Current Bioactive Compounds 2008, 4, 33-35
33
Inconsequential Study on Larvicidal Efficacy of Anise and Celery Seed
Extracts Indicates that Standards in Bioinsecticide Screening are
Necessary
E. A. Shaalan1 and D. V. Canyon2,*
1
Zoology Department, Aswan Faculty of Science, South Valley University, Aswan 81528, Egypt, 2Anton Breinl Centre
for Public Health and Tropical Medicine, James Cook University, Townsville, Australia
Abstract: The larvicidal activity of Pimpinella anisum (anise) and Apium graveolens (celery) seed extracts was evaluated
against 4th instar Aedes aegypti larvae under laboratory conditions as per WHO methodology. Preliminary screening
showed anise seed extracts to be more active than celery seed extracts and hexane produced the best extract of anise seeds
giving a mildly potent lethal concentration of 403.84 mg/L. Other researchers have reported higher efficacies which
cannot be explained by differences in extraction method or choice of solvent. Other probable causes of efficacy variability
in phytochemical potency studies are discussed and bioinsecticide screening standards are recommended.
Keywords: Bioinsecticide, screening standards, botanical extracts, anise, celery, mosquitoes.
INTRODUCTION
Aedes aegypti is a well known vector of several
important vector-borne diseases, such as dengue and yellow
fever, to which a considerable portion of the global population is exposed [27]. Since there are no effective vaccinations available for many arboviruses, vector control has
been the primary measure used to decrease the incidence
related diseases. The effectiveness of this approach has been
warning due to the lack of novel insecticides, the high cost of
synthetic insecticides, and concern for the environment and
increasing insecticide resistance on a global scale. Interest in
botanical alternatives to synthetic products has thus increased in recent decades and it is possible that phytochemicals
have a future in the insecticide industry. Research has found
hundreds of plant species that cause a range of acute and
chronic toxic effects [20, 25].
As would be expected, the larvicidal activity of extracted
oils varies considerably according to mosquito species, plant
species and plant part. The choice of extraction solvent is
also critical factor that affects larvicidal efficacy. There is a
converse relationship between the extract effectiveness and
solvent polarity [12]. For instance, successive extraction of
thyme (Thymus capitatus) by solvents of increasing polarity
showed that potency against the larvae and adults of Culex
pipiens was highly attributed to the non-polar fraction (e.g.
petroleum ether) [10]. Despite these known variables,
researchers fail to standardise when it comes to botanical
screening with the result that the results from most studies
can hardly be compared.
This research initially focused on an evaluation of Anise,
Pimpinella anisum, and celery, Apium graveolens, which are
widely used as popular aromatic herbs and spices. Certain
bioactive compounds that have been isolated from anise have
exhibited antibacterial activity [3, 23], antifungal activity [8],
*Address correspondence to this author at the Anton Breinl Centre for
Public Health and Tropical Medicine, James Cook University, Townsville,
Qld 4811, Australia; Tel: +61-8-9385 9415; Fax: +61-8-9385 9415; E-mail:
deoncanyon@gmail.com
1573-4072/08 $55.00+.00
acaricidal activity [9], pediculicidal activity [13] and
insecticidal activity [14]. Extracts from celery have shown
circarcidal activity [21], antifungal and nematicidal activity
[19]. Previous studies have also demonstrated some larvicidal activity by anise [17] and celery [4, 15]. This paper
thus reports on the larvicidal activity of essential oils
extracted from anise and celery, and compares the efficacy
of extracting methods to those used by some of the
previously mentioned scientists, but more interestingly, it
then identifies a short-coming that is common to all
bioinsecticide studies and recommends changes.
MATERIALS AND METHODS
Ae. aegypti mosquitoes were obtained from a colony
initiated from mosquitoes collected in 2002 from Townsville, Australia. Mosquitoes were maintained at conditions of
27 ± 2 Cº and 70 % ± 5 RH under 14L - 10D cycles. Ae.
aegypti larvae were kept in plastic buckets half filled with
tap water and fed on goldfish flakes. Water in rearing
containers was refreshed every 2 d. Adult mosquitoes were
maintained on a 10% sugar solution while females were
additionally fed on rat blood.
P. anisum seeds were bought from Egyptian herb markets
and A. graveolens seeds were provided by Arthur Yates &
Co Ltd (21A Richmond Road, Homebush, NSW 2140,
Australia). Seeds were washed with running water, dried at
room temperature, ground, bottled and refrigerated until
extraction. Essential oils of seeds were extracted according
to the method of Stein and Klingauf [24]. A quantity of
ground seeds (5 - 15 g) was extracted for 4 hr with 150 - 300
ml of solvent (ethanol, hexane or methanol) in a Soxhlet
apparatus. The crude extracts, comprised primarily of
essential oils, were separated under vacuum by using a rotary
evaporator at temperatures equivalent to the boiling points of
the solvents used.
Standard WHO methodology [28] was followed to determine the dose-response curve and lethal concentrations for
50 % of the population (LC50). Crude extracts were first
screened at doses of 500 and 100 mg/L to identify the lowest
© 2008 Bentham Science Publishers Ltd.
34
Current Bioactive Compounds 2008, Vol. 4, No. 1
Shaalan and Canyon
dose that kills 100 % of mosquito larvae. About 25 newly
moulted 4th instar larvae were released into glass beakers
containing 99 ml de-ionized water. Botanical extract (1 ml)
was added to each beaker while 1 ml of ethanol was added to
the control. Four replicates for each concentration were
conducted. Larvae in both test and control were selected
from different buckets [26] to avoid effects resulting from
rearing differences between batches of larvae. Percentage
mortality was recorded after 24 h and Abbott’s formula [1]
was used to correct the percentage mortalities if the control
mortality was between 5% and 20 %.
SPSS version 11 (SPSS Inc.) was used to determine the
LC50, LC90 and the corresponding 95 % confidence intervals
of the extracts.
RESULTS AND DISCUSSION
The initial results of the present study, presented in Table
1, show that out of the three extracts tested, only ethanol and
hexane extracts of P. anisum produced 100 % mortality
when tested against Ae. aegypti larvae at 500 mg/L. In
contrast, larval mortality caused by extracts of A. graveolens
at the same concentration ranged from 12-59%. This indicated that an extremely high, unpractical concentration
would be needed to produce 100 % mortality. Accordingly,
A. graveolens was considered non effective against Ae.
aegypti larvae and its dose-response curve was not evaluated.
At 100 mg/L, the P. anisum, extracts produced a mortality of
1.35 - 11.12 %, indicating a LC50 between 100 and 500
mg/L. Because the hexane extract produced the highest
mortality at 100 mg/L, its dose-response curve was
determined. It was shown to be marginally effective with an
LC50 of 251.14 mg/L (Table 2).
Our results showed lower efficacy than those reported by
Prajapati et al. [17] for P. anisum (LC95 = 115.7 mg/L), by
Choochote et al. [4] for A. graveolens (LC50 = 81 mg/L), and
for A. graveolens (LC50 = 40.07 mg/L) by Pitasawat et al.
[15]. Although it is tempting to dismiss studies that show
such large variations, there are some very good explanations.
These differences are, in part, influenced by the extracting
method and the solvent [20]. In the present study, a Soxhlet
apparatus was used to extract essential oils with 3 solvents
whilst hydro distillation [17], steam distillation [15] and
ethanol maceration [4] were the methods used for extraction
in the other studies. However, this does not explain all since
Chahad and Boof [2] reported that Piper nigrum extracts
obtained using the Soxhlet method produced higher
mortality against Cx. quinquefasciatus larvae and reduced
lethal time than the extract obtained by maceration. Thus,
our results should have shown lower lethal concentrations
than those obtained by other methods, but this was not the
case and further explanation was sought.
When it comes to comparing the results from different
studies, in addition to keeping extraction methods identical,
other biological factors must be taken into account. In the
same way that viruses vary in virulence, the quantity of
active phytochemical in any given plant will vary due to a
whole range of spatial, temporal and biological factors. The
origin of plant material, nutrition, accumulation of bioactive
Table 1. Preliminary Screening of Pimpinella anisum and Apium graveolens Extracts Against Newly Moulted 4 th Instar Larvae of
Aedes aegypti
Botanical extracts
Pimpinella anisum
Apium graveolens
500 Mg/L
100 Mg/L
500 Mg/L
100 Mg/L
Ethanol extract
100
1.35
41
2
Hexane extract
100
11.12
59
0
Methanol extract
89
3.25
12
3
Control
0
0
0
0
Table 2. LC50, LC90, and Slope Data of Pimpinella anisum Hexane Extract Tested Against Newly Moulted 4th Instar Larvae of Aedes
aegypti
Conc.
Mean
LC50
LC90
Slope ± SE
(ppm)
mortality %
Mg/L
95 % FL
Mg/L
95 % FL
450
96.0
251.14
233.11-267.95
403.84
381.56-431.42
400
86.0
300
73.0
200
28.3
50
5.5
Control
0
0.0083 ± 0.00062
Inconsequential Study on Larvicidal Efficacy of Anise
Current Bioactive Compounds 2008, Vol. 4, No. 1
compounds during growth and development and influence of
harvest time may all have a significant impact on the quality
and quantity of phytochemicals [18, 6]. For instance, Policegoudra et al. [16] found that growth and developmental
stages of mango ginger, Curcuma amada, rhizomes influenced the accumulation of bioactive compounds, particularly
difurocumenonol, a bioactive terpenoid compound, and
phenolics. Harvest time was found to affect the quantity of
oil-based bioactive compounds in sea buckthorn berries,
Hippophae rhamnoides L. ssp. Sinensis [5]. November-harvested berries yielded the highest carotenoid content in the
fruit fraction (p < 0.05) and September yielded significantly
higher (p < 0.05) levels of major compounds, -tocopherol
and -sitosterol, in the fruit fraction. Indeed, such variables
may go further in explaining differences in research results
than normally considered variables. Accordingly, it is not
realistic or sensible to compare the results from studies in
which the spatial, temporal and/or environmental conditions,
particular to the tested botanical material, have not been
standardised. Furthermore, the rearing conditions of mosquitoes are well known to influence results and the WHO
[26] even recommended that test populations be drawn from
several different batches to minimise this effect. Why is it
that plant materials are not subject to the same scientific
rigour? Articles on promising botanical insecticides should
thus include plant rearing data and future studies need to
determine the ideal growing conditions required to produce
the maximum potency and quantity of active product.
Identification of superior strains of species with potential
would also be of benefit.
ACKNOWLEDGEMENTS
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