Journal of the Chilean Chemical Society
versión On-line ISSN 0717-9707
J. Chil. Chem. Soc. v.52 n.2 Concepción jun. 2007
J. Chil. Chem. Soc, 52, Nº 2 (2007) págs.: 1142-1144
SECONDARY METABOLITES IN THE FLOWER HEADS OF HAPLOPAPPUS BERTERII (ASTERACEAE) AND ITS RELATION WITH INSECT-ATTRACTING MECHANISMS
ALEJANDRO URZÚA*, ROCÍO SANTANDER, JAVIER ECHEVERRÍA, MARCOS C. REZENDE
Universidad de Santiago de Chile, Facultad de Química y Biología, Departamento de Ciencias del Ambiente, Laboratorio de Química Ecológica, Casilla- 40, Correo- 33, Santiago, Chile.
The chemical compositions of the flower heads of H. berterii and of Chrysantemum coronarium L., visited by a varied entomofauna, were compared in the search of possible correlations that might explain why different plants are visited by the same insects. Though some similarities were observed in the flavonoid contents of both species, their overall composition was dramatically different, pointing to the existence of rather complex mechanisms of insect attraction by these species. Our results thus represent a cautionary remark to interpretations of such mechanisms based solely on the chemical composition of the volatile components of flowers in the field.
keywords: Haplopappus berterii; Asteraceae; Monoterpenes; Sesquiterpenes; Flavonoids; Insect-attracting stimuli
The Chilean littoral rock formations (limit of IV and V Regions), Los Molles (V Region, Chile 32º 30S, 71º 30W), is the habitat of Haplopappus berterii Phil. (Asteraceae) an endemic evergreen shrub with yellow flowers of 2.5 cm of diameter 1).
Flower heads of H. berterii are visited by a varied entomofauna and although no systematic studies have been performed, it is known that they are the host of Trupanea Schrank (Diptera: Tephritidae)2) species. In addition Arthrobracus sp. (Coleoptera: Melyridae) use the flower heads as diet, and one Chilean social bee Diasiadae sp. (Himenoptera: Apiadeae) and the butterfly Vanessa carye (Lepidoptera: Nymphalinae) also visit the flowers.
These species have also been identified as visitors of Chrysanthemum coronarium L. another Asteracea with yellow flowers like H.berterii, that grows near this Haplopappus species.
In this communication we report the composition of the volatile compounds, epicuticular chemistry and flavonoids of H. berterii flower heads, and make a comparison with the chemistry of Chrysanthemum coronarium L. flowers in order to establish if there are some chemical similarities that might explain why these two different species are visited by the same insects.
Haplopappus berterii Phil. (Asteraceae) flower heads, were collected in November 2004 in Los Molles (V Region, Chile 32º 30S, 71º 30W). Voucher specimens were deposited in the Herbarium of the National Museum of Natural History , Santiago, Chile.
Fresh flower heads of H. berterii (275 g) were extracted by dipping the plant material in 1.5 L of cold CH2Cl2 for 60 s. The extraction was repeated twice. The material exhausted with CH2Cl2 was dried in an oven at 50°, milled and submitted to percolation in 95 % cold EtOH (1.5 L) for 24 h. The procedure was repeated twice. The EtOH extracts were concentrated and partitioned between water and CHCl3. The organic layer was discarded and the water layer was extracted with AcOEt.
Column chromatography separation of the extracts
The CH2Cl2 extract (1.2 g, 0.44 %) was fractionated by CC (silica gel) using pentane CH2Cl2 and CH2Cl2 MeOH step gradients to afford 4 fractions. The AcOEt extract (1.4 g, 0.51 %) was fractionated by CC (silica gel) using a CHCl3 MeOH step gradient to afford 80 fractions.
TLC study of the extracts and fractions
TLC of the extracts and fractions was performed on silica gel 60 F254 pre-coated plates from Merck. Specific spray reagents were used for detection of different families of compounds 3) : anisaldehyde-H2SO4, phosphomolibdic acid and vainillin-H3PO4 for terpenoids and diphenylboric acid-β-ethylamino ester-4000(PEG) for flavonoids.
GC-EM analysis of the CH2Cl2 extract
The fraction eluted with pentane, from the CH2Cl2 extract, were analyzed in triplicate in a GC-MS ( gas chromatograph: Hewlett-Packard model using a HP5891; mass spectrometric detector with integrated data system: Hewlett-Packard model HP5972). Separation was performed using an Ultra-2 H.P. capillary column (15 m x 0.25 mm). The temperature of the injector was 295 ºC, and the temperature of the column was programmed, starting at 45 ºC, for 2 min, followed by a rise to 200 ºC at 10 ºC /min and to 300 ºC at 20 ºC / min-1. The temperature was kept constant al 300 ºC for 20 min. Helium was the carrier gas at 10 lb. psi. Detection was done using QI and EI.
All NMR experiments were performed on a Bruker-400 Avance spectrometer using DMSO-d6. Two-dimensional spectra were obtained using standard Bruker software. FTIR spectra were obtained on a Perkin Elmer spectrophotometer in KBr.
Nomenclature of compounds
Names of monoterpenes, sesquiterpenes, and flavonoids are given according to the Handbook of terpenoids 4) and Flavonoids Chemistry, Biochemistry and Applications 5).
Yields of fractions and compounds
The yield of extracts and compounds were calculated in relation to the fresh plant material. The percentage of different families and individual compounds was calculated from the peak areas of the chromatograms.
RESULTS AND DISCUSSION
The CH2Cl2 extract (1.2 g, 0.44 %) was fractionated by CC (silica gel) using pentane CH2Cl2 and CH2Cl2 MeOH step gradients. Fraction A eluted with pentane, (377 mg, 0.137%) was submitted to extensive GC-MS analysis. The AcOEt extract (1.4 g, 0.51 %) was fractionated by CC (silica gel) using a CHCl3 MeOH step gradient to afford 80 fractions regrouped after TLC in four new fractions: B (0.025 g), C: (0.027 g), D (0,12 g) and E (0,31 g).
Monoterpenes (0.0015 %): α-pinene (1), β-pinene (2), β-myrcene (3), limonene (4).
Sesquiterpenes (0.013 %): 3-cubebene (5), 4(15)-cubebene (6), 1(10)-aristolene (7), copaene (8), isocaryophyllene (9), α-caryophyllene (10), 4,9-bulgaradiene (11), 4,10(14)-bulgaradiene (12), 4,11-amorphadiene (13), 1(10),4-cadinadiene (14), 2,5,5-trimethyl-1,3,4,5,6,7-hexahydro-2H-2,4a-ethanonaphthalene (15).
n-Alkanes (0.11 %): C10H22; C11H24; C12H26; C13H28; C14H30; C15H32; C16H34; C17H36; C18H38; C19H40; C20H42; C21H44; C22H46; C23 H48; C24H50; C25H52 ;C26H54; C27H56; C28H58; C29H60; C30H62; C31H64; C32H66; C33H68.
Miscellaneous alkanes (0.001 %): 2-methyldacaline; 2,4,6-trimethyloctane; 2,6-dimethylundecane; 4,6-dimethylundecane and 2,10-dimethylundecane.
The compounds identified in the fractions obtained from the CC separation of the AcOEt extract were: Flavonoids (0.51 %): diosmetin (5,7,3′- trihydroxy-4′-methoxyflavone) (0.004 %) (16), tamirexin (3,5,7,3′- tetrahydroxy-4′-methoxyflavone) (0.006 %) (17) , luteolin (5,7,3′4′-tetrahydroxyflavone) (0.043 %)(18) and quercetin (5,6,7,3′,4′-pentahydroxyflavone) (0.089 %) (19).
Identification of the compounds
The identification of compounds in the chromatographic profiles was achieved by comparison of their mass spectra with a library data base (NIST 1998) using a reverse search technique which verified that main peaks in the reference spectrum were present in the unknown spectrum 6). Spectra were considered coincident if the similarity index was higher than 95% 6).
Preliminary identifications were confirmed by the observation of peak enhancements upon coinjection of standards. When standards were not available, the mass spectra were compared with published spectra of authentic compounds 7,8,9). Also, Kovats index of the peaks were compared with values from the literature.
Fractions B, C, D and E were respectively crystallised from CHCl3-EtOH (85:15). Compound E, yellow crystals (230 mg) and compound D, yellow amorphous solid (89 mg),were respectively identified as quercetin (19) and luteolin (18) by direct comparison (FTIR, TLC, 1H-NMR) with authentic samples obtained from Aldrich.
Compound C, yellow amorphous solid (17 mg) shows an 1H-NMR spectrum that indicate the structure of a methyl derivative of quercetin (3,5,7,3′,4′-pentahydroxyflavone) (19). Bidimentional NMR experiments (HMBC and HSQC) provide support for the determination of position of the methoxy group at C-4′ in ring B. The compound was identified as tamirexin (3,5,7,3′- tetrahydroxy-41-methoxyflavone) (17) and data were in agreement with those reported in the literature 10).
Compound B, yellow amorphous solid (14 mg) shows an 1H-NMR spectrum that indicate the structure of a methyl derivative of luteolin (5,7,3′4′-tetrahydroxyflavone) (18). Bidimentional NMR experiments (HMBC and HSQC) provide support for the determination of position of the methoxy group at C-4′ in ring B. The compound was identified as diosmetin (5,7,3′-trihydroxy-4′-methoxyflavone) (16) and data were in agreement with those reported in the literature 10).
The composition of the flower extracts of H. berterii may be compared with that of other Chilean Haplopappus. The hydrocarbon fraction accounted for 93.39 % of the total chromatogram peak area with n-alkanes (81.71 %) comprising the major group of compounds, represented mainly by C29H60 (42 %), C31H62 (18.6 %) and C27H56 (13.1 %). Although this hydrocarbon profile is similar to that of other Haplopappus species, a remarkable contrast in the yields of this fraction is observed. The epicuticular compounds of species growing in the mountains comprise a 25-50% content of hydrocarbons 8,9) whereas H. berterii only yielded 0.11 %, a value even lower than that of H. foliosus DC.(8%) growing in coast areas of the V region 8) and H. bustillosianus Remy (1.9 %) growing in the coast rock formations of Villarrica lake 11) .
Only trace amounts (0.0015%) of monoterpenes 1-4 were found in H. berterii. Although in the epicuticular compounds of Haplopappus, the presence of monoterpenes seems to be erratic, compounds 1-4 have been identified in H. velutinus Remy and H. illinitus Phil. while H. foliosus DC. contains limonene (4) 8,9) and H. bustillosianus Remy contains α-pinene (1) and β-pinene (2) 11). By contrast, no monoterpenoids have been identified in: H. cuneifolius (Ness), H. uncinatus Phil. and H. shumanni (OK.) Br. et Clark 9) .
Finally, an overview of the sesquiterpene composition of H. berterii is in agreement with the results found for the epicuticular chemistry of other Haplopappus species. Even though some of these molecules and structural families are repeated among species, a clear sesquiterpene pattern common to the genus Haplopappus could not be found.
The flower heads of Asteraceae are visited by various insects. The visitors obtain shelter, abundant food and are found everywhere on the flower heads. Even in the same eco-system, the Asteraceae insect visitors differ during the course of a day. Different species visit the flower heads in early hours of the morning, mid-morning, noon and afternoon.
In the specific case of H. berterii, a large entomofauna may be observed on its flower heads. Some of the visitors include the Trupanea Schrank (Diptera: Tephritidae) species, whose larvae have been found within the florets eating away the ovaries 2) .The Arthrobracus sp. (Coleoptera: Melyridae) uses the flower heads as part of their diet, eating the disc florets. Finally, one Chilean social bee, Diasiadae sp. (Hymenoptera: Apiadeae) and the butterfly Vanessa carye (Lepidoptera: Nymphalinae) are also regular visitors to the flower heads. Bees visitors play only a minor role as pollinators and are mostly pollen robbers in Asteraceae 12).
These four species were also identified on the yellow flower heads of Chrysanthemum coronarium L., another Asteracea that grew near the H. berterii flowers. Our field observations took place during mid-morning, between 11:00 and 13:00 h, for two days a week in November and December, for nine weeks. Although field conditions prevented a rigorous quantification of the insect visits, there was apparently little difference regarding the insect preferences between the two plants.
Our observations led us to search for a common set of stimuli in both plants that might be responsible for the attraction of the same insects. We hypothesized that the flower heads of both Asteraceae might elicit similar chemical stimuli, and that this could be verified by a comparison of the compositions of their volatile constituents.
In a recent communication, the surface and volatile compounds of flower heads of Chrysanthemum coronarium was described 13). In the fraction of volatile compounds the only sesquiterpene identified was α-farnesene. The monoterpenes camphor, bornyl acetate and trans-chrysanthenyl acetate accounted for more than 60% of the volatile components. None of these compounds were present in H.berterii. The only common compound in both plants was limonene (4).
As regards flavonoids, large amounts of luteolin (18) and quercetin (19) have also been found in the flower heads of C. coronarium 14) .Thus we may conclude that, with the exception of the common presence of low concentrations of limonene (4), the compositions of the volatile components of H. berterii and C. coronarium, responsible for eventual odorous stimuli to insects, are dramatically different. Flowers of these species share in common, besides similar shape and size, the yellow flavonoids luteolin (18) and quercetin (19), responsible for their common colour. This might be taken as an indication that optical cues are more important than chemical stimuli in determining the choice of these insects for the two flowers. Because of the limitations of field observation, this conclusion should be regarded with caution. Chemical and optical attractants to insects are difficult to measure and quantify in the field. It has been suggested that there is little orientation when an insect is at some distance from its host, and that searching for a host plant is essentially a random process 15). Once the insect finds a suitable host, this random search is followed by a learning process after which the insect is able to respond positively to optical and chemical stimuli by the plant, and recognize it to feed and for oviposition 16).
These considerations only emphasize the difficulties involved in field studies, and the cautious nature of any conclusions drawn from them. Our results revealed little or no correlation between the chemical composition of the volatile components of H. berterii and C. coronarium flowers and the preferences of some of their guest insects. Because of the complex interplay of factors determining this preference in the field, such absence of correlations does not rule out the existence of odorous stimuli in the interaction between the studied insects and these plants. Such correlations might be obscured by the essentially random learning process undergone by an insect in the field. Nevertheless, our results also represent a word of caution to interpretations of insect-plant interactions based solely on the presence of volatile chemical constituents in the latter.
Financial support from DICYT (USACH) is gratefully acknowledge.
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