Genetics and Plant Physiology – 2013, Volume 3 (1–2), pp.

90–97
©2013 Published by the Institute of Plant Physiology and Genetics – Bulgarian Academy of Sciences
Available online at [Link]

ESTIMATION OF SALICYLIC ACID IN EUCALYTPUS LEAVES USING

SPECTROPHOTOMETRIC METHODS

Warrier R . R.*, M. Paul, M. V. Vineetha

Institute of Forest Genetics and Tree Breeding, Forest Campus, PB 1061, R. S. Puram
Coimbatore-641002, Tamilnadu, India

Received: 27 May 2013 Accepted: 09 October 2013

Summary: A simple and reliable procedure for spectrophotometric determination of salicylic

acid (SA) in Eucalyptus leaves is described. The procedure is based on the formation of Fe(H2O)63+
ion which has an intense violet colour when SA in an aqueous form reacts with Fe (III). The
absorbance of the complex was measured at 540 nm. The procedure was tested with different
solvents and varying sample sizes to optimize the extraction protocol and study the interference
of oils.

Citation: Warrier R. R., M. Paul, M. V. Vineetha, 2013. Estimation of salicylic acid in Eucalyptus
leaves using spectrophotometric methods. Genetics and Plant Physiology, 3(1–2): 90–97.

Keywords: Spectrophotometry; salicylic acid; Eucalyptus; ferric chloride.

INTRODUCTION

Plants have been subjected to attack by metabolic products. These secondary

an array of organisms like viruses, bacteria, metabolites play a major role in defense
fungi, insects, nematodes and herbivores mechanisms against herbivores.
for centuries. In their long association with Salicylic acid (SA) or ortho-hydroxy
pests and pathogens, plants have evolved benzoic acid and related compounds belong
an impressive array of defensive tools. The to a diverse group of plant phenolics.
evolution of chemical defenses in plants Salicylates from plant sources have been
is linked to the emergence of chemical used in medicine since antiquity. Using
substances that are not involved in the modern analytical techniques it has been
essential photosynthetic and metabolic found that salicylates are distributed in
activities. These substances, secondary many important agricultural plant species.
metabolites, are organic compounds that SA is a phenolic phytohormone playing
are not directly involved in the normal a role in plant growth and development,
growth, development or reproduction of photosynthesis, transpiration, ion uptake and
organisms, and are often produced as by- transport. SA also induces specific changes
products during the synthesis of primary in leaf anatomy and chloroplast structure.

*Corresponding author: rekhawarrier@[Link], rekha@[Link]

 Spectrophotometric assay of salicylic acid 91

The discovery of flower-inducing Wilson, 1978), gas chromatography-mass

action and bud formation in tobacco cell spectrometry (Meuwly et al., 1995);
cultures by Eberhard et al. (1989) was HPLC-based method involving extraction
the first indication of a physiological of SA in organic solvents, evaporation
effect of SA. The stimulatory effect of of organic solvents, chromatographic
SA on flowering was later demonstrated purification and detection by fluorescence
in other plant species forming the basis spectroscopy (Verberne et al., 2002;
for suggesting that SA can function as Abdoul-Sood et al., 2004); biosensor-
an endogenous regulator of flowering based method and HPLC-based approach
(Cleland and Ajami, 1974). (Marek et al., 2010); gas chromatograph
SA is involved in endogenous with electron capture detection method
signaling, mediating plant defense against (Meher et al., 2011) are available, but they
pathogens. It plays a role in the resistance are extremely costly and time consuming.
to pathogens by inducing the production Eucalyptus clones are commercially
of pathogenesis-related proteins. It planted because of their valuable wood
is involved in the systemic acquired and fiber properties which have been
resistance (SAR) in which a pathogenic exploited in the pulp and paper industry.
attack on one part of the plant induces Although Eucalyptus trees are generally
resistance in other parts. The signal can disease tolerant, they can do succumb
also move to nearby plants by SA being to diseases caused by a wide range of
converted to a volatile ester, methyl pathogens (Wingfield et al., 2008). Jacob
salicylate. The highest levels of SA were et al., (2007) reported a severe outbreak
determined in the inflorescence with of an invasive insect, Leptocybe invasa,
necrotizing pathogens (Raskin, 1992). presumed to have made an entry from
SA is a relatively polar, poorly Australia, on eucalypts plantations and
aqueous soluble material. The salt form, nurseries all over the country. This tiny
however, is water soluble. Further, it wasp has been reported to induce galls on
can be easily extracted into water at shoot terminals, on petioles and midribs in
high temperatures. Its presence can be saplings and trees of eucalypts (Mendel et
detected in very minute quantities of 1 al., 2004). A report on the mode of action
part to 100,000. A colorimetric method of the wasp by Krishnakumar and Jacob
by ferric chloride, comparing the depth of (2010) suggested biotrophic pathogenesis
color from a known solution of SA, was which triggered the SA pathway for
given by Muter (1876) for food samples. defense mechanisms. A stepping stone
SA, with ferric chloride, has also been for improving our understanding of
proposed by Weiske (Cohn, 1899) as an Eucalyptus responses to pathogen
indicator in acidimetry. Routine analysis defenses would be to quantify the levels
of the compound has been carried of SA in these plants. Since no standard
out in food, dairy and pharmaceutical protocols are available for quantitative
industry (AOAC, 1967; Venema et al., estimation of SA in tree species, the aim of
1996; FSSAI, 2012). However, in plants this study was to standardize an extraction
sophisticated methods like the thin-layer and estimation protocol for free SA in the
densitometric method (Chrastil and leaves of Eucalyptus trees.

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92 Warrier et al.

MATERIALS AND METHODS was added to the solvent to make

100 ml of the solution. This solution
SA easily forms complexes with contained 1000 ppm (µg/ml).
minute traces of ferric salts and hence, All stock and standard solutions may
can be used as a very useful method for stay for at least 6 months if stored in a
detection of the compound. It forms a refrigerator.
violet complex due to the formation of
a ligand and the extinction of the ferric Optimization of extraction protocols
complex can be determined at 540 nm. Leaf samples of Eucalyptus were
This principle was adopted in the present frozen in liquid nitrogen and ground
study for the detection of SA in Eucalyptus. to powder. Samples were left at room
temperature to thaw. Varying aliquots (10
– 1000 mg) of the sample were extracted
in 1.0 ml of different solvents to assay
the solubility of SA from tissues in the
presence of interfering substances. Eight
clones of Eucalyptus were chosen to
Standard solutions study the pattern of variation in levels
SA reacts with almost all solvents and of SA following the optimized sample
has high solubility. It is soluble in many weight and extraction volume. This was
solvents. One gram can be dissolved in 460 done to optimize the solvent to be taken
ml of water at room temperature, 15 ml up for extraction. This was followed by
boiling water, 2.7 ml alcohol, 3 ml acetone, extraction of SA into the optimized solvent
42 ml chloroform, 135 ml benzene, 60 ml by varying the volumes of the solvents for
glycerol, etc. (Rainsford, 2004). Standards 50 mg and 100 mg tissue.
of varying concentrations were prepared In all experiments, samples were
in chloroform, amyl alcohol, ether, ethanol swirled well in the solvent followed by
and water to check the suitability of the centrifugation at 10,000 g for 10 min.
solvents for extraction of SA. The supernatant was stored on ice for SA
A. Water as solvent - Stock SA solution: measurement. 100 µl of the supernatant
100 mg SA was dissolved in sufficient was mixed with 0.1% freshly prepared
water to make 100 ml of solution. This ferric chloride. The volume of the reaction
solution contained 1000 ppm. Working mixture was made up to 3.0 ml and the
Stock solution: 1.0 ml of the stock complex formed between Fe3+ ion and SA,
solution was added to sufficient water which is violet in colour was determined
to make 10 ml of the solution. This by spectrophotometry, measuring the
solution contained 100 ppm (µg/ml). absorbance of the complex in the visible
B. Organic Solvents (chloroform, amyl region (at 540 nm).
alcohol, ether, ethanol) - Stock SA SA measurements were carried out
solution: 100 mg of SA was dissolved with different clones of Eucalyptus using
in 100 ml of solvent. This solution the standardized protocols and the amount
contained 1000 ppm. Working Stock of SA in the leaf samples was determined
solution: 1.0 ml of the stock solution accordingly.

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 Spectrophotometric assay of salicylic acid 93

Statistical analysis ranging from 10 mg to 1.00 g were

A single-factor analysis of variance ground in 1.0 ml of different solvents
(one-way ANOVA) was adopted to and the absorbance was measured
investigate the effect of different solvents at 540 nm (Fig. 2). It was observed
on the extraction of SA and compare that although the standard curves
any significant differences between obtained for SA dissolved in organic
them. Experimental results were means solvents showed intense colour at low
of five parallel measurements and were concentrations, these solvents were not
analyzed by Microsoft Excel 2010 and able to effectively extract SA from the
SPSS 16.00 for Windows. Differences plant leaf samples. Among the different
were considered significant at p < 0.05. organic solvents, ethanol showed
slightly improved results. However, it
RESULTS AND DISCUSSION was observed that after extraction of free
SA in water (aqueous extract), maximum
Development of standard curves SA in the samples was detected. There
Five different solvents which have was a steady and linear increase in the
been reported as solvents for SA were levels of SA in the aqueous extract
used for the generation of the standard from 10 mg to 50 mg which plateaued
curves. It was observed that it took at 60 and 70 mg. This was followed by
longer time to dissolve SA in water a gradual reduction in the levels of SA
which could be easily dissolved by suggesting that with increasing sample
slight warming of the solution. SA was size, the efficiency of SA extraction
dissolved rapidly in organic solvents. For into the solvent was reduced. A similar
the development of the standard curves, trend was also observed in ethanol.
it was observed that water as a solvent With increasing concentrations in the
was aliquoted in 100, 200,…500 µg sample, the solvent got saturated with
series while in case of organic solvents, SA resulting in a decline in the levels of
detection was observed in 20, 40, …100 SA with increasing sample size.
µg series. Standard curves of SA using Eight clones of Eucalyptus were
the five different solvents and the curve selected for extraction of SA using
of best fit by regression analysis were different solvents with the standardized
worked out. The calibration curve, curve sample weight and volume. ANOVA
of best fit and regression coefficients are analysis revealed that significant
presented in Fig. 1 A–E. All standard differences were observed in the levels
graphs had an r2 value closer to 1 of SA extracted from the different
indicating strong correlation between clones using different solvents (Table
the x and y axis data. 1). Maximum extraction was observed
in an aqueous solution followed by
Development of extraction protocols ethanol. Extractions in acetone, amyl
Once the calibration equations for alcohol and chloroform were on par.
the five solvents were developed, they This suggests interference of fats/oils
were used to estimate the concentration in the extraction of SA in the organic
of SA in Eucalyptus samples. Samples solvents tested.

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94 Warrier et al.

Figure 1. Standard curves of SA using different solvents.

Optimization of the extraction methods in the amount of SA from 100 to 1000 µl

To fine tune the quantity of samples which declined with further increasing
required for effective extraction of SA the volume of water whereas in the case
from the leaves of Eucalyptus, two of 100 mg sample, SA was found to be
samples (50 and 100 mg) were taken constant till the volume reached 1000 µl
and extracted in varying volumes of followed by a drastic reduction. T-test at
water ranging from 100 µl to 10.0 ml 5% level for comparing the two samples
(Fig. 3). It was observed that in the 50 revealed that the variations were
mg sample, there was a steady increase insignificant suggesting that 50–100 mg

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 Spectrophotometric assay of salicylic acid 95

Figure 2. Development of protocols for extraction of SA from Eucalyptus

leaves using five different solvents.

Table 1. ANOVA for SA estimation in different clones of Eucalyptus using different solvents.
Source of Variation SS df MS F P-value
Clones Between Groups 64727.3 7 9246.76 0.151 0.993
Within Groups 1965842 32 61432.6
Total 2030569 39
Solvents Between Groups 1727085 4 431771 49.795 0.000
Within Groups 303484 35 8670.97
Total 2030569 39

Figure 3. Optimization of sample size and extraction volume for optimal

extraction of SA from Eucalyptus leaves.

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96 Warrier et al.

sample could be the optimal sample size 157–160.

for extraction of SA from Eucalyptus Eberhard, S., N. Doubrava, V. Marta, D.
leaves. Mohnen, A. Southwick, A. Darviell,
P. Albersheim, 1989. Pectic cell wall
CONCLUSIONS fragments regulate tobacco thin-cell
layer explant morphogenesis. Plant
1. In this study, a rapid method for Cell, 1: 747–755.
determination of SA in Eucalyptus FSSAI, 2012. Food Safety and Standards
leaves based on its reaction with ferric Authority of India. pp. 43–47.
chloride, which reacts with minute Jacob, J. P., Devaraj, R. and Natarajan,
traces of SA to yield a purple coloured R., 2007, Outbreak of the invasive
complex was presented. gall inducing wasp Leptocybe invasa
2. The effect of some possible interfering on eucalypts in India. Newsltr.
substances, mainly oils, which are Asia–Pacific Forest Invasive Species
present abundantly in Eucalyptus, Network, 8: 4–5.
rendered extraction of SA in organic Krishnakumar, N. and Jacob, J. B., 2010.
solvents ineffective. Eucalyptus gall – A recent invasive
3. For extraction, 50-100 mg sample in in man-modified forest ecosystem in
1.0 ml of water provided repeatable Andhra Pradesh. Karnataka J. Agric.
results. Sci., 23 (1): 217–219.
4. In comparison with the well known Mendel, Z., Protasov, A., Fisher, N. and
sophisticated methods like HPLC, La Salle, J., 2004, Taxonomy and
GC, MS where extraction procedures biology of Leptocybe invasa gen. &
are very cumbersome and time- sp. n. (Hymenoptera: Eulophidae), and
consuming, the described method is invasive gall inducer on Eucalyptus.
simple, fast, reliable and accurate. Aust. J. Ent., 43: 101–113.
Muter, J. 1876. Note on a simple
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