J. ChiL Chern. Soc.

, 52, N 2 (2007)

SECONDARY METABOLITES IN THE FLOWER HEADS OF HAPLOPAPPUS BERTERII

(ASTERACEAE) AND ITS RELATION WITH INSECT-ATTRACTING MECHANISMS
ALEJANDRO URZUA", Rocfo SANTANDER, JAVIER ECHEVERRiA, MARCOS C. REZENDE
Universidad de Santiago de Chile, Facultad de Quimica y Biologia, Departamento de Ciencias del Ambiente.
Laboratorio de Quimica Ecologiea, Casilla- 40, Correo- 33, Santiago, Chile.

ABSTRACT
The chemical compositions of the flower heads of H. bertedi and of Chrysantemum coronarium 1., 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. OUf 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; Asteraccae; Monoterpcnes; Sesquiterpenes; Flavonoids; Insect-attracting stimuli

INTRODUCTION
The Chilean littoral rock formations (limit of IV and V Regions), Los
MoUes (V Region, Chile 32" 30'S, 71 0 30'W), is the habitat of Haplopappus
bcrterii Phil. (Astcraceae) an endemic evergreen shrub with yellow flowers of
2.5 em of diameter I).
Flower heads of H. berterii are visited by a varicd entomofauoa and
although no syslematic studics have been pcrformed, it is known that they
are the host of Trupanea Schrank (Diptera: Tephritidae)2l species. In addition
Arthrobracus sp. (Coleoptera: Melyridac) 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 Asteracca with yellow flowers like H.berterii, that
grows near this Haplopappus species.
In this communication we report the composition oflhe 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 IQ establish ifthere are some chemical similarities that might explain why
these two different species are visited by the same insects.

EXPERfMENTAL
Plant material
Haplopappus berterii Phil. (Asteraceae) flower heads, were collected in
November 2004 in Los MoUes (V Region, Chile 3230'S, 71 0 30'W). Voucher
specimens were deposited in the Hcrbarium onhe National Museum ofNatural
History, Santiago, Chile.
Plant extraction
Fresh flower heads of H. be11erii (275 g) were extracted by dipping the
plant material in 1.5 L of cold CH 2Cl 2 for 60 s. The extraction was repeated
twice. The material exhausted with CH 2Cl 2 was dried in an oven aL50, 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 CHCl]. The organic layer was discarded and the watcr layer
was extracted with AcOEt.
Column chromalography separation of the extracts
The CH 2Cl 2 extract (1.2 g, 0.44 %) was fractionated by CC (silica gel)
using pentane - CH 1Cl 2 and CH 2CI 2 - McOH step gradients to afford 4
fractions. The AcOEI extract (1.4 g, 0.51 %) was fractionated by CC (silica
gel) using a CHC~ - MeOH step gradienllo afford 80 fractions.
TLC study of the extracts and fractions
TLC of
extracts and fractions was performed on silica gel 60 F25-1 precoated plares from ~lerck. Specific spray reagents were used for detection of
diffetenl families of compounds 3l : anisaldehyde-~SO~, phosphomolibdic
acid znd \"3illiI.I.in-H.!O... for terpenoids and diphenylboric acid-~thylamino
~~EG I fur ih"llDOids.

GC-EM analysis ofthe CH 1CI 2 extract

The fraction eluted with pentane, from the CH1.C!2 extract, werc analyzed
in triplicate in a GC-MS ( gas chromatograph: Hewlett-Packard model using
a HP5891; mass spectrometric detector with integrated data system: Hew1eUPackard model HP5972). Separation was performed using an Ultra-2 H.P.
capillary column (IS m x 0.25 mm). The temperature of tile injector was 295
C, and the temperature of the column was programmed, starting at 45C, for
2 min, followed by a rise to 200 C at 10C /min and to 300 C at 20 C / min l .
The temperature was kept constant al 300C for 20 min. Helium was the carrier
gas at 10 lb. psi. Detection was done using QJ and EI.
Spectroscopy
All NMR experiments were perfonned on a Bruker-400 AV3nce
spectrometer using DMSO-d6 Two-dimensional spectra were obtained using
standard Bruker software. FTIR spectra were obtained on a Perkin Elmer
spectrophotometer in KEr.
Nomenclature of compounds
Names ofmonoterpenes, sesquiterpenes, and flavonoids are given according
Lo the Handbook of terpenoids 4} and Flavonoids Chemistry, Biochemistry and
Applications'l.
Yields of fractions and compounds
The yield ofextracts 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 DISCUSSfON

Chemical composition
The CH 2CI 2 extract (1.2 g, 0.44 %) was fractionated by CC (silica gel)
using pentane - C~CI2 and CH 2Cl 2 - MeOH stcp gradients. Fraction A eluted
with pentane, (377 mg, 0.137%) was submiUed to extensive GC-MS analysis.
The AcOEt extract (1.4 g, 0.51 %) was fractionated by CC (silica gel) using a
CHell - MeOH step gradient to afford 80 fractions regrouped after TLC in four
new fraction" B (0.025 g), C: (0.027 g), 0 (0,12 g) and E (0,31 g).
Monoterpenes (0.0015 %): a.-pinene (1), p-pinene (2), p-myrcene (3),
limonene (4).
Sesquiterpenes (0.013 %): 3-cubebene (5), 4(15)-cubebene (6), 1(10)aristolene (7), copaene (8), isocaryophyllene (9), a.-caryophyllene (10), 4,9bulgaradiene (II), 4,1O(14)-bulgaradiene (12), 4,11-amorphadiene (13),
1(I 0),4-cadinadiene (14), 2,5,5-trimethyl-1 ,3,4,5,6,7-hexahydro-2H-2,4aethanonaphthalene (15).

me

Miscellaneous alkanes (0.00 I %): 2-methyldacaline; 2,4,6-trimethyloctanej

J. Chi!. Chern. Soc., 52, N2 (2007)

2,6-dimethylundecane; 4,6-dimethylundecane and 2,IO-dimethylundecane.

The compounds identified in the fractions obtained from the CC separation
of the AcOEt extract were: Flavonoids (0.51 %): diosmetin (5,7,3'- trihydroxy4'-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 oftbe compounds
The identificalion 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 confinned by the obsel\'ation of peak
enhancements upon coinjection of standards. When standards were not
available, the mass spectra were compared with published spectra of authentic
compounds 7,~.9). Also, Kovats index of the peaks were compared with values
from the literature.
Fractions B, C, 0 and E were respectively crystallised from CHCI3-EtOH
(85:15). Compound E, yellow crystals (230 mg) and compound D, yellow
amorphous solid (89 mg),were respectively identified as qucrcctin (19) and
[uteolin (18) by direct comparison (FTIR, TLC, IH-NMR) with authentic
samples obtained from Aldrich,

R,

;?'
HO

"""
#'
OH

R,

1" RI =: R2 =: H , _3 =: OC::=:.::
17 R. =:R2 =OE, R]=0CE3
18 Rl=H; R2=R3=OE:

R,

1. R 1 =R2 = R 3 =OE

Compound C, yellow amorphous solid (17 mg) shows an IHNr-,.m

spectrum that indicate the structure of a methyl derivative of quercetin
(3,5,7,3',4'-pentahydroxyflavone) (l9). Bidimentional NMR experiments
(HMBC and HSQC) provide support for the determination of position of the
Illcthoxy group at C-4' in ring B. The compound was identified as tamirexin
(3,5,7,3'- tetrahydroxy-4'-melhoxyflavone) (17) and data were in agreement
with those reported in the literature 10),
Compound B, yellow amorphous solid (14 mg) shows an IH_NMR
spectrum that indicate the structure of a methyl derivative ofluteolin (5,7,3'4'tetrahydroxyflavone) (i8). 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'-merhoxyflavone) (16) and data were in agreement with those
reported in the literature 10).
The composition of the flower extracts of H. bertcrii may be compared
with that of other Chilean Haplopappus. The hydrocarbon fraction accounted
for 93.39 % of the total chromatogram peak area with nalkanes (81.71 %)
comprising the major group of compounds, represented mainly by C29H60
(42 %), C3lH62 (18.6 %) and C27H56 (13.1 %). Although Ihis hydrocarbon
profile is similar to thai 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% coment of hydrocarbons l,9)
whereas H. bertel'ii only yielded 0.11 %, a value even lower than that of H.
foliosus DC.(8%) growing in coast areas ofthe V region 5J and H. bustillosianus
Remy (1.9 %) growing in the coast rock formations ofVillarrica lake II).
Only trace amounts (0.0015%) of monoterpencs 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 a-pinene (i) and ~-pinene (2) II).
By contrast, no monolcrpenoids have been identified in: H. cuneifolius (Ness),
H. uncinatus Phil. and H. shumalUli (OK.) Br. etClark 9).
Finally, an overview of the sesquiterpene composition of H. berterii is

in agreement with the result'> 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 ro
the genus Haplopappus eouid not be found.
10

II

"

14

IJ

15

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. Oi ffercnt species visit the flower heads in early hours of the
morning, mid-morning, noon and afternoon.
In the specific case ofH. 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 Z) .The Artbrobracus 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 1.
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 if! November and December, for nine weeks.

1143

1. Chi!. Chern. Soc., 52, N' 2 (2007)

ACKNOWLEDGEMENTS

AJthougb field conditions prevented a rigorous quantification of the insect

visilS, there was apparently little difference regarding the insect preferences

Financial support from DICYT (USACH) is gratefully acknowledge.

between the two plants.

Our observations led us to search for a common set ofstimuli in both plants
that might be responsible for lheattraction aCthe same insects. We hypothesized
that the flower heads of both Asternceae 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 Chrysanl.hemum coronarium was described Il). In the metion of

volatile compounds the only sesquiterpene identified was a-famesene. The

monOlcrpenes camphor, hornyl acetate and tnms-chrysanlhenyl acetate
accounted for more than 60010 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 (I8) and quercetin
(19) have also been found in the flower heads ofe. 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 bc taken as an indication
that optical cues are more import'ant than chemical stimuli in detcnnining
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 linle orientation when an insect is at
some distance from its host, and that searching for a host plant is essentially
a random process 151. 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 [61.

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 detennining 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 correlation's 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.

1144

REFERENCES
1.-

Hoffmann, 1. A., 1995. Flora Silvestre de Chile, Zona Central. 3 Ed.

Edicioncs Claudio Gay, Santiago Chile pp. 238-239.
2.- Frias, D., 1985. Rev. Bras. Zool. 2, 363.
3.- Wagner H., Bladt S., Zgainski E.M., 1984. "Plant Drug Analysis"
Springer-Verlag, Berlin.
4.- Connolly J.D., Hill R.A., 1992. "Dictionary of Terpenoids Vol I. Mono
and Sesquiterpenoids" Chapman and Hall, London, UK.
5.- Andersen O. M., Markham, K. R., 2006." Flavonoids: Chemistry,
Biochemistry and Applications" Taylor and Francis Group, Boca Raton,
FL, USA.
6.- Pesyna, G. M., Venkataraghavan, R., Dayringer H. E., McLafferty, F. W.,
1976 Anal. Chem. 48, 1362.
7.- Swingar, A. A., Silverstein, R. M., 1981. Monoterpenes Infrared, Mass,
I H NMR, and U C NMR Spectra, and Kovats Indices. Aldrich Chemical
Company, Inc., Milwaukee USA.
8.- Urz\ia, A., 2004. J. Chil. Chern. Soc. 49, 137.
9.- Urzua, A., Contreras, R., Jara, P., Avila, F., Suazo, M., 2004 Biochem.
Syst. Ecol. 32, 215.
10.- Harbome, J., B., 1996 " The fIavonoids Advances in Research Since
1986" Chapman and Hall, London, UK., pp. 295-330.
11.- UrziJa, A., Iturra, 8., Sebastian, B., Munoz, M., 2007 Biochem. SySL

Eoo l. in press.
12.- Mani, M., S., Saravanan, J., M., 1999. "Pollination Ecology and Evolution
in Compositac (Aslcraceae). Science Publishers, Inc. New Hampshire,
USA, pp. 12-23.
13.- Urzita, A., Sebastian, 8., Vines, M., 2006 Flavour and Fragrance J. 21,
783.
14.- Urzua, A., Wlpublished results.
15.- Schoonhoven, L. M., A van Loon J. J., Dicke, M., 2005. "Insect-Plant
Biology" (Chapler 6, Host-plant selection: how to find a host plant).
Oxford University Press, New York, USA, pp. 135-160.
16. Lewis, A. C., Lipani, G., 1990. Learning and flower use in bUllcrfiies:
hypothesis from honey bees. In Bcmays, E. A., "Insect-plant interaction,
vol 2". CRC Press, Boca Raton, FL., pp. 95-110