A study was undertaken to explore the content and composition of volatile oil from decaying leaves of lemon-scented eucalypt (Eucalyptus citriodora Hook.) not analyzed earlier. GC and GC-MS analysis of the oil (yield 0.6%) revealed the monoterpenoid nature with citronellal (52.2%), citronellol (12.3%) and isoisopulegol (11.9%) as the major constituents. Overall, 17 components were identified that accounted for over 94% of the decaying leaf oil. Surprisingly, the decaying leaf oil contained nearly 1.8% of trans-rose oxide, which is generally absent in eucalypt essential oil. Decaying leaf oil and its major 2 components (citronellal and citronellol) inhibited the germination and root elongation of two weeds Ð Cassia occidentalis (broad-leaved) and Echinochloa crus-galli (grassy weed). Based on the dose-response studies, I 50 values were determined for decaying leaf oil and the effect was more on germination only of broad-leaved weed (C. occidentalis), whereas that of citronellal and citronellol were on germination as well as root length of E. crus-galli (grassy weed). Based on I 50 values it was observed that citronellal was more phytotoxic and germination inhibiting in nature, whereas citronellol was a more potent root inhibitor, thereby indicating a possible different mode of action. The study concludes that decaying leaf oil hold a good commercial value for exploitation as weed management agent

Daizy R Batish

Harminder Pal Singh

Nidhi Setia

Ravinder K Kohli

Shalinder Kaur

CiteSeerX

Chemical Composition and Inhibitory Activity of Essential Oil from
Decaying Leaves of Eucalyptus citriodora
Daizy R. Batisha, Harminder Pal Singhb,c,*, Nidhi Setiaa, Shalinder Kaura, and
Ravinder K. Kohlia,b
a Department of Botany, Panjab University, Chandigarh 160014, India
b Centre for Environment and Vocational Studies, Panjab University, Chandigarh 160014,
India. E-mail: hpsingh_01@yahoo.com
c Academic Staff College, Panjab University, Chandigarh 160014, India
* Author for correspondence and reprint requests
Z. Naturforsch. 61c, 52Ð56 (2006); received July 25, 2005
A study was undertaken to explore the content and composition of volatile oil from decay-
ing leaves of lemon-scented eucalypt (Eucalyptus citriodora Hook.) not analyzed earlier. GC
and GC-MS analysis of the oil (yield 0.6%) revealed the monoterpenoid nature with citronel-
lal (52.2%), citronellol (12.3%) and isoisopulegol (11.9%) as the major constituents. Overall,
17 components were identified that accounted for over 94% of the decaying leaf oil. Surpris-
ingly, the decaying leaf oil contained nearly 1.8% of trans-rose oxide, which is generally
absent in eucalypt essential oil. Decaying leaf oil and its major 2 components (citronellal and
citronellol) inhibited the germination and root elongation of two weeds Ð Cassia occidentalis
(broad-leaved) and Echinochloa crus-galli (grassy weed). Based on the dose-response studies,
I50 values were determined for decaying leaf oil and the effect was more on germination only
of broad-leaved weed (C. occidentalis), whereas that of citronellal and citronellol were on
germination as well as root length of E. crus-galli (grassy weed). Based on I50 values it was
observed that citronellal was more phytotoxic and germination inhibiting in nature, whereas
citronellol was a more potent root inhibitor, thereby indicating a possible different mode of
action. The study concludes that decaying leaf oil hold a good commercial value for exploita-
tion as weed management agent.
Key words: Decaying Leaf Essential Oils, I50 Values, Germination Inhibitor
Introduction
Eucalyptus L’Herit (Myrtaceae), a native plant
of Australia, is a unique genus of tall trees and
shrubs comprising of around 800 species widely
cultivated in various parts of the world (Brooker
and Kleinig, 2004). These are commonly called
gum trees as these exude a gum and are known
world over for insect-repellant properties. The eu-
calypt trees are characterized by evergreen foliage
that is variably fragrant due to the presence of vol-
atile essential oils. These find an extensive use in
perfumery and pharmaceutical industry and their
amount and fragrance varies with the species. In
India, eucalypt was first introduced in 1792 and is
now one of the major trees of the over 32.6 Mha
area under the forest plantations (FAO, 2001). Be-
sides, these are also cultivated along roadsides,
parks, and gardens mainly for aesthetic value and
along agricultural fields as windbreaks and shelter-
belts. Some of the species commonly grown in In-
0939Ð5075/2006/0100Ð0052 $ 06.00 ” 2005 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D
dia are red gum (E. camaldulensis), lemon-scented
gum (E. citriodora), Tasmanian blue gum (E. glo-
bulus) and Cider gum (E. tereticornis). Among
these, E. citriodora is a large, quick-growing tree
with smooth and white bark and lemon-scented
leaves. It is extensively planted and coppiced for
the extraction of essential oil that is rich in citron-
ellal and used in perfumery and as flavouring
agent. The oil is known to possess a wide spectrum
of biological activities including fungicidal (Rame-
zani et al., 2002), insecticidal (Isman, 2000), nema-
ticidal (Pandey et al., 2000) and allelopathic prop-
erties (Kohli, 1990). For various industrial uses the
oil is generally extracted from the juvenile and
adult foliage. However, no attempt has ever been
made to explore the essential oil from the decay-
ing leaves that form a matrix on the floor of its
plantations owing to the evergreen nature of the
tree and their slow decomposition rate. Further,
there are reports that the floor of E. citriodora
lacks any vegetation (Kohli, 1990); this is generally
D. R. Batish et al. · Essential Oil from Decaying Leaves of Eucalyptus citriodora 53
correlated with the emission of volatile oils from
the foliage. However, nothing is known regarding
the role of decaying leaves/litter fallen on the floor
with reference to the amount of essential oil, its
chemical constitution and role as germination in-
hibitor. This information can serve as an important
resource for exploring its commercial utilization.
With this objective, a study was therefore planned
to determine the content, composition and phyto-
toxicity of essential oil in the decaying leaves of
E. citriodora.
Materials and Methods
Plant material and oil extraction
Decaying leaves were collected from the floor
of the nearly 25-year-old trees of lemon-scented
eucalypt (Eucalyptus citriodora Hook.) growing
on Panjab University campus, Chandigarh, India
in April 2004.
The essential oil was extracted by steam-distilla-
tion using Clevenger’s apparatus. Nearly 250 g of
decaying leaves were mixed into 1 l of distilled wa-
ter in a 2 l round bottom flask fitted with a con-
denser. After boiling contents for 2 h, the oil was
collected from the nozzle of the condenser, dried
over sodium sulphate and its amount was meas-
ured to determine yield. It was stored at 4 ∞C for
further identification and bioassay.
Analysis of oil
The essential oil was analyzed by GC and GC-
MS. GC was performed on a Shimadzu GC-14B
gas chromatograph with a flame ionization de-
tector and using a Supelco wax column (60 m ¥
0.25 mm i.d., film thickness 0.25 µm) and N2 as
carrier gas. The oven temperature was pro-
grammed from 40 ∞C, initially held isothermal for
4 min, to 220 ∞C at the rate of 4 ∞C/min and held
for 5 min. Relative amounts of different constitu-
ents were determined by computer based calcula-
tion of peak area normalization without any cor-
rection factor. Peaks obtained were compared
with data obtained from GC-MS.
GC-MS was done on a Q Mass 910 Perkin-
Elmer Mass Spectrophotometer equipped with
fused silica (BP 21) capillary columns. The analysis
was carried out using a BP-21 column (30 m ¥
0.25 mm i.d., film thickness 0.25 µm). The injector
and detector temperature were fixed as 230 ∞C and
250 ∞C, respectively, and He was used as carrier
gas. The oven temperature was programmed from
40 ∞C, initially held isothermal for 7 min, to 190 ∞C
at the rate of 5 ∞C/min and finally held isother-
mally for 20 min.
Identification of components
The compounds were identified on the basis of
computer matching of mass spectra using the li-
brary search system HP-5872 (Hewlett-Packard),
consulting data bases viz. Wiley 275 and NBS 75K
libraries (McLafferty, 1989), NIST 98 (Stein,
1990), and compilation by Adams (1995).
Dose-response study and inhibitory activity
The role of the essential oil from decaying
leaves and its two major monoterpenes (citronellal
and citronellol) as germination inhibitor was ex-
plored against two weedy species viz. Cassia occi-
dentalis (broad-leaved) and Echinochloa crus-galli
(grassy weed). For this, seeds of each test weed
were divided into 22 groups (7 each for essential
oil, citronellal, and citronellol, and one for con-
trol) of 100 each and dipped in distilled water for
24 h for imbibition prior to germination. These
were then equidistantly placed in 15 cm diameter
petri dishes (20 seeds each in 5 petri dishes as rep-
licates), lined with a single layer Whatman no. 1
filter circle moistened with 7 ml distilled water.
The filter paper was treated with eucalypt oil or
citronellol or citronellal so as to have a concentra-
tion of 0.06, 0.12, 0.24, 0.60, 1.2, 2.4 and 6.0 mg/ml.
The concentrations used were in mg/ml instead of
mm or µm since the molecular weight of oil was
not known. After the treatment, petri dishes were
sealed with a paraffin film to avoid loss of oil on
vaporization. Treatment without oil or pure mo-
noterpenes in a similar manner served as control.
Petri dishes were placed in a growth chamber at
(24 ð 3) ∞C and a 16 h/8 h light/dark photoperiod,
a photon flux density of approximately 150 µmol
mÐ2 sÐ1 and relative humidity of around 75%.
After a week, the number of seeds that germi-
nated was counted and the root length of emerged
seedlings was measured.
The significance of observed values in response
to treatment was determined over control by 2-
sample t-test at p < 0.05 whereas differences within
treatments were analyzed by one-way ANOVA
followed by separation of means using the SPSS
package (version 10).
54 D. R. Batish et al. · Essential Oil from Decaying Leaves of Eucalyptus citriodora
Results and Discussion
The yield of volatile essential oil from the de-
caying leaves of E. citriodora was 0.60% (v/w). GC
and GC-MS analysis of this oil revealed its mono-
terpenoid nature including 16 monoterpenes and
Table I. Composition of the decaying eucalypt leaf oils
as revealed by GC and GC-MS analysis.
Constituent Retention time Amount
[min]
α-Pinene 5.9 0.19
Myrcene 6.0 0.02
Linalool 11.22 0.04
trans-Rose oxide 14.95 1.77
trans-Pulegol 15.53 0.99
Citronellal 19.44 52.23
Isopulegol 21.50 5.02
Isoisopulegol 21.72 11.9
α-Terpineol 22.47 1.04
Isolimonene 23.08 0.51
-Citronellene 24.10 6.12
Linalool acetate 25.17 0.49
Citronellol 26.60 12.31
Geraniol 27.36 0.36
Eugenol 30.29 0.35
Citronellyl acetate 30.82 0.17
-Caryophyllene 31.76 1.51
Total 94.21
1 sesquiterpene (Table I). These were isolated at
retention times between 5 to 35 min, and 17 con-
stituents were identified accounting for 94.21% of
the oil. The monoterpenes included four hydrocar-
bons (2 cyclic Ð α-pinene and isolimonene; 2 acyc-
lic Ð myrcene, -citronellene), seven alcohols (3
acyclic Ð linalool, citronellol, geraniol; 4 cyclic Ð
isopulegol, isoisopulegol, α-terpineol, trans-pule-
gol), one of each of acyclic aldehydes (citronellal),
cyclic ethers (trans-rose oxide) and allylphenyl
monoterpenes (eugenol), and two acyclic mono-
terpene esters (citronellyl and linalool esters). The
only sesquiterpene identified was -caryophyl-
lene Ð a cyclic one (Table I). Among the consti-
tuents, five major ones were monoterpenes viz.
Treatment Germination Root elongation
C. occidentalis E. crus-galli C. occidentalis E. crus-galli
Decaying leaf oil 1.09 1.45 0.31 0.35
Citronellal 0.55 (3.56) 0.14 (0.91) 0.27 (1.75) 0.13 (0.84)
Citronellol 1.67 (10.64) 0.24 (1.54) 0.13 (0.83) 0.09 (0.58)
Table II. I50 values (in mg/
ml) of E. citriodora decay-
ing leaf oil and its two ma-
jor components for germi-
nation and root elongation
in C. occidentalis and E.
crus-galli. Values in paren-
thesis indicate I50 in mm.
citronellal (52.2%), citronellol (12.3%), isoisopu-
legol (11.9%), -citronellene (6.1%) and isopule-
gol (5.0%), and accounted for over 87% oil con-
tent. The rest of the 12 constituents were nearly
or less than 7%. Though, the composition of oil is
in conformity with that reported hitherto in fresh
leaves of E. citriodora (Dethier et al., 1994; Chal-
chat et al., 2000), yet the relative amount of indi-
vidual constituents differed. Surprisingly, the de-
caying leaf oil contained nearly 1.8% of trans-rose
oxide (an important compound in flavour fragrant
industry), which is generally absent or present in
very low amount in eucalypt essential oils. Citron-
ellal though observed was a major constituent in
decaying leaf oil, but its amount (52%) was not
sufficient to make it a potential resource for per-
fumery industry that normally requires 65Ð80%.
Nevertheless, other monoterpenes like trans-rose
oxide, citronellol and pulegol isomers are commer-
cially important and find extensive use in flavour
and fragrance industry. Furthermore, the decaying
leaf oil can be explored for weed management, as
the essential oils and their constituent monoterpe-
nes are presently being viewed as excellent weed
suppressants (Singh et al., 2003). However, the de-
caying leaf oil has not been assessed for such an
activity and its phytotoxicity is unknown. Studies
in this regard reveal that decaying leaf oil and its
two major constituents, citronellal and citronellol,
inhibited the germination and initial growth of test
weeds (Figs. 1, 2).
A significant reduction in germination and root
length of test weeds was observed with eucalypt
oil, citronellal and citronellol, though the magni-
tude of inhibition differed (Figs. 1, 2). Based on
the dose-response curves, I50 values were calcu-
lated (Table II). In general, the effect of decaying
leaf oil was more on the germination of broad-
leaved weed C. occidentalis compared to grassy
weed E. crus-galli indicating the species-specificity.
However, the effect was similar on the root length
of both the weeds. In contrast the effect of both
citronellol and citronellal was on germination as
D. R. Batish et al. · Essential Oil from Decaying Leaves of Eucalyptus citriodora 55
C. occidentalis
0.06 0.6 6
0
20
40
60
80
100
I
50  
Values
 Decaying Leaf Oil
 Citronellal
 Citronellol
P
er
ce
n
t 
G
er
m
in
at
io
n
Concentration (mg/ml)
E. crus-galli
0.06 0.6 6
0
20
40
60
80
100
I
50  
Values
 Decaying Leaf Oil
 Citronellal
 Citronellol
P
er
ce
n
t 
G
er
m
in
at
io
n
Concentration (mg/ml)
Fig. 1. Effect of decaying eucalypt leaf oil and its two
major constituent monoterpenes on the percent germi-
nation of test weeds measured after one week. Data
present mean ð SE.
well as root length of E. crus-galli. Based on I50
values it was observed that citronellal was more
phytotoxic and germination inhibiting in nature,
whereas citronellol was a more potent root inhibi-
tor (Table II), thereby indicating a possible differ-
ent mode of action. The variability in the effect of
crude oils compared to the two major monoterpe-
nes is probably due to the fact that oil is a compos-
ite mixture of a number of monoterpenes and
these two compounds could not account for the
observed inhibitory pattern of crude oils. Probably
some other components, though present in lesser
C.  occidentalis
0.06 0.6 6
0
20
40
60
80
100
I
50  
Values
 Decaying Leaf Oil
 Citronellal
 Citronellol
P
er
ce
n
t 
R
o
o
t 
L
en
g
th
Concentration (mg/ml)
E. crus-galli
0.06 0.6 6
0
20
40
60
80
100
I
50  
Values
 Decaying Leaf Oil
 Citronellal
 Citronellol
P
er
ce
n
t 
R
o
o
t 
L
en
g
th
Concentration (mg/ml)
Fig. 2. Effect of decaying eucalypt leaf oil and its two
major constituent monoterpenes on the percent root
length of test weeds measured after one week. Data
present mean ð SE.
amount, are also responsible for the observed inhi-
bition. Such an inhibitory effect of essential vola-
tile oils is not surprising as the essential oils and
their monoterpenoid constituents are known alle-
lochemicals (Asplund, 1968; Vaughn and Spencer,
1993). At present, due to their germination inhibi-
tory effects, these essential oils are being screened
as possible candidates for the control of weed spe-
cies (Singh et al., 2003) since they offer a number
of novel characteristics such as biodegradability,
unique modes of action and very little or no mam-
malian toxicity (Isman, 2000). Based on the
56 D. R. Batish et al. · Essential Oil from Decaying Leaves of Eucalyptus citriodora
present study, it is thus concluded that decaying
eucalypt leaf oil suppresses the germination and
root growth of weed species and thus hold a good
promise for future exploitation for weed manage-
ment purposes.
Adams R. P. (1995), Identification of Essential Oils Isman M. B. (2000), Plant essential oils for pest and dis-
Components by Gas Chromatography and Mass Spec- ease management. Crop Prot. 19, 603Ð608.
trometry. Allured Publishers Corp., Carol Stream, Illi- Kohli R. K. (1990), Allelopathic Properties of Eucalyp-
nois. tus. MAB-DOEn Report. Ministry of Environment
Asplund R. O. (1968), Monoterpenes: relationship be- and Forests, New Delhi, India.
tween structure and inhibition of germination. Phyto- McLafferty F. W. (1989), Registry of Mass Spectral Data,
chemistry 7, 1995Ð1997. 5th Ed. John Wiley and Sons, New York.
Brooker M. I. H. and Kleinig D. A. (2004). Field Guide Pandey R., Kalra A., Tandon S., Mehrotra N., Singh
to Eucalypts. Vol. 3. Northern Australia. Blooming H. N., and Kumar S. (2000), Essential oils as potent
Books, Victoria, Australia. sources of nematicidal compounds. J. Phytopathol.
Chalchat J.-C., Garry R.-P., Sidibe L., and Harama M. 148, 501Ð502.
(2000), Aromatic plants of Mali (V): Chemical com- Ramezani H., Singh H. P., Batish D. R., and Kohli R. K.
position of essential oils of four Eucalyptus species (2002), Antifungal activity of the volatile oil of Euca-
implanted in Mali: Eucalyptus camaldulensis, E. citrio- lyptus citriodora. Fitoterapia 73, 261Ð262.
dora, E. toreliana and E. tereticornis. J. Essent. Oil Singh H. P., Batish D. R., and Kohli R. K. (2003), Allelo-
Res. 12, 695Ð701. pathic interactions and allelochemicals: new possibili-
Dethier M., Nduwimana A., Cordier Y., Menut C., and ties for sustainable weed management. Crit. Rev.
Lamaty G. (1994), Aromatic plants of tropical central Plant Sci. 22, 239Ð311.
Africa. XVI. Studies on essential oils of five Eucalyp- Stein S. E. (1990), National Institute of Standards and
tus species grown in Burundi. J. Essent. Oil Res. 6, Technology (NIST). Mass Spectral Data Base and
469Ð473. Software, Ver. 3.02. Gaithersburg, Maryland, USA.
FAO (Food and Agricultural Organization). (2001), Vaughn S. F. and Spencer G. F. (1993), Volatile monoter-
Global Forest Resources Assessment 2000 Main Re- penes as potential parent structures for new herbi-
port. FAO Forestry Paper 140. Food and Agriculture cides. Weed Sci. 41, 114Ð119.
Organization of the United Nations, Rome, Italy.
Acknowledgements
The financial support from UGC (Daizy R. Ba-
tish), DST (Harminder Pal Singh), and CSIR
(Nidhi Setia and Shalinder Kaur) is gratefully
acknowledged.


Chemical composition and inhibitory activity of essential oil from decaying leaves of Eucalyptus citriodora

http://citeseerx.ist.psu.edu/viewdoc/download;jsessionid=F3C40A233DE5AB9681EE705579C16D7E?doi=10.1.1.1055.8003&rep=rep1&type=pdf

Chemical composition and inhibitory activity of essential oil from decaying leaves of Eucalyptus citriodora

Abstract

Similar works

Full text

Available Versions

CiteSeerX