Research Article

Received: 28 December 2008 Revised: 27 February 2009 Accepted: 2 March 2009 Published online in Wiley Interscience:

(www.interscience.wiley.com) DOI 10.1002/jsfa.3582

Chemical composition and insecticidal

properties of Cinnamomum aromaticum (Nees)
essential oil against the stored product beetle
Callosobruchus maculatus (F.)
Rezuanul Islam,a,b Rejaul Islam Khan,b Sharif M Al-Reza,a Yong Tae Jeong,a
Chi Hyun Songa and M Khalequzzamanc∗

Abstract
BACKGROUND: Cinnamomum aromaticum is a widely used cooking ingredient in South Asian countries. In this study the
essential oil of C. aromaticum was tested against the stored product beetle Callosobruchus maculatus. The objective was to
identify the natural compounds with insecticidal properties in the essential oil of C. aromaticum with a view to its potential use
as an alternative to synthetic pesticides.

RESULTS: The chemical composition of the hydrodistilled bark essential oil of C. aromaticum was analysed by gas
chromatography/mass spectrometry, and cis-cinnamaldehyde (53.90%) was found to be the principal constituent. The surface
film and fumigation toxicities and repellency activity against C. maculatus were evaluated. The extracted oil showed 94.44%
mortality against adult C. maculatus through the surface film bioassay. The LD50 values were 27.56 and 23.16 µg cm−2 after
24 and 48 h of exposure respectively. The regression equations were calculated as Y = 0.39 + 3.20X and Y = 1.25 + 2.75X
respectively. In the fumigation bioassay the LD50 value was 434.69 µg cm−2 after 24 h of exposure, with the regression equation
Y = 0.87 + 1.57X. It was also found that the extracted oil contained compounds that had a dose-dependent protective effect
on egg hatching and adult emergence.

CONCLUSION: The results obtained from this study suggest that the toxicity and insecticidal activity of C. aromaticum are
attributable to its essential oil, which could be used as a biodegradable and natural bioprotectant for controlling stored product
pests.

c 2009 Society of Chemical Industry

Keywords: essential oil; Cinnamomum aromaticum; Callosobruchus maculatus; insecticidal activity; cinnamaldehyde

INTRODUCTION respectively), with trans-cinnamyl acetate and β-caryophyllene as

The genus Cinnamomum comprises about 250 species distributed
throughout Asia and Australasia.1 Different varieties of cinnamon
oil contain cinnamaldehyde, eugenol, cinnamic acid, cinnamyl
acetate, benzaldehyde, methyl salicylate, hydrocinnamaldehyde,
o-methyl-coumaraldehyde, salicyaldehyde, cuminaldehyde, α-
pinene, 1,8-cineol, linalool and α-terpineol.2 The leaf essential
oil of Cinnamomum osmophloeum grown in Taiwan contained
mainly trans-cinnamaldehyde (79.85%) and showed excellent
antibacterial, antitermite, antimite, antimildew, antipathogenic,
antifungal and anti-inflammatory activities.3 – 5 The bark essential
oil of Cinnamomum zeylanicum contained 13 compounds, with (E)-
cinnamaldehyde as the major component along with δ-cadunene
(0.9%), α-copaenene (0.8%) and α-amorphene (0.5%).6 The major
components of cinnamon leaf grown in Little Andaman, India
were eugenol (76.6%), linalool (8.5%) and pipertone (3.3%),7
whereas the stem-distilled volatile oil of cinnamon fruit grown
at Karnataka and Kerala, India consisted of hydrocarbons (32.8 and
major components.8,9
The search for new strategies or natural products to control
destructive insect pests and vectors of diseases is desirable owing
to the prevalent occurrence of vector resistance to synthetic
insecticides and the problem of toxic non-biodegradable residues
contaminating the environment and having undesirable effects on
non-target organisms.10,11 The practice of adding a little vegetable
oil to stored rice or legumes for protection against insect pests is
well known and well established in oriental countries such as China,
∗ Correspondence to: M Khalequzzaman, Department of Zoology, Rajshahi
University, Rajshahi 6205, Bangladesh. E-mail: kzaman@ru.ac.bd
a Department of Biotechnology, Daegu University, Gyungsan City, Gyungbuk
712-714, Korea
b Department of Biotechnology and Genetic Engineering, Islamic University,
Kushtia 7003, Bangladesh
1241

20.8% respectively) and oxygenated compounds (63.7 and 73.4% c Department of Zoology, Rajshahi University, Rajshahi 6205, Bangladesh

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c 2009 Society of Chemical Industry
 www.soci.org
100 e e
24h exposure
80 48h exposure
d were injected manually in splitless mode. The relative proportions
Mortality (%)
60
d normalisation.
c
40
c
b and NIST MS Search 2.0 according to the literature.15 The relative
20 b
aa
0
62.85 31.43 15.71 7.86 Control
Concentration of essential oil (µg cm-2)
Figure 1. Toxicity effect of Cinnamomum aromaticum essential oil against
adult Callosobruchus maculatus after 24 and 48 h of treatment in surface
film bioassay. Each data point represents mean ± SD of ten replicates, each
comprising ten adult insects and five separate experiments. Data that do
not share the same letter are significantly different at P < 0.05 (based on
DMRT).
Bangladesh, India and Indonesia. Aromatic species, particularly
those of the family Labiatae (or Lamiaceae), are among the most
widely used plants in insect pest control.12,13
Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) is a major
pest of several pulses, including cowpea, chickpea, lentil, soya
and haricot bean. Among the insects attacking stored products,
Bruchidae and especially Callosobruchus spp. have attracted the
attention of many scientists as experimental model insects.
Nowadays, methods such as storage in airtight plastic or
steel containers, application of chemical insecticides, gamma
irradiation, freezing the pulses or heating them are some of the
additional possibilities. However, most of these methods require
high inputs, often unavailable and unaffordable for subsistence
farmers. In this study we examined the chemical composition of the
bark essential oil of C. aromaticum by gas chromatography/mass
spectrometry (GC/MS) and tested the efficacy of the extracted oil
in controlling the pulse beetle C. maculatus during storage.
MATERIALS AND METHODS
Plant material and essential oil extraction
Mature bark of C. aromaticum was purchased from a local market
in Dhaka, Bangladesh in June–July 2007. The collected sample
was identified by Professor ATM Nadiruzzaman, Department of
Botany, University of Rajshahi, Bangladesh. Air-dried bark from the
sample (200 g), in triplicate, was subjected to hydrodistillation in
a modified Clevenger-type apparatus for 3 h.14 The oil was dried
over anhydrous Na2 SO4 , preserved in a sealed vial and stored in a
refrigerator at 4 ◦ C until further analysis.
GC/MS analysis
GC/MS analysis of the essential oil was performed using a
SHIMADZU (GC-17A, Kyoto, Japan) gas chromatograph/mass
spectrometer (GC/MS) equipped with a ZB-1 fused silica capillary
column (30 m × 0.25 mm i.d., film thickness 0.25 µm). For GC/MS
detection an electron ionisation system with an ionisation energy
of 70 eV was used. Helium was employed as the carrier gas at a
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constant flow rate of 1 mL min−1 . The injector and MS transfer
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c 2009 Society of Chemical Industry
R Islam et al.
line temperatures were set at 220 and 290 ◦ C respectively. The
oven temperature was programmed from 50 to 150 ◦ C at 3 ◦ C
min−1 , then held isothermal for 10 min and finally raised to 250 ◦ C
at 10 ◦ C min−1 . Diluted samples (1 : 100 v/v in methanol) of 1 µL
of oil constituents were expressed as percentages by peak area
Quantification was performed using percentage peak area
calculations, and the identification of individual components was
done using the Wiley/NBS Registry of Mass Spectral Database
concentration of each compound in the essential oil was quantified
according to the peak area integrated by the analysis program.
Insecticidal activity
Insects
Callosobruchus maculatus specimens (insects) were obtained from
an agricultural product storehouse. Mass cultures were maintained
in sterilised earthen pots and subcultures in plastic pots or beakers
with the food medium. The beakers were kept in an incubator
at 30 ± 0.5 ◦ C without light or humidity control. Black lentil
(Lens esculentus) was used as the food medium throughout the
experiment.
Surface film bioassay
The surface film bioassay was conducted using groups of ten adult
insects to evaluate the mortality effect. An appropriate quantity of
cinnamon oil was diluted in acetone to obtain each test solution.
A pilot experiment was carried out to determine the oil doses
at which the mortality rate was between 10 and 90% for 4–10-
day-old insects. The oil was applied by the surface film method.
A 500 µL aliquot of test solution containing 62.85, 31.43, 15.71,
7.86 or 0 (control) µg cm−2 oil was poured onto a petri dish (6 cm
diameter). The treated petri dishes were kept in an incubator at
30 ± 0.5 ◦ C. Insect mortality was recorded after 24 and 48 h of
treatment. Each experiment was replicated five times and the
insecticidal activity of the oil was expressed as % mean mortality
of adult insects.
Fumigation bioassay
Glass vials (5.5 cm × 2.5 cm diameter) capped with polypropylene
stoppers were used for the fumigation bioassay. Insects were
transferred to the vials in groups of ten adults. The vials were
covered with fine nylon cloth secured with adhesive tape. The
doses of cinnamon oil used were 95.45, 63.63, 47.72, 31.82 and
0 (control) µg cm−2 . The vials containing the insects were turned
upside down over the vials containing the oil such that the oil
vapour saturated the atmosphere of the containers. The vials were
kept at 30 ± 0.5 ◦ C with a photoperiod of 16 h light/8 h dark.
Mortality counts were made at 24 and 48 h after treatment. Ten
replications were used for each combination of dose and exposure
time.
Repellency test
The repellency test consisted of experimental units of 20 pairs
of insects enclosed in a plastic box (18 cm × 18 cm × 7 cm) in
which two glass petri dishes (5 cm diameter) containing 40 lentils
each and placed at opposite corners of the box were offered. A
200 µL aliquot of test solution containing 62.85, 31.43, 15.71 or
7.86 µg cm−2 oil was applied to a filter paper disc (2.1 cm diameter)
and placed on one petri dish, while a filter paper disc without oil
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Bioactive properties of cinnamon oil www.soci.org

B
A 6.5
6.3 Y = 0.39 + 3.20X Y = 1.25 + 2.75X

Probit Mortality
5.5
5.3

4.3 4.5

3.3 3.5
0.5 1 1.5 2 0.5 1 1.5 2
Log dose (µg cm-2) Log dose (µg cm-2)

Figure 2. Probit mortality line of Cinnamomum aromaticum oil log dose (µg cm−2 ) against adult Callosobruchus maculatus after (A) 24 and (B) 48 h of
treatment in surface film bioassay.

16 Treatment of eggs with aromatised powder

e Two lentils together with a pair of newly emerged insects were
14
24h, exposure placed in each of 60 glass bottles. After 3 days the insects were
12 d removed, the eggs were counted and 20 eggs were left in each
10 c
bottle (the others were removed using a needle). The eggs were
Mortality (%)

then sprinkled with 0.5 mg of kaolin powder aromatised with C.

8 aromaticum essential oil at concentrations of 0, 10, 20, 30, 40 and
6 b 50 µL g−1 . For each concentration applied to the lentils, ten bottles
were used, with five replicates per treatment. The control consisted
4 of eggs that were not sprinkled with the aromatised powder but
2 were held under the same conditions (number, replications and
a
environment). The eggs were examined after 5 days to distinguish
0
those that had developed (opaque and yellowish) from those that
had not (translucent and yellow). The bottles were covered and
95.45 63.63 47.73 31.82 Control checked again after 30 days to compare the number of emerged
Concentration of essential oil (µg cm-2) insects with the number of eggs that had been deposited.
Figure 3. Toxicity effect of Cinnamomum aromaticum essential oil against
adult Callosobruchus maculatus after 24 h of treatment in fumigation Statistical analysis
bioassay. Each data point represents mean ± SD of ten replicates, each
comprising ten adult insects and five separate experiments. Data that do The mortality percentage was corrected using Abbott’s
not share the same letter are significantly different at P < 0.05 (based on formula:16,17
DMRT).
Pt = [(Po − Pc )/(100 − Pc )] × 100

4 Y = 0.87 + 1.57X where Pt is the corrected mortality (%), Po is the observed mortality
Probit Mortality

(%) and Pc is the control mortality (%).

3.6 The observed data were then subjected to probit analysis
according to Finney18 and Busvine17 using software developed
3.2 in the Department of Agricultural and Environmental Science,
University of Newcastle Upon Tyne, UK, which adapts the
traditional calculations to automatic computation. Heterogeneity
2.8
1.4 1.6 1.8 2 is tested by a χ 2 test. If the probability is greater than 5%, an
-2 automatic correction of heterogeneity is introduced. The program
Log dose (µg cm )
also calculates the confidence limits for LD50 . These data are
Figure 4. Probit mortality line of Cinnamomum aromaticum oil log dose entered into a linear regression program, which fits a regression
(µg cm−2 ) against adult Callosobruchus maculatus after 24 h of treatment line on the probit log dose concentration graph. Percentage
in fumigation bioassay. mortality and dose concentration can be determined from this
graph using the probit transformation table.19

was placed on the opposite petri dish as control. After 48 h the

number of eggs laid on each lentil was counted. The repellency RESULTS
capacity was quantified by comparing the number of eggs laid Chemical composition of essential oil
on lentils in the petri dish with cinnamon oil against the number
Distillation of C. aromaticum bark yielded 1.12% (w/w) essential
of eggs laid on lentils in the corresponding petri dish without oil
oil based on dry weight. The results of GC/MS analysis of the C.
(control).
aromaticum bark essential oil are listed in Table 1 according to
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their elution order on the ZB-1 capillary column. The essential

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 www.soci.org R Islam et al.

120

Table 1. Chemical composition of essential oil isolated by hydrodis-

Egg hatched & Adult emerged (%)

a a
tillation from bark of Cinnamomum aromaticum 100

Number RIa Compound Peak area (%)b

80 Hatched, cinnamon oil
Adult emerged,Cinnamon oil
1 982 Benzaldehyde 0.47
2 1005 1,8-Cineol 0.88 60
3 1017 Acetophenone 0.10
4 1076 Linalool 0.12 40 b
b
5 1090 Myrcenol 0.10
c b
6 1136 Phenylethyl alcohol 0.14 20 d c
7 1159 α-Terpineol 0.97 e c,d e d
8 1161 2-Bornanol, 2-methyl 0.58 0
9 1171 Benzaldehyde, 2-methoxy 0.18 -10 0 10 20 30 40 50 60
10 1178 Chavicol 0.31 Concentration of essential oil (µl g-1)
11 1181 trans-Cinnamyl alcohol 0.78
Figure 5. Effect of Cinnamomum aromaticum essential oil on Calloso-
12 1189 cis-Cinnamaldehyde 53.90
bruchus maculatus egg hatching and adult emergence. Each data point
13 1220 p-Anisaldehyde 0.29 represents mean ± SD of ten replicates, each comprising 20 eggs and
14 1290 p-Cuminol 0.12 five separate experiments. Data that do not share the same letter are
15 1320 β-Farnesene 0.10 significantly different at P < 0.05 (based on DMRT).
16 1345 α-Cubenene 1.99
17 1372 Isoledene 1.65
oil contained 52 different compounds representing 92.22%
18 1378 Methyl cinnamate 0.47
of the total oil. The major components detected in the oil
19 1380 Eugenol 5.36
were cis-cinnamaldehyde (53.90%), eugenol (5.36%), α-cadinol
20 1386 Azulene 0.58
(2.67%), caryophyllene (2.23%), viridiflorol (2.06%), α-cubenene
21 1395 Eugenol methyl ether 0.14
(1.99%), ledol (1.90%), isoledene (1.65%), β-cedren (1.59%) and
22 1404 β-Cedren 1.59
caryophyllene oxide (1.25%). α-Terpineol (0.97%), 1,8-cineol
23 1438 Aromadendrene 0.45
(0.88%), trans-cinnamyl alcohol (0.78%) and α-bisabolol (0.76%)
24 1441 Isosativene 0.18
were found as minor components.
25 1447 α-Humulene 0.42
26 1475 α-Curcumene 0.50
Surface film bioassay of essential oil on adult C. maculatus
27 1492 Germacrene 0.59
28 1494 Caryophyllene 2.23
The essential oil of C. aromaticum exhibited a moderate to high
29 1538 α-Calacorene 0.53
toxic effect on adult C. maculatus in a dose-dependent manner. As
30 1552 Acetyl eugenol 0.22
shown in Fig. 1, the oil concentration of 62.85 µg cm−2 produced
31 1556 Cuparene 0.24
the highest insect mortality (94.44%) after 24 h of treatment.
32 1561 Caryophyllene oxide 1.25
The LD50 values, 95% confidence limits, regression equations and
33 1564 Nerolidol 0.56
χ 2 values are given in Table 2. They show a variable degree of
34 1568 Viridiflorol 2.06
insect mortality. The LD50 value was calculated as 27.56 µg cm−2
with 95% confidence limits from 20.98 to 36.21 µg cm−2 and
35 1580 Ledol 1.90
the regression equation was obtained as Y = 0.39 + 3.20X after
36 1610 Tetradecenal 0.08
24 h of treatment. After 48 h of exposure the LD50 value was
37 1626 α-Cadinol 2.67
calculated as 23.16 µg cm−2 with 95% confidence limits from 17.01
38 1628 Cubenol 1.09
to 31.53 µg cm−2 and the regression equation was obtained as
39 1639 Patchouli alcohol 0.24
Y = 1.25 + 2.75X. The significant χ 2 values indicate the goodness
40 1682 α-Bisabolol 0.76
of fit of the regression lines presented in Figs 2A and 2B.
41 1697 cis,trans-Farnesol 0.75
42 1733 Benzyl benzoate 0.30
43 1769 Tetradecanoic acid 0.48 Fumigation bioassay of essential oil on adult C. maculatus
44 1800 Hexadecenal 0.38 The essential oil had a lower fumigation effect on adult insects as
45 1832 Pentadecanoic acid 0.43 shown in Fig. 3. The LD50 values, 95% confidence limits, regression
46 1854 Hexadecanol 0.15 equations and χ 2 values are given in Table 2 and the regression line
47 1968 n-Hexadecanoic acid 1.16 is shown in Fig. 4. The LD50 value was calculated as 434.69 µg cm−2
48 2009 Eicosane 0.57 with 95% confidence limits from 55.80 to 3386.30 µg cm−2 after
49 2045 Phytol 0.18 24 h of treatment. The regression equation was obtained as
50 2183 9,12-Octadecadienoic acid 0.36 Y = 0.87 + 1.57X with a χ 2 value of 1.09 at two degrees of
51 2300 Tricosene 0.54 freedom (2DF). According to the results presented in Fig. 4, we
52 2704 Phthalic acid 0.13 observed similar trends to the surface film toxicity of C. aromaticum
Total identified components 92.22 essential oil.
a Retention index relative to n-alkanes on ZB-1 capillary column.
b Repellency capacity of essential oil on adult C. maculatus
Compound percentage.
A dose-dependent effect of the essential oil on C. maculatus
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oviposition was observed. At concentrations of 62.85, 31.43, 15.71

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c 2009 Society of Chemical Industry J Sci Food Agric 2009; 89: 1241–1246
Bioactive properties of cinnamon oil
Table 2. LD50 values, 95% confidence limits, regression equations and χ 2 values of Cinnamomum aromaticum essential oil against adult
Callosobruchus maculatus in surface film and fumigation bioassays
95% confidence limits
Treatment time (h) LD50 (µg cm−2 )a Lower (µg cm−2 )
Surface film bioassay
24 27.56 ± 3.78 20.98
48 23.16 ± 3.76 17.01
Fumigation bioassay
24 434.69 ± 32.56 55.80
a Each value represents mean ± standard deviation of ten replicate bottles and five separate experiments.
and 7.86 µg cm−2 the oil reduced egg laying by 60.0, 43.3, 33.3
and 11.7% respectively compared with the non-treated control.
Statistically significant results were obtained, with 40% more eggs
being found on control lentils than on oil-treated lentils.
Treatment of eggs with aromatised powder
The essential oil from C. aromaticum had a significant (P < 0.05)
and concentration-dependent effect on both egg hatching and
adult emergence as shown in Fig. 5. The oil might hamper the
generation of insects resident in stored products and could be
useful to reduce losses during storage.
DISCUSSION
The use of natural products can be considered as an important
alternative for the control of stored product pests. The results
from this study indicated that the essential oil of C. aromaticum
exhibited effective toxicity to C. maculatus in all aspects (surface
film and fumigation bioassays, repellency, egg hatch and adult
emergence). Essential oils can affect insects in several ways: they
may disrupt major metabolic pathways and cause rapid death, act
as fumigants,12 contact insecticides,20 repellents,21 antifeedants,22
attractants, deterrents or phagostimulants, or modify oviposition.
They may also retard or accelerate development or interfere with
the life-cycle of insects in other ways.23 From our findings in
the surface film bioassay, cinnamon oil proved to be an effective
biocontrol agent. In the fumigation bioassay, moderate insect
toxicity was recorded after 24 h of treatment. The larvae that hatch
from the eggs of Callosobruchus spp. must penetrate the food
medium to survive, but they are unable to do this unless the egg
is attached to the food surface. Eggs on oil-treated lentils were
found to be less firmly attached than those on control lentils,
suggesting that the oil may inhibit successful larval penetration
into the food medium. While oil used alone can effectively protect
pulses from the pulse beetle, it can leave a persistent odour
that can be unpleasant when eating the product. Similar results
were obtained with the essential oils of Tagetes minuta L., Hyptis
suaveolens Poit., Ocimum canum L., Ocimum basilicum and Piper
guineense Schum.24 The principal component of C. aromaticum
essential oil is cis-cinnamaldehyde, which has been found to
play a key role in controlling the pulse beetle.5 In general, the
cytotoxic activity of essential oils is mostly due to the presence of
phenols, aldehydes (cinnamaldehyde) and alcohols.25 The other
major component of cinnamon oil, eugenol, may also induce an
increase in cell membrane permeability and consequently growth
inhibition due to its action on intracellular enzymes.26 It is also
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c 2009 Society of Chemical Industry
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Upper (µg cm−2 ) Regression equation χ 2 (at 2DF)
36.21 Y = 0.39 + 3.20X 2.27
31.53 Y = 1.25 + 2.75X 1.14
3386.30 Y = 0.87 + 1.57X 1.1
possible that various minor components may be involved in some
type of synergism with other active components.27
The results of the present study suggest the possible use
of C. aromaticum essential oil in research for selecting new
biodegradable and natural biocontrol components, because
cinnamon oil has potential insecticidal activity. However, further
investigations on the insecticidal mode of action of the oil, its
effect on non-target organisms and field evaluation are needed.
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