Biochemical Systematics and Ecology 27 (1999) 1 10

Anti-insect activity of plant extracts from the wild

flora in southeastern Spain
Mar a J. Pascual-Villalobos *, Antonio Robledo

Consejer& a de Medio Ambiente, Agricultura y Agua, Centro de Investigacio& n y Desarrollo Agroalimentario,
Estacio& n Seric& cola, 30150 La Alberca, Murcia, Spain

Ingemisa, Plaza de Sandoval no. 5, 30004 Murcia, Spain
Received 26 January 1998; accepted 27 May 1998

Abstract
Plant extracts of a group of 57 species from 21 different botanical families, harvested from the
wild in southeastern Spain, were screened for antiinsect activity using the stored grain pest
ribolium castaneum Herbst. (Coleoptera: Tenebrionidae). Echium creticum, epidium
graminifolium, Anacyclus clavatus, Galactites duraei, Helichrysum decumbens, Senecio vulgaris,
Pallenis spinosa, Ecballium elaterium, Haplophyllum linifolium and Solanum bonariense gave
activity in most tests performed. Ballota hirsuta produced growth inhibition in larvae. Ononix
natrix, avatera cretica and rginea maritima (bulb) showed contact toxicity in pupae (100%
mortality). Compositae species had a tendency to induce either growth inhibition (with or
without mortality) or repellency on the target insect. Overall, 70% of the extracts tested showed
some activity and 21% of them (including endemisms) were more active and newly identified.
 1988 Elsevier Science Ltd. All rights reserved
Keywords: Screening; Plant extracts; Botanical insecticides; Anti-insect activity; ribolium castaneum

1. Introduction
In recent years, agrochemical companies have focussed on the study of natural
products for the development of new insecticides (Addor, 1995). The discovery of
active compounds that are more selective and less persistent will be beneficial for both
the environment and agricultural product consumers, although natural products
cannot automatically be assumed to be without risk.
The protection of agricultural products in storage against attack by pests is
essential in many countries suffering from inadequate storage facilities and/or climatic
*Corresponding author. Tel.: 68 840150; fax: 68 844802, e-mail: mjpascual@readysoft.es.
0305-1978/98/$19.00  1998 Elsevier Science Ltd. All rights reserved.
PII: S0 30 5 - 19 7 8( 9 8 )00 0 51 -9

M.J. Pascual-Villalobos, A. Robledo/Biochemical Systematics and Ecology 27 (1999) 110

conditions that favor deterioration of food commodities (e.g. in the Middle East).
The application of chemical pesticides appart from environmental concerns may not
be sufficiently effective since insects develop resistance to them. The search for
environmentally safe grain storage methods and products is in this context very
relevant.
The interaction between plants and insects is chemically mediated by secondary
metabolites. Bioactive plant products have been used by man since ancient times,
especially in cultures with a strong herbal tradition (Secoy and Smith, 1983; Yang and
Chang, 1988).
Many authors have undertaken the screening of plant species for pesticidal activity.
Hoffmann et al. (1993) evaluated 300 plant species from southwestern USA for
antimicrobial activity, Gonzalez-Coloma et al. (1994a,b) detected antifeedant and
insecticidal effects in plants from the Canary Islands and Japanese Lauraceae while
Cun at et al. (1990) and March et al. (1991) have studied biocidal and antimicrobial
activities in plants from the Valencian region in Spain.
According to Alcaraz et al. (1997) the Murcia Region of Spain has at least 2500 wild
species. Here, we report the results of the antiinsect activity found in a group of 57
species belonging to 21 families harvested from the wild in southeastern Spain. Several
plant extracts obtained with solvents of different polarity were tested for each species
by a range of bioassays using the stored grain pest ribolium castaneum Herbst.
(Coleoptera: Tenebrionidae) as a model insect.

2. Material and methods

2.1. Plants
We studied a group of 57 species belonging to a number of plant families (Table 1).
Aerial parts were harvested from the wild during 1994 or 1995, mainly when
plants were at the flowering stage. Voucher specimens were deposited in the Centro
de Investigacion y Desarrollo Agroalimentario (Murcia, Spain) Herbarium
(CIDAHERB). In some cases, a species was sampled at different times or its different
parts separated for independent processing. The material was freeze dried and ground
to 1 mm diameter particle size with a mill and stored at room temperature in
hermetically sealed plastic bags prior to extraction.
2.2. Preparation of crude extracts
Each plant sample (1 g) was extracted with organic solvents of increasing polarity at
room temperature ("20C): 48 h with hexane (10 ml), 24 h with acetone (10 ml) and
48 h with 50% aqueous methanol. All three extracts from each sample were stored at
5C. For testing, extracts were evaporated to dryness and the residue was weighed and
redisolved, in the same solvent, at a concentration of 2 mg of crude extract/ml of
solvent.

M.J. Pascual-Villalobos, A. Robledo/Biochemical Systematics and Ecology 27 (1999) 110

Table 1
Plant material harvested from the wild in southeastern Spain
Family and species
Boraginaceae
Echium creticum
Capparidaceae
Capparis spinosa
Cistaceae
Cistus monspeliensis
Helianthemum almeriense
Compositae
Achillea santolinoides
Anacyclus clavatus
Artemisia barrelieri
Artemisia herba-alba
Artemisia lucentica
Aster sedifolius
Atractylis cancellata
Calendula arvensis
Centaurea melitensis
Conyza sumatrensis
Dittrichia viscosa
Galactites duraei
Helichrysum decumbens
Senecio malacitanus
Senecio vulgaris
Pallenis spinosa
Cruciferae
Carrichtera annua
Diplotaxis erucoides
Diplotaxis ilorcitana
epidium graminifolium
obularia maritima
Moricandia arvensis
Cucurbitaceae
Ecballium elaterium
Cupresaceae
etraclinis articulata
Cyperaceae
Cyperus rotundus
Chenopodiaceae
Suaeda vera
Geraniaceae
Erodium malacoides
Graminaceae
Stipa tenacissima
Gutiferae
Hypericum perforatum
Labiatae
Ballota hirsuta
Ballota hirsuta

Plant material

Collection date

Plant stage

Whole plant

20/04/1995

Flowering

Leaves and stems

09/11/1994

Vegetative

Leaves
Leaves and flowers

09/11/1994
09/11/1994

Vegetative
Flowering

Leaves
Leaves
Whole plant
Whole plant
Whole plant
Whole plant
Whole plant
Whole plant
Whole plant
Whole plant
Whole plant
Leaves
Leaves
Whole plant
Whole plant
Whole plant

09/11/1994
06/03/1995
09/11/1994
09/11/1994
09/11/1994
09/11/1994
20/04/1995
09/11/1994
20/04/1995
09/11/1994
09/11/1994
09/11/1994
09/11/1994
09/11/1994
09/11/1994
20/04/1995

Plantlet
Flowering
Flowering
Flowering
Flowering
Flowering
Flowering
Pre-flowering
Flowering
Flowering
Flowering
Plantlet
Vegetative
Flowering
Flowering
Flowering

Leaves and stems

Whole plant
Whole plant
Whole plant
Whole plant
Whole plant

09/11/1994
09/11/1994
09/11/1994
09/11/1994
09/11/1994
09/11/1994

Plantlet
Flowering
Flowering
Flowering
Flowering
Flowering

Leaves

09/11/1994

Ripening

Leaves

09/11/1994

Vegetative

Whole plant

09/11/1994

Flowering

Leaves

09/11/1994

Vegetative

Leaves

09/11/1994

Vegetative

Whole plant

09/11/1994

Vegetative

Leaves

09/11/1994

Ripening

Leaves
Leaves

09/11/1994
20/04/1995

Vegetative
Pre-flowering
continued

M.J. Pascual-Villalobos, A. Robledo/Biochemical Systematics and Ecology 27 (1999) 110

Table 1continued
Family and species

Plant material

Collection date

Plant stage

avandula dentata
avandula multifida
avandula stoechas
Nepeta sp.
Rosmarinus officinalis
Salvia officinalis (rose flower)
Salvia officinalis (purple flower)
Sideritis marminorense
eucrium carthaginense
hymus hyemalis
Leguminosae
Calicotome intermedia
Ononix natrix
Psoralea bituminosa
Liliaceae
rginea maritima
rginea maritima
Malvaceae
avatera cretica
Papaveraceae
Glaucium corniculatum
Poligonaceae
Poligonum equisetiforme
Rutaceae
Haplophyllum linifolium
Ruta angustifolia
Solanaceae
Nicotiana glauca
Solanum nigrum
Solanum nigrum
Solanum bonariense
hitania frutescens
Thymeleaceae
hymelaea hirsuta

Whole plant
Whole plant
Whole plant
Leaves
Leaves
Leaves
Leaves
Whole plant
Whole plant
Whole plant

20/04/1995
20/04/1995
20/04/1995
02/05/1995
02/05/1995
02/05/1995
02/05/1995
20/04/1995
20/04/1995
20/04/1995

Flowering
Flowering
Flowering
Flowering
Vegetative
Flowering
Flowering
Flowering
Flowering
Flowering

Whole plant
Whole plant
Leaves

20/04/1995
20/04/1995
30/06/1995

Flowering
Flowering
Ripening

Leaves
Bulb

09/11/1994
09/11/1994

Post-ripening
Post-ripening

Leaves

09/11/1994

Vegetative

Whole plant

20/04/1995

Ripening

Whole plant

09/11/1994

Flowering

Leaves
Leaves

09/11/1994
09/11/1994

Vegetative
Vegetative

Leaves and flowers

Whole plant
Whole plant
Leaves and flowers
Leaves

09/11/1994
09/11/1994
20/04/1995
09/11/1994
09/11/1994

Flowering
Ripening
Ripening
Flowering
Flowering

Whole plant

09/11/1994

Flowering

2.3. Insects
Larvae, pupae and adults of . castaneum were obtained from same age cultures
kept on artificial diet of white wheat flour and beer yeast (95 : 5) at a constant
temperature of 30C and darkness (24 h) in the laboratory.
2.4. Bioassays
Each of the extracts was assayed for antiinsect activity using the following tests:
(1) Insect growth and/or feeding inhibition (FI). Extracts were mixed with the diet
(the extract solution was incorporated and the solvent was allowed to evaporate at

M.J. Pascual-Villalobos, A. Robledo/Biochemical Systematics and Ecology 27 (1999) 110

37C during 24 h) at concentrations of 0.05% and offered to 10 day old larvae

(1.82.0 mm in length) or adults (3 day old and unsexed) in five replications of two
insects and 100 mg of diet each. A control was prepared in the same way but extract
application was omitted. Mortality and length of larvae after 10 days and mortality in
adults after 15 days were recorded and compared with the control.
(2) Contact toxicity (CT). 250 ll of a solution (2 mg/ml) of each extract were applied
to filter paper disks (diam. 5 cm) and solvent was allowed to evaporate in a warm
room at 40C for 24 h, this gave a concentration of 25.5 lg/cm. Five replications per
extract and per control treatments were included. Ten newly emerged pupae (less than
24 h old) were placed on each filter paper inside a glass Petri dish and kept at 30C
and darkness. After 6 days, mortality and insect growth regulation activity was
observed and recorded according to the scale proposed by Mandava (1985): 0"normal adult, 1 and 2"adult with normal appearance but alterations in the genitalia,
3"intermediate adult-pupae forms (inviable adultoids) and 4"pupae. If larvae were
used for this test, only mortality after 72 h was recorded.
(3) Topical applications (TA). 1.5 ll of acetone solutions (2 mg/ml) of each extract
was topically applied (3 lg) to each insect (larva, pupa or adult) singly placed inside an
uncovered glass tube with 10 replications per treatment and control. After 72 h for
larvae and adults and 6 days for pupae at standard conditions (30C and darkness),
mortality was recorded and also insect growth regulation activity (if the test was run
with pupae) with the same scale as for the CT bioassay.
(4) Repellency bioassay (R). Extracts were mixed with the diet at 0.05%. A choice
bioassay with 10 replications (dishes) offering treated and untreated diet was set up
using 10 larvae (25 days old) per dish released in the middle of a plastic Petri dish
(diam. 10 cm). After 2 and 24 h the number of larvae present at each amount of treated
or control diet was counted. Repellent index was calculated as RI"(C!/C#)
;100 being C"number of larvae on control diet and "number of larvae on
treated diet.
2.5. Statistics
Scale depending on percentage of insects (0100%) in which activity was detected
was used. Also, for each bioassay variables were recorded and quantified as previously
mentioned, e.g. larva length, percentage of mortality, index of growth regulation
activity or repellent index. When appropriate, comparisons of treatment and control
were done using the non-parametric MannWhitney U test.

3. Results
3.1. Botanical families
The number of species tested were over 10 for Compositae and Labiatae, from two
to six for the families Cistaceae, Cruciferae, Leguminosae, Rutaceae and Solanaceae
and just one for all other families (Table 1). Activity was found for all plants screened

M.J. Pascual-Villalobos, A. Robledo/Biochemical Systematics and Ecology 27 (1999) 110

Table 2
Larval growth inhibition and mortality of . castaneum caused by active acetonic plant extracts at
concentration of 0.05% in diet
Species

Ballota hirsuta
Echium creticum
epidium graminifolium
eucrium carthaginense

Length (mm) of larvae

Mortality

(%)

3.80
5.53
3.68
3.80

5.35
4.64
4.67
4.20

12.0**

18.5**
20.0n.s.

20
70
0
10

Average values after 10 days, "treatment, C"control

Mann-Whitney U test (if n(5 U statistic omitted). n.s. 0.05( p, **0.001(p(0.01.
0% mortality in control

in at least one extract and/or bioassay (130 extracts out of 183 tested were active in at
least one assay). Tables 24 summarize results of promising species.
Some species gave activity in most bioassays performed such as: Echium creticum,
epidium graminifolium, Anacyclus clavatus, Galactites duraei, Helichrysum decumbens,
Senecio vulgaris, Pallenis spinosa, Ecballium elaterium, Haplophyllum linifolium and
Solanum bonariense. In other instances, a plant extract was very active in only one test,
e.g. Ballota hirsuta produced growth inhibition (FI) in larvae and Ononix natrix,
rginea maritima (bulb) and avatera cretica showed contact toxicity (CT) in pupae.
No ecological trends relating species distribution and bioassay outcome could be
drawn.
Extracts of Compositae produced growth inhibition in larvae and/or repellency.
Overall, the anti-insect activity detected in Labiatae species was not very strong
although some extracts induced a clear growth inhibition in insects.
Variations were found when material from the same plant at different harvesting
times and/or seasons was tested, e.g. the acetone extract of Ballota hirsuta leaves was
very active in the sample harvested in November 1994 but not in that from April 1995.
In other cases, the plant part evaluated had an influence on results such is the case of
rginea maritima in which the bulb toxicity was higher than that of the leaves.
Different populations of Solanum nigrum harvested at different times produced variation in activity.
3.2. Insect growth and/or feeding inhibition
Acetone extracts of Ballota hirsuta and epidium graminifolium produced a significant growth inhibition in larvae (p(0.01) although the mortality after 10 days was
low. On the other hand Echium creticum produced a high mortality (70%) but not
a reduction in length of surviving larvae. In other cases, such as that of eucrium
carthaginense (an endemic), statistically significant differences were not observed

M.J. Pascual-Villalobos, A. Robledo/Biochemical Systematics and Ecology 27 (1999) 110

Table 3
Growth regulation and toxic effects in larvae or pupae caused by active plant extracts at doses of 20 lg/cm
on discs or three topical lg/insect
Species

Extract

Insect stage Application

Mortality

(%)

Hormonal activity
(index 04)

Atractylis cancellata
Carrichtera annua
Centaurea melitense
Conyza sumatrensis
Ecballium elaterium
Hypericum perforatum
avandula stoechas
avatera cretica
Ononix natrix
Senecio malacitanus
Sideritis marminorense
Solanum bonariense
Solanum bonariense
Solanum nigrum
hymelaea hirsuta
rginea maritima (bulb)
hitania frutescens

A
M
A
A
A
M
M
A
A
A
A
A
M
H
A
A
A

Pupae
Larvae
Larvae
Pupae
Pupae
Larvae
Larvae
Pupae
Pupae
Pupae
Larvae
Larvae
Pupae
Larvae
Pupae
Pupae
Pupae

80
59.4
80
80
100
84.6
80
100
100
51.7
80
78.7
51.4
45
80
100
80

2.2 ns

3.2 ns
3.8 ns

3.2 ns
4**
1.93*

1.83*

2.4 ns
3.4 ns
2.4 ns

Topical
Disc
Topical
Topical
Topical
Disc
Topical
Topical
Topical
Disc
Topical
Disc
Disc
Disc
Topical
Topical
Topical

Extracts obtained using sequentially hexane"H, acetone"A and 50% aqueous methanol"M

0% mortality in control
significance of MannWhitney U test for comparison between treatments and control (ns 0.05(p,
0.01(p(0.05, 0.001(p(0.01); the index is only applicable to pupae.

although the reduction of growth in larvae (3.8 mm) was very clear in comparison
with the control (4.2 mm) at the time of observation (Table 2).
3.3. Contact toxicity and topical applications
Table 3 shows results of contact applications of the extracts to the insects. Most
plants listed produced mortality values of 80100% after topical or disc applications
on insects. Ecballium elaterium, avatera cretica, Ononix natrix and rginea
maritima (bulb) produced 100% mortality in pupae after topical application. The
values of the index of growth regulation activity indicated no significant activity
except for Ononix natrix (p(0.01), Senecio malacitanus (p(0.05) and Solanum
bonariense (p(0.05).
3.4. Repellency
Table 4 shows repellent effects at 2 and 24 h of treated diet on larvae. In most cases,
there was a consistency in both observations e.g. Artemisia barrelieri and Stipa
tenacissima had values of RI'30 at 2 h and '70 after 24 h. In Carrichtera annua,

M.J. Pascual-Villalobos, A. Robledo/Biochemical Systematics and Ecology 27 (1999) 110

Table 4
Repellent activity in larvae caused by active plant extracts at doses of 0.05% in diet
Species

Artemisia barrelieri
Aster sedifolius
Carrichtera annua
Conyza sumatrensis
Dittrichia viscosa
Echium creticum
Galactites duraei
Helichrysum decumbens
Nicotiana glauca
Nicotiana glauca
Pallenis spinosa
Senecio vulgaris
Solanum bonariense
Stipa tenacissima
Suaeda vera

Extract

A
A
A
A
A
A
A
A
A
M
A
A
A
A
A

Repellent index

2h

24 h

37.3
46.7
2.7
36.6
37.3
38.7
53.3
!5.3
0
12
!25.3
50.7
52
37.3
20

87.5
!30.0
72.0
45.3
36
29
59.2
66.7
80
20
46.7
45.3
56
73.3
40

Extracts obtained using secuentially organic solvents of increasing polarity: H"hexane, A"acetone and
M"50% aqueous methanol

Repellent index RI"(C!/C#);100; average values of five replications; C"no. of larvae in
control; "no. of larvae in treatment

Helichrysum decumbens and Nicotiana glauca the repellent effect was only clear after
24 h.
Nevertheless, in some cases, the activity was reversed. For example, Aster sedifolius
was repellent after 2 h exposure but was attractant after 24 h; volatilization of
compounds or mixing of treated and control diet by the larvae themselves could
partly explain this result. On the other hand, Pallenis spinosa (endemic) was attractive
after 2 h and repellent after 24 h which could be explained if the reason for such
behaviour is a response to antifeedant or deterrent compounds.

4. Discussion
Twenty one percent of the active extracts were newly identified (27 out of 130)
including 5% of endemic species.
Only a few species included in our study have been reported to be active by other
authors. Cun at et al. (1990) published that fractions of Dittrichia viscosa and epidium
graminifolium were insecticidal against Oncopelthus fasciatus.
Compositae species of the genera Artemisia, Aster, Centaurea and Conyza have
been reported to show antimicrobial activity (Hoffman et al., 1993; March et al., 1991).
It is also documented that Artemisia, Solanum and Senecio species posses pesticidal

M.J. Pascual-Villalobos, A. Robledo/Biochemical Systematics and Ecology 27 (1999) 110

properties (Yang and Tang, 1988). Our contribution in such cases is the identification
of either more promising species of the same genera or new activities. A number
of these species or close relatives have been studied, for pharmaceutical uses,
and compounds isolated, although those compounds might not be responsible for
the insecticidal activity. For example, the repellency shown by Artemisia barrelieri
extracts to . castaneum larvae in our experiments may be unrelated to artemisin
in Artemisia annua, a compound with antimalarial activity (Charles and Simon,
1990).
However, literature citations may be helpful in other instances. The genus eucrium
synthesizes clerodane diterpenoids showing antifeedant activity (Simmonds et al.,
1989) and it could be worth-while to study the active compounds of eucrium
carthaginense accordingly. Scilla glycosides are the major toxicants used as rodenticides from . maritima bulbs (Elliot, 1995) but research concerning insecticidal compounds from this source has not been undertaken.
Our screening has been performed using only one insect and therefore the activity
found is restricted only to tests carried out in the conditions described. Also,
plant species included represent only a small sample of all species present in Southeastern Spain. The identification of active compounds is a difficult task and testing
biological activity with insects has problems because in a batch there is always
variability in insect responses. Testing promising species against other pests of
economic importance and chemical studies to isolate active compounds need to be
undertaken.

Acknowledgements
We are grateful to N. Alburquerque and M.Mart nez for their help in running
routine bioassays. This research has been funded by the National Institute for
Agricultural Research in Spain (project INIA SC94-039) and also supported by
a student grant of the Consejer a de Agricultura from the Murcia Region.

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