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Journal of Environmental Science and Health, Part B (2013) 48, 585–589
Copyright C
Taylor & Francis Group, LLC
ISSN: 0360-1234 (Print); 1532-4109 (Online)
DOI: 10.1080/03601234.2013.774933
Insecticidal activity of raw ethanolic extracts
from Magnolia dealbata Zucc on a tephritid pest
NORMA FLORES-EST ´
EVEZ, SURIA G. VASQUEZ-MORALES, TOM ´
AS CANO-MEDINA, L ´
AZARO R.
S´
ANCHEZ-VEL ´
ASQUEZ, JUAN C. NOA-CARRAZANA and FRANCISCO D´
IAZ-FLEISCHER
INBIOTECA, Universidad Veracruzana, Xalapa, Veracruz, Mexico
Ethanolic extracts from Magnolia dealbata (Zucc.) (Magnoliaceae); leaves, bark, seeds, sarcotesta and flowers were evaluated for
insecticidal activity against adults of the Mexican fruit fly, Anastrepha ludens (Loew) (Diptera: Tephritidae). Using feeding bioassays
composed from sugar-extract mixtures, only the extract from sarcotesta indicated insecticidal activity against the flies. The extracts
from the other four plant tissues (leaves, bark, seeds and flowers) did not manifest any biological activity. The most effective extract
was obtained from oven-dried sarcotesta, whereas extracts from fresh sarcotesta were inactive. Our results suggest that M.dealbata
sarcotesta contains secondary metabolites with insecticidal activity against A. ludens adults. These metabolites are as potent as natural
pyrethins and represent a potential substance for controlling this type of pest.
Keywords: Magnolia dealbata,Anastrepha ludens, botanical insecticides, bioprospection, raw extracts.
Introduction
The continuous use of conventional synthetic chemical
insecticides for crop protection represents a serious
hazard for both human and environmental health, by
provoking harmful effects for natural enemies as well as
non-target insects. Furthermore, this has led to increasing
pest resistance to insecticides.[1] Synthetic insecticides are
also banned in organic crop production systems creating
the need for alternative tools for controlling devastating
pests that attack crops.[1] Despite the generally negative
effects on non-target insects, spraying killing agents on
crops is one of the best ways to reduce pest populations.
Thus, bioprospecting for new and natural insecticidal
compounds that are derived from plants, represent reduced
toxicity for warm-blooded animals and are less persistent
in the environment might be a viable alternative.[2] Plants
are a natural source for insecticides as the constant
interaction with insect herbivores promotes the develop-
ment of defensive strategies, including the synthesis of
large numbers of secondary metabolites with insecticidal
properties. Botanical insecticides are one of an extensive
group of alternative pest-control agents that are generally
Address correspondence to F. D´
ıaz-Fleischer, INBIOTECA, Uni-
versidad Veracruzana, Apartado Postal 250, Xalapa, Veracruz
91090, Mexico; E-mail: fradiaz@uv.mx
Received September 18, 2012.
more biodegradable, less toxic to warm-blooded animals
and also less antagonistic to the pest’s natural enemies.[2,3]
Some plants are especially attractive for the study of
chemical properties because they are rich in secondary
metabolites. For example, ethanolic extracts from the
perennial herb horseradish (Armoracia rusticana Gaertner,
Meyer et Scherb) and the common garlic (Allium sativum
L., Liliaceae) exhibited insecticidal activity against Aedes
albopictus (Skuse) larvae.[4] Certain species of Magnolia
such as M. officinalis, M. salicifolia, M. virginiana and M.
ovovata have demonstrated bactericidal and mosquitoci-
dal effects.[5,6,7] The foliage from Sweetbay magnolia (M.
virginiana) contains at least two biologically active phenyl-
propanoid compounds (magnolol and a biphenyl ether),
both of which are toxic to a number of generalist insect
herbivores.[8,9]
Magnolia dealbata Zucc. is a deciduous tree that grows
up to 25 m in height, with large (20 cm) white flowers and
leaves of up to 60 cm long. The altitudinal distribution
range of M. dealbata is 1200–1500 m. It is an endemic tree
species found in the cloud forest in Mexico and has been
cataloged as being in danger of extinction by the IUCN
Redlist.[10] Two secondary metabolites (hoinokiol and mag-
nolol) have been reported in ethanolic extracts from leaves
and bark.[11,12]
In this study, ethanolic extracts of different tissues of
M.dealbata plants were prepared and their insecticide ef-
fects were tested on the Mexican fruit fly Anastrepha ludens
Loew (Diptera: Tephritidae). The polyphagous Mexican
fruit fly is a serious citrus and mango pest with distributions
586 Flores-Est´
evez et al.
ranging from the South of Texas to Costa Rica. Anastrepha
ludens is currently chemically controlled by spraying with
malathion, baited with hydrolyzed protein or with bacteri-
ally derived bioinsecticide GF-120.[13,14]
The amount of secondary metabolites may vary among
plant tissues, since most plants tend to assign greater
amounts of specific metabolites to reproductive tissues.[15,16]
Therefore, the decision was taken to obtain and test extracts
from: leaves (mature and immature), bark, fruit, sarcotesta
and seeds.
Materials and methods
Raw extracts
Six mature trees from M.dealbata were sampled at Coy-
opolan, Municipality of Ixhuac´
an de los Reyes, Veracruz,
M´
exico. Samples of leaves (both mature and developing),
bark, fruits, seeds and flowers were collected and immedi-
ately taken to the laboratory of INBIOTECA, at the Uni-
versidad Veracruzana in Xalapa, Veracruz.
All plant parts were air-dried at 30–45◦C for 96 h and
then ground in a coffee grinder. In the case of the sar-
cotesta, both fresh and dried specimens were ground with
a mortar. One hundred grams of each plant part sample
was extracted with 95% ethanol (1:5 w v−1(50 g 250−1),
by soaking for 72 hours. The extracts were filtered with
a column (Whatman No. 1) and centrifuged at 2500 ×g.
Total volume was reduced to 10 mL in a vacuum using a
rotary evaporator (BUCHI Mod. R-210, at 40◦Cand56
a 58 cmHg vacuum). The dry extracts were suspended in
90 mL of distilled water and transferred to vials. The ex-
tracts were stored in a refrigerator at 5◦C and removed only
when required for assay.
As a positive control to ensure that the extraction method
was viable, extracts of flowers from Chrysanthemum gran-
diflorum Kitam (a source of pyrethrin), bought at a local
flower shop were obtained using the same method described
above.
Insects
All flies came as pupae from the A. ludens mass rearing
facility at the MoscaFrut-MoscaMed Program, located in
Metapa de Dominguez, Chiapas, Mexico. Around 1500
pupae were placed in 5 cages of 900 cm3made of wood and
cotton mesh (approximately 300 pupae per cage). Water
and food (3:1 mixture of sugar and hydrolyzed yeast protein
[ICN Biochemicals, Aurora, OH]) were provided ad libitum
until being bioassayed.
Treatments and Bioassay
The experimental units consisted of cages with 25 pairs of
flies between 10 and 25 days old (25 females and 25 males).
About 24 h prior to the beginning of the experiments, the
flies were deprived of food and water. During testing, ex-
tracts mixed only with sugar were available to the flies. Tests
were carried out in the laboratory at 25 ±1◦C, 70 ±10%
RH and 12h photophase.
Ten milligrams of sugar mixed with 10 mL of each one of
the extracts (leaves, bark, flowers, seeds, sarcotesta and C.
grandiflorum flowers) were used to feed the flies (1:1 weight
vol−1). As a control, a mixture of sugar and 10 mL of 10%
ethanol was used. Treatments were applied on 0.5 mg of
cotton to reduce stickiness. The number of dead flies was
recorded every 24 hours during three days. Those treat-
ments that showed insecticidal effects were tested again for
a longer period (four days). Each treatment was replicated
at least four times. Additionally, three decimal dilutions of
active extract were tested: 0.1, 0.01, 0.001 mg mL−1.
Statistical analysis
A complete random design was used and a one-way
ANOVA followed by a LS Means difference Tukey HSD
post-hoc test were used for the analysis. Percent data was
arcsin transformed prior to analysis.[17] Survival analyses
were conducted according to the Kaplan-Meier method
and survival characteristics were compared using log rank
tests.[18] Data were corrected for control mortality using
Abbott’s formula[19] (Equation 1).
CM(%) =n.i.dead after treatment −n.i.dead in the Co
100 −n.i.dead in the Co
(1)
where CM =corrected mortality, n.i. =number of insects,
Co =control
Results
There were significant statistical differences in fly survival
depending on the different extract treatments after 3 days
of exposure (F8,55 =16.59; P<0.0001). However, only
the dry sarcotesta extracts presented an insecticidal effect
comparable to that of pyrethrin extracts. The Abbott index
indicates that both active extracts (C. grandiflorum and dry
sarcotesta) were equally effective for killing flies (F7,43 =
67.89; P<0.0001). Nevertheless, when flies were exposed
for 4 days to dry sarcotesta and C. grandiflorum extracts,
there were significant differences between them in terms
of survival (F2,54 =64.5; P<0.0001) and also in efficacy
(F1,36 =5.33; P<0.023) (Table 1).
The survival analysis indicated that fewer flies survived
when exposed to C. grandiflorum extracts than to dry
sarcotesta extracts or a sugar control (Log-Rank, Chi-
square =875.28; DF =2; P<0.0001). Differences were
more evident during the first two days of observation
(Fig. 1).
In the case of dilutions, statistical differences were ob-
served among the three concentrations as dry sarcotesta
Insecticidal activity of raw ethanolic extracts 587
Table 1. Percentage survival and Abbott indices for A.ludens
flies exposed during 3 and 4 days to seven ethanolic extracts of
different plant tissues of M. dalbata and a positive (C. grandiflo-
rum) and a negative control (sugar) treatments. Active extracts
are presented in bold.
Survivals % Abbott index
Treatments (mean ±SE) (mean ±SE)
3 days
Bark 90.0±1.1b −0.9±3.8b
C. grandiflorum 16.0±4.7a 83.1±4.8a
Flower 83.0±2.5b −2.1±4.5bc
Immature leaf 87.0±2.0b −3.7±3.5cd
Mature leaf 87.5±1.7b −4.2±2.8de
Fresh sarcotesta 99.6±0.4b −1.7±1.2e
Dry sarcotesta 12.8±8.0a 86.8±8.3a
Seeds 77.0±5.2b 17.9±2.9b
Sugar 89.7±2.7b
4 days
C. grandiflorum 7.4±5.0a 90.5±6.8a
Dry sarcotesta 13.0±3.5b 85.1±3.8b
Sugar 89.5±1.9c
Means within a column followed by the same letter are not different at a
0.05 level of significance.
extract of 0.1 mg mL−1killed more flies than other extracts
(F4,24 =42.16; P<0.0001). Efficacy was significantly high
for dry sarcotesta extract of 0.1 mg mL−1and C. grandi-
florum, but not for other treatments (F3,16 =20.30; P<
0.0001) (Table 2). With respect to survival, only the 0.1
dry sarcotesta extract reduced the survival of the fly in a
way similar to C. grandiflorum (Log-Rank, Chi-square =
429.08; DF =4; P<0.0001) (Fig. 2).
1234
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Probability of Survival
Da
y
s
Sugar
Dry sarcotesta
C. grandiflorum
Fig. 1. Kaplan-Meier survival plot for A. ludens flies exposed to
ethanolic extracts of C. grandiflorum and dry sarcostesta of M.
dealbata.
Table 2. Percent of survival and Abbott indices for A.ludens flies
exposed to three concentrations (mg/mL) of dry sarcotesta ex-
tracts, a positive (C. grandiflorum) and a negative control (sugar)
treatments during 4 days. Active extracts are presented in bold.
Survivals % Abbott index
Treatments (mean ±SE) (mean ±SE)
4days
C. grandiflorum 9.6 ±2.7 b 83.0 ±5.8 a
Dry sarcotesta 0.1 3.6 ±2.2 a 96.1 ±2.3 a
Dry sarcotesta 0.01 66.4 ±8.3 c 32.1 ±7.7 b
Dry sarcotesta 0.001 78.0 ±2.8 c 19.8 ±2.5 b
Sugar 97.2 ±1.3 c
Discussion
As expected, not all the plant tissue extracts had insectici-
dal effects on flies. Dry sarcotesta extracts were unique in
demonstrating clear insecticidal activity with efficacy that
wascomparabletoourpyrethrinsource.
Accumulation of secondary metabolites with protective
properties in plant reproductive tissues is very com-
mon among a variety of plants.[16,17,20] Thepresenceof
compounds with insecticidal activity in the sarcotesta is
probably related to a defensive response on the part of
the plant to attack by pre-dispersal predators and seed-
dispersing animals, in order to avoid or reduce the damage
to immature seeds. It is well-known that plants invest
in many physical, chemical and phenological barriers to
protect seed from fungus and bacteria and also against
pre-dispersal seed predators[21] and this seems to be the
function of the chemicals in M.dealbata’s sarcotesta. How-
ever, a question remains with respect to our results. Why
does sarcotesta exhibit insecticidal activity when dried but
not in fresh condition? It was expected that fresh sarcotesta
12345
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Probability of Survival
Sugar
0.001
0.01
0.1
C. grandiflorum
Days
Fig. 2. Kaplan-Meier survival plot for A. ludens flies exposed to
ethanolic extracts of C. grandiflorum and dry sarcostesta of M.
dealbata at three different concentrations.
588 Flores-Est´
evez et al.
extracts would be more active than extracts from dried sar-
cotesta. In nature this barrier is fresh, as is the case among
other fruit species. For example, in the case of methanolic-
petroleoum ether citrus peel extracts, fresh peel extracts
actively kill olive flies, Bactrocera oleae (Gmelin) but this
was not the case with dried peel extracts.[20] These authors
suggest that toxic compounds, mainly essential oils and
aldehydes are reduced, volatilized or denaturalized during
the drying process. In the case of M. dealabata extracts, it is
possible that dried sarcotesta extracts increase the concen-
tration of toxic compounds, not only because of the water
loss during the process but also because there is an increase
in the weight-volume ratio in dried sarcotesta extracts.
Leaves and bark extracts of M.dealbata did not in-
dicate any toxic effects on flies. Allelochemicals such as
biphenyl ether and the natural phenolic phenylpropanoid
magnolol found in leaves of Magnolia species are perhaps
selected to protect the plant against immature insects, such
as the larvae of some lepidopterans and against some phy-
topathogens.[7–9] However, it seems that these compounds,
present in M.dealbata, do not have sufficient insecticidal
activity to combat adult flies. Importantly, some phenyl-
propanoids, such as methyl eugenol,[22] a compound found
in the leaves, flowers, or fruits of plants in over 10 fami-
lies, act as a powerful attraction for males of the Oriental
Fruit Fly, Bactrocera dorsalis Hendel and for males of B.
philippinensis (Drew and Hancock).[23,24] Additionally, wild
males from B.dorsalis that ingested methyl eugenol exhib-
ited increased signaling effort, signal attractiveness, and
mating success compared with males not given access to
the lure.[25,26] It seems that adult fruit flies possess the phys-
iological machinery to degrade or even assimilate certain
phenylpropanoids.
Conclusion
These results clearly indicate the presence of insectici-
dal metabolites with similar biological activity of natural
pyrethins in the sarcostesta of M.dealbata seeds. These
metabolites represent renewable resources in terms of bioin-
secticides for agriculture. The isolation of these chemicals
by using liquid chromatography represents the next stage
in our research.
Acknowledgments
WewouldliketothankPabloMontoyaandDineshRaofor
their comments, as well as Rogelio Lara Gonz´
alez for tech-
nical help. Caroline Sarah Karslake revised English spelling
and grammar. Ing. Jos´
e Manuel Guti´
errez Ruelas, Director
de la Campa˜
na Moscas de la Fruta (SAGARPA-DGSV)
provided support and encouragement. This research was
partly sustained by the FOMIX-Conacyt-Veracruz Project
37502.
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