowed with insecticidal activity against Spodoptera littoralis caterpillars.
2. Materials and methods 2.1. Plant material C. macrotum (D.R.) Ball. leaves were collected in 2007 during its flowering stage in April from the region of Zaghouan (Tunisia). The sampling was made on the rocky mountain slopes (36◦ 23 13.15 N, 10◦ 07 51.49 E) at about 305 m above the sea level which belong to the sub-humid bioclimatic stage. Voucher specimens were deposited in the National Gene Bank of Tunisia. Leaves were dried in open air in the shade.
2.2. Chemical study One hundred gramme of the dried powdered leaves of C. macrotum were extracted at room temperature with MeOH (1 l × three times during 3 days). After filtering, extracts were combined and dried at reduced pressure. 45 g of residues were re-dissolved in methanol–water (3:1) and partitioned in a separatory funnel with EtOAc and BuOH, in the order. After solvents removal at reduced pressure, respective EtOAc (8.7 g), BuOH (3.6 g) and MeOH–H2 O (32.4 g) extracts were obtained. After bio-insecticidal activity evaluation of these three crude extracts, it was established that the most effective was the EtOAc one. Thus, a portion of this latter extract (7 g) was re-dissolved in MeOH and submitted to size-exclusion chromatography on a Sephadex LH-20 column, eluting with 100% MeOH. According to TLC (Merck Kieselgel 60 F254) analysis, chromatograms were visualized under UV light at 254 and 366 nm and/or sprayed with cerium sulphate or Naturstoffereagenz A-PEG reagents. Collected fractions were combined in nine homogeneous fractions (ACI –ACIX ). All of them were tested for their insecticidal activity. Only ACI and ACII (0.98 g) fractions resulted effective and were further fractionated on a silica gel 60 column, eluting with CHCl3 : MeOH mixtures (9:1, 8.5:1.5, 7:3, 5:5, to 100% MeOH). After TLC analysis, 21 homogeneous fractions (ACS1 –ACS24 ) were obtained. Finally, a pure secondary metabolite (1, 3.5 mg), endowed with a high insecticidal activity was isolated (Flamini et al., 1997, 2004; Song et al., 2005). Structural determination of (1) was performed by spectroscopic analysis. Melting points (uncorrected) were determined with Kofler apparatus; 1H and 13C NMR spectra were obtained with a Bruker Avance II 250 spectrometer in CDCl3 , using TMS as internal standard. All experiments were performed using the standard Bruker library of microprograms. Known compounds were identified by comparison of their spectral data to those of the literature (Flamini et al., 1997, 2004).
2.3. Insects Insects were obtained from a culture of S. littoralis and maintained under standard conditions of temperature (27 ± 1 ◦ C), photoperiod (L16:D8) and relative humidity (60–70%). caterpillars were reared in Petri dishes with small artificial diet cubes based on wheat germ (Poitout and Bues, 1974), while adults with a 15% honey water solution. The culture was continuously supplemented with wild moths captured with a light trap in High Institute of Agronomy, Tunisia.
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2.4. Insect assay 2.4.1. Extracts and fractions effects on insect survival Thirty 3rd instar caterpillars were individually placed in glass Petri dishes (1 cm high and 9 cm in diameter) and provided with appropriate artificial diet added with 0 (control), 0.1, 1 and 10 mg/g of C. macrotum leaves methanol crude extract, or 1 mg/g of semipurified BuOH, EtOAc or MeOH–H2 O extracts. Fractions obtained from the Sephadex column were tested at 1 mg/g, while those from silica gel at 0.1 mg/g. All fractions are dried and well mixed with the artificial diet after preparation and cooling according to the procedure of Martinez and Van Emden (2001). Insect mortality was recorded in the end of experiment and adjusted for control using Abbott’s correction (Abbott, 1925). Mc =
Mo − Me × 100 100 − Me
Mo = mortality rate of treated insects (%); Me = mortality rate of control (%); Mc = corrected mortality rate (%). 2.4.2. Extracts and fractions effects on food consumption and utilization The effect of extracts and fractions on food consumption and utilization by third instar caterpillars was investigated using reared caterpillars on control diet after the second molt (<24 h). They were weighed and individually placed in Petri dishes. They were fed with known weights of diets containing 0, 0.1, 1 and 10 mg/g of extract (n = 30 and five replications for each concentration) and left to feed for 2 days, a period slightly shorter than instar duration. At the end of the experiment, caterpillars and faeces were weighed and food consumption was determined. Nutritional indices, namely relative consumption rate (RCR), relative growth rate (RGR), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD) and approximate digestibility (AD) were calculated as follows: RCR = I/Ba T RGR = B/Ba T ECI = (B/I) × 100 AD = [(I − F)/I] × 100 ECD = [B/(I − F)] × 100 where: I = weight of consumed food; Ba = arithmetic mean of insect weight during the experiment = [(PF − PI)/log (PF/PI)]; PF = caterpillars final weight (mg); PI = caterpillars starting weight (mg); T = feeding period in hours; B = change in body weight; F = weight of faeces produced during the feeding period (Waldbauer, 1968; Farrar et al., 1989). 2.5. Statistical analyses Nutritional indexes obtained after treatments with methanol and ethyl acetate extracts and fractions from sephadex column, as well as mortality, for all different treatments were compared using analysis of variance (ANOVA) followed by Duncan test for multiplecomparison when significant differences were observed at P = 0.01. Nutritional indexes resulting on caterpillars treated on fractions obtained from silica gel column were analyzed using the principal compound analysis (Tanagra version 1.4.38). 3. Results 3.1. Effect of methanol extract After preliminary study related to the insecticidal activity of flowers and leaves Chrysanthemum powders and their methanolic extracts against S. littoralis caterpillars (Haouas et al., 2008; Haouas,
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Table 1 Effect of C. macrotum methanolic extract on nutritional index and toxicity of the 3rd instar S. littoralis caterpillars at concentration of 0.1; 1 and 10 mg/g.a
Control 0.1 mg/g 1 mg/g 10 mg/g
RCR (mg/mg/day)
AD (%)
± ± ± ±
94.87 95.14 96.41 97.70
0.1064 0.1262 0.1357 0.1047
0.03b 0.02a 0.04a 0.006b
ECD (%) ± ± ± ±
1.47c 1.32c 0.94b 1.21a
8.12 4.39 2.40 2.66
± ± ± ±
ECI (%) 2.93a 1.19b 0.80c 0.91c
7.69 4.18 2.31 2.59
± ± ± ±
RGR (mg/mg/day) 2.76a 1.18b 0.76c 0.89c
0.0077 0.0051 0.0029 0.0027
± ± ± ±
0.0019a 0.0007b 0.0007c 0.0008c
Mtotb (%) 22.9 ± 1.2b 97.0 ± 3.2a 100.0 ± 0.0a
RCR, relative consumption rate; AD, approximate digestibility; ECD, efficiency of conversion of digested food; ECI, efficiency of conversion of ingested food; RGR, relative growth rate; Mtot, total mortality. a Means in the same column followed by the same letters are not significantly (P < 0.01) according to Duncan test. b Values were adjusted for control mortality using Abbott’s correction (Abbott, 1925).
2010), obtained results showed that C. macrotum leaves methanol extract was the most active one. For this raison it was selected to continue its chemical study. The incorporation of the MeOH extract of C. macrotum leaves in artificial diet of S. littoralis caterpillars at concentrations of 0.1, 1 and 10 mg/g affected positively or negatively all nutritional indexes. In fact, these doses of extract stimulate: (i) the relative consumption rate for caterpillars at concentrations of 0.1 and 1 mg/g and (ii) the approximate digestibility when employed at 10 mg/g. The comparison of RGR for each concentration showed that this parameter was affected at low concentration (0.1 mg/g). We noted a significant difference (P < 0.01) between caterpillars treated at 0.1 mg/g and control (respectively 0.0051 ± 0.0007 mg/mg/day and 0.0077 ± 0.0019 mg/mg/day). Concerning the RCR we noted an increasing effect of crude extract at the concentration of 1 and 0.1 mg/g. The mortality analysis showed a dose–effect correlation. In fact, at 0.1 mg/g, mortality was 22.9% and it increased to 100% at 10 mg/g (Table 1).
ACVIII fractions is noted, whereas fraction ACIII increases significantly the same parameter. In addition, approximate digestibility decreases significantly when ACII fraction is added to the artificial diet of Spodoptera caterpillars. The same result is also obtained for the ECD after adding ACIII and ACVI fractions to the caterpillars’ diet. Moreover, statistical analysis of the relative growth rate of treated caterpillars shows that ACIX and ACV fractions slow significantly the insect growth. In contrast the ACIV fraction is responsible for the growth perfection rate. These results highlight the specific effect of each fraction on nutritional indexes (Table 3). However, the effect of fractions is notably important on insects’ survival. Indeed, the addition of ACI and ACII fractions in artificial diet contribute to the mortality of 88% of treated caterpillars (Table 3). Knowing that ACI and ACII are collected successively from the sephadex column, have the highest rate of mortality, the same effect on nutritional indexes and practically the same TLC chromatograms, we suggest combine and simplified them using Silica gel column. 3.4. Activity of the fractions from Silica gel column chromatography
3.2. Effects of EtOAc, BuOH and MeOH–H2 O extracts
ACI and ACII fractions were combined and loaded on a silica gel column, obtaining 21 homogeneous fractions that were screened for their insecticidal activity. Results of principal compound analysis shows, on one side, an opposition between the effect of fractions on ECD, ECI and RGR (high positive correlation with the first axis) and their effect on RCR and AD (significant negative correlation with the first axis) on another side. Axis one is an axis of high fractions effect on ECD, ECI and RGR but it is low on RCR and AD. Axis two is an axis of high fractions effect on AD (Fig. 1). Statistical analysis of total mortality evidenced those fractions ACS9 , ACS10 , ACS11 , ACS12 , ACS16 , ACS1789 and ACS21 cause more than 50% death rate (Fig. 2). We remark that most died insects present molt difficulties and lethal malformation of new chrysalides.
These three extracts were tested for their insecticidal activity against S. littoralis caterpillars. Statistical analysis of relative consumption rate demonstrates that there are no significant differences between treatments and control and within different extracts. However, the analysis of the ECD, ECI and AD showed that the BuOH extract do not significantly affect the performance of the caterpillars. The most significant parameter that differenced the three extracts was the mortality rate. In fact, the EtOAc extract caused death to 84.4% of treated caterpillars. We remark also that many insects are died during molting and chrysalides formation. All combined results oriented us to choose the EtOAc extract for further bio-guided insecticidal assays (Table 2). 3.3. Effect of ethyl acetate fractions
3.5. Compound identification The ethyl acetate extract was chromatographed over Sephadex LH-20 column and the obtained fractions were added to the artificial diet of S. littoralis caterpillars (1 mg/g). By calculating nutritional indexes, a disturbing action, either as improvement or degradation, was evidenced for most fractions. Indeed, a significant reducing in the relative consumption rate caused by ACVI and
After insects assays the remaining amount of tested fractions are very low (ACS9 = 6.9 mg, ACS10 = 3.5 mg, ACS11 = 13.2 mg, ACS12 = 13.9 mg, ACS16 = 7.0 mg, ACS1789 = 23.5 and ACS21 = 1.6 mg). The TLC results of the fractions ACS9 , ACS11 , ACS12 , ACS16 and ACS1789 and ACS21 show a mixture of compounds, but taking in
Table 2 Action of ethyl acetate, buthanol and methanol extracts of C. macrotum on nutritional index and toxicity of the 3rd instar S. littoralis caterpillars at concentration of 1 mg/g.a
Control EtOAc BuOH MeOH–H2 O
RCR (mg/mg/day)
AD (%)
± ± ± ±
95.07 95.10 94.49 95.99
0.1160 0.1248 0.1334 0.1336
0.05a 0.02a 0.04a 0.024a
ECD (%) ± ± ± ±
1.63b 1.19b 1.33b 0.89a
7.92 6.13 7.85 5.50
± ± ± ±
ECI (%) 3.22a 1.60b 2.82a 1.46b
7.51 5.83 7.41 5.27
± ± ± ±
RGR (mg/mg/day) 3.03a 1.51b 2.67a 1.39b
0.0077 0.0070 0.0091 0.0068
± ± ± ±
0.0019b 0.0012b 0.0019a 0.0012b
Mtotb (%) 84.7 ± 2.0 a 18.8 ± 1.4c 42.0 ± 2.1b
RCR, relative consumption rate; AD, approximate digestibility; ECD, efficiency of conversion of digested food; ECI, efficiency of conversion of ingested food; RGR, relative growth rate; Mtot, total mortality. a Means in the same column followed by the same letters are not significantly (P < 0.01) according to Duncan test. b Values were adjusted for control mortality using Abbott’s correction (Abbott, 1925).
D. Haouas et al. / Industrial Crops and Products 34 (2011) 1128–1134
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Table 3 Results obtained on nutritional index and toxicity after treatment of the 3rd instar S. littoralis caterpillars with ethyl acetate fraction at 1 mg/g.a
Control ACI ACII ACIII ACIV ACV ACVI ACVII ACVIII ACIX
RCR (mg/mg/day)
AD (%)
± ± ± ± ± ± ± ± ± ±
95.07 93.79 93.25 94.95 94.05 94.55 93.55 94.40 93.78 95.08
0.1160 0.1245 0.1219 0.1545 0.1269 0.1137 0.0934 0.1048 0.0947 0.1067
0.05bcd 0.03bc 0.03bcd 0.02a 0.02b 0.03bcd 0.02e 0.02de 0.02e 0.02cde
ECD (%) ± ± ± ± ± ± ± ± ± ±
1.63a 2.35ab 3.71b 1.42a 1.35ab 1.87ab 3.81ab 1.44ab 2.27ab 1.71a
7.92 6.66 6.99 4.77 6.96 4.75 7.74 6.58 8.24 5.89
± ± ± ± ± ± ± ± ± ±
ECI (%) 3.22a 2.56ab 3.29ab 1.85c 2.48ab 2.85c 3.65ab 1.87abc 2.92a 2.54bc
7.51 6.20 6.43 4.51 6.52 4.47 7.19 6.20 7.67 5.58
± ± ± ± ± ± ± ± ± ±
Mtotb (%)
RGR (mg/mg/day) 3.03a 2.20ab 2.82ab 1.68c 2.26ab 2.64c 2.84ab 1.71ab 2.57a 2.36bc
0.0077 0.0073 0.0071 0.0067 0.0079 0.0046 0.0065 0.0063 0.0070 0.0057
± ± ± ± ± ± ± ± ± ±
0.0019ab 0.0012abc 0.0017abc 0.0020abcd 0.0020a 0.0021e 0.0012bcd 0.0013 cd 0.0017abc 0.0023d
88.9 88.9 22.2 22.2 33.2 66.7 44.3 44.4 55.6
± ± ± ± ± ± ± ± ±
2.6a 3.1a 3.2f 2.4f 3.1e 2.0b 2.4d 2.2d 2.6c
RCR, relative consumption rate; AD, approximate digestibility; ECD, efficiency of conversion of digested food; ECI, efficiency of conversion of ingested food; RGR, relative growth rate; Mtot, total mortality. a Means in the same column followed by the same letters are not significantly (P < 0.01) according to Duncan test. b Values were adjusted for control mortality using Abbott’s correction (Abbott, 1925).
Fig. 1. Principal compound analysis of the fractions effect on nutritional indexes of S. littoralis.
account the remaining amount, only fractions ACS11 , ACS12 and ACS1789 are loaded on a silica gel column. The quantities of obtained fractions are very small but not pure. So these fractions are not sufficient to continue insects’ tests not to identify the compound structure (Fig. 3). However, ACS10 TLC relieved a simple chromatogram. Nuclear magnetic resonance study of ACS10 led to the structure determination of the active compound (Table 4). The results were in agreement with those of literature for a C13 H10 O3 spiroketal enol ether derivative, in particular it was identified as (E)-7-(2,4-hexadiynylidene)-1,6-dioxaspiro[4.4]nona-2,8-dien4-ol (Fig. 4) (Zeisberg and Bohlmann, 1974).
4. Discussion The reduction in growth rate of caterpillars accurately reflects a reduction in the efficiency of conversion into biomass of the ingested and digested food. Similar results were reported by Pavela and Chermenskaya (2004) and Pavela (2004), when S. littoralis caterpillars were fed on Achillea ptarmica L., Ambrosia artemisiifolia L., Artemisia vulgaris L., Taraxacum officinal Weber and Cnicus benedictus L. methanol extracts. The sesquiterpene lactones isolated from Parthenium species showed a growth inhibition activity on Heliothis zea and Spodoptera exigua (Isman and Rodriguez, 1983). But the combination between toxicity and growth reduction could
Table 4 Chemical shift of hydrogen and carbon (CDCl3 , 250 MHz (1 H) and 62.5 MHz (13 C)). 1
Fig. 2. Mortality rate of S. littoralis caterpillars treated with fractions eluted from silica gel column.
1 2 3 4 5 6 7 8 9 10 11 12 13
H
1.92, 3H, d, J = 1.1 Hz – – – – 5.03, 1H, brs – 6.7, 1H, d, J = 5.7 Hz 6.28, 1H, dd, J = 5.7 and 1.9 Hz – 5.66, 1H, dd, 2.0 and 2.0 Hz 5.13, 1H, dd, 3.0 and 3.0 Hz 6.53, 1H, dd, 3.0 and 2.0 Hz
Coupling
13
H6 – – – – H1, H8, H9 – H6, H9 H6, H8 – H12, H13 H11, H13 H12, H11
4.6 80.3 65.3 77.4 70.1 83.0 168.6 133.7 125.8 117.2 76.0 106.6 147.0
C
DEPT CH3 C C C C CH C CH CH C CH CH CH
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Fig. 3. Chromatographic steps led to the isolation of compound (1).
1
CH3
2
C
4
3
C
C (1)
8
5
C H
9
6
C
E
10
7
O
Fig. 4. Structure of the spiroketal enol ether derivative.
O OH 11
13
12
D. Haouas et al. / Industrial Crops and Products 34 (2011) 1128–1134
be due to another compound, such as the chromene encecalin, isolated from Encelia genus responsible for mortality (77%) and growth inhibition of Peridroma saucia (Noctuidae) caterpillars (Isman and Proksch, 1985). The ingestion of C. macrotum EtOAc, BuOH and the MeOH–H2 O extracts affected the nutritional indexes. The follow-up of insects mortality treated with these same extracts showed that the EtOAc one causes an important toxicity (84.7%) in treated caterpillars. Similar results were obtained by Faini et al. (1997) which showed that Flourensia thurifera leaves and stems extracts were endowed with insecticidal activity against S. littoralis caterpillars because of their content in coumarins and chromenes. Fractions collected from the Sephadex column showed different activities against S. littoralis caterpillars, suggesting a different distribution of the active compounds. Most toxic fractions (ACI and ACII ) were further simplified and tested against S. littoralis caterpillars again. In this way, four of the nine fractions maintained a high mortality (more than 50%), that can be due to the perturbation of nutritional indexes. These observations allow affirming that nutritional imbalance can be one factor contributing the caterpillars mortality. A compound responsible for this toxicity is identified as a spiroketal enol polyacetylene, in particular as (E)-7-(2,4hexadiynylidene)-1,6-dioxaspiro[4.4]nona-2,8-dien-4-ol. In the Chrysanthemum genus, this compound was isolated for the first time by Wrang and Lam (1975) from C. leucanthemum L., roots, but without any further study on its biological activity. This compound is characterized by the presence of oxygen atoms, both as alcoholic groups and heterocyclic, chiral centers and an apolar part, typical characteristics of a potential insecticidal natural product, as reported by Ujváry (2002). Natural polyacetyenes are found as C10 –C17 derivatives. Compounds with 13 carbon atoms, such as this active spiroketal, are widely distributed in the Asteraceae family. Bohlmann and Zdero (1975) identified from C. macrotum polyacetylenes with 12 and 13 carbons without reported any activity for them. Later, Bohlmann and Fritz (1979) isolated a new sulfurcontaining polyacetylene in C. coronarium. Other studies performed by Gao et al. (1996) highlight the presence of spiroketal enol ether from C. segetum and C. coronarium endowed with an anti-feeding activity against Pieris brassicae (Gao et al., 1998; Yin et al., 2003, 2004; Chen et al., 2004, 2005). Phytochemical studies on some Asteraceae species (A. vulgaris L., C. leucanthemum Centautia spp. and Dahlia spp.) contribute to the isolation of 14 polyacetylenes compounds and some thiophene derivatives (Wat et al., 1981). Most of these compounds were characterized by a high toxic activity against Aedes aegypti, Simulium vitatum larvae and Caenorhabditis elegans nematodes. The toxicity of these polyacetylenics compounds can be attributed to different mode of actions. In absence of light the polyacetylenes are antifeedant to insects (Champagne et al., 1986). This can explain our results concerning the perturbation of nutritional indexes on S. littoralis caterpillars. In presence of light, there are two hypotheses explaining the insect toxicity. The first one can owe photocatalic cycle of single oxygen generation and other excited state molecule that leads to rapid lipid peroxidation and cell death. The second one can contribute to a photogenotoxicity, in which compounds intercalate into DNA, and upon irradiation with near UV light form mono- and bi-functional adducts with pyrimidine bases (Arnason and Bernards, 2010). Phototoxic compounds can lead to redding and thinning of cuticle in lepidopteran caterpillars exposed to dietary furanocoumarins (Berenbaum, 1987). They can also lead to the appearance of black cuticular lesions and prevent a normal molt and died before metamorphosing in the case of Manduca sexta (tobacco hornworm) when it was exposed to oral or topical of thiophene, poyine or hypericin (Downum et al., 1984; Champagne et al., 1986; Samuels and Knox, 1989).
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