Arabian Journal of Chemistry (2016) 9, S840–S845

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
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ORIGINAL ARTICLE

Toxicity, antimicrobial and antioxidant activities of

methyl salicylate dominated essential oils of
Laportea aestuans (Gaud)
Ganiyat K. Oloyede *

Department of Chemistry, Natural Products/Medicinal Chemistry Unit, University of Ibadan, Nigeria

Received 26 March 2011; accepted 10 September 2011

Available online 20 September 2011

KEYWORDS Abstract The colourless essential oils obtained by hydro-distillation from the whole plant of
Methyl salicylate; Laportea aestuans (Gaud) were analysed by GC and GC/MS. The major constituents in the oil were
2,2-Diphenyl-1- methyl salicylate (54.50%), fenchol (10.59%), 1, 2-cyclohexanedione dioxime (9.40%), 1, 4-octadi-
picrylhydrazyl; ene (8.86%) and linalool (3.26%). The toxicity results obtained from brine shrimp lethality test gave
Toxicity; LC50 value of 367.1805 lg/ml indicating that the oil is toxic. The oils showed appreciable antimi-
Antimicrobial; crobial activity against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas
Antioxidant; aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Candida albicans, Rhizopus stolon, Aspergillus
Laportea aestuans;
niger and Penicillium nonatum at 200 mg/ml when compared to standards; gentamicin for bacteria
Urticaceae
and tioconazole for fungi. The oil was however very active against the fungi R. stolon and A. niger at
25–200 mg/ml. While the in vitro antioxidant activities of the oils determined by scavenging effect
on 2, 2-diphenyl-1-picryl hydrazyl radical method showed that the oils have promising antioxidant
activity as a free radical scavenger. At 0.1 and 0.2 mg/ml, the % inhibition of the essential oil
(84.46% and 86.87%, respectively) was discovered to be higher than the % inhibition of a-tocoph-
erol (15.4% and 12.4%).
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1. Introduction and benefits of the complementary systems. The medicinal ef-

fect of plants results from the active constituents or secondary
Increasing interest in herbal medicine is as a result of appre- metabolites such as alkaloids, flavonoids, mucilage, bitters,
hension concerning the toxicity and safety of modern drugs glycosides, essential oils and terpenoids (Burkill, 1985; Trease
and Evans, 1987; Newmann et al., 2000). Essential oil is any
* Tel.: +234 803 562 2238. concentrated hydrophobe containing volatile aroma com-
E-mail address: oloyedegk@gmail.com. pounds from plants. Studies have revealed that essential oils
Peer review under responsibility of King Saud University. serve as powerful antioxidants that produce adverse environ-
ment for damaging free radicals thus preventing mutations
and oxidants in cells. They therefore function as scavengers
for free radicals (Hanasaki et al., 1994; Potterat, 1997). Oxida-
Production and hosting by Elsevier tion reactions can produce excess free radicals which in turn

http://dx.doi.org/10.1016/j.arabjc.2011.09.019
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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Toxicity, antimicrobial and antioxidant activities of methyl salicylate dominated essential oils S841

start chain reactions that damage cells. Antioxidants terminate slopes and kept in a refrigerator at 4 C. Hundred millilitres
these chain reactions by removing free radical intermediates of aliquots of nutrient broth were inoculated with the culture
and inhibit other oxidation reactions. They do this by being of test micro-organisms using a loop and then incubated at
oxidized themselves, so antioxidants are often reducing agents 37 C for 24 h.
such as thiols, ascorbic acid or polyphenols (Gordon, 1996;
Namiki, 1990; Mensor et al., 2001; Jovanovic et al., 1994). 2.1.2.2. Reference standards. Gentamicin at 5 mg/ml for bacte-
Laportea aestuans, of the family Urticaceae is an herbaceous ria and tioconazole (70%) for fungi (both for antimicrobial
plant which appears as weed in new cultivations and fallows in activity); ascorbic acid, butylated hydroxyanisole (BHA) and
West India, Africa and Asia. It is used traditionally as antimi- a-tocopherol for antioxidant activity.
crobial, anti-inflammatory, abortifacient, febrifuge, laxative,
pain-killer, in pulmonary and stomach troubles amongst others 2.1.3. Major equipments used
(Boye and Ampofo, 1990; Nadine, 2004; Sofowora, 2008). UV–visible spectrophotometer (Unico1200 & Perkin Elmer
The aim of this research work is to determine the chemical lambda 25 models), GC–mass spectrophotometer (Agilent
composition of the essential oils of L. aestuans by carrying out Technologies), hydro distiller – Clavenger apparatus.
gas chromatography and gas chromatography–mass spectrom-
etry analysis, to determine the toxic level of the essential oils 2.2. Method
using Brine shrimp lethality test and subject the oil to antimi-
crobial and antioxidant screening. Brine shrimp lethality test is 2.2.1. Isolation of essential oils
a bench top bioassay for the discovery and purification of The oil was obtained by hydro-distillation on a Clavenger type
bioactive natural products and they are an excellent choice apparatus for 4 h in accordance with the British pharmaco-
for elementary toxicity investigations of consumer products poeia specifications (1980). The essential oil was collected
(Dvorack et al., 1999; Meyer et al., 1982; Keddy et al., and stored at 4 C until analysis. The oil yield was calculated
1995). The antimicrobial effect is carried out in others to esti- relative to the dry matter.
mate the potency of the plant on certain micro-organisms as
the plant is claimed to be an effective antimicrobial agent in 2.2.2. Analysis of the essential oils
traditional medicine practice. The activity is compared with 2.2.2.1. Gas chromatography. GC–MS analysis of the essential
antimicrobial standards, gentamicin and tioconazole. The anti- oil was carried out on an Agilent Technologies 7890A GC sys-
oxidant activity of L. aestuans oil was determined by in vitro tem coupled to a 5975C VLMSD mass spectrometer with an
assessment on DPPH (2,2-diphenyl-1-picrylhydrazyl) radical. injector 7683B series device. An Agilent (9091)-413:325 C
The main characteristic of an antioxidant is its ability to trap HP-5 column (30 m · 320 lm · 0.25 lm) was used with helium
excess free radicals which can initiate degenerative disease as the carrier gas at a flow rate of 3.3245 ml/min. The GC oven
(Bors and Saran, 1991; Arouma, 1996; Koppenol, 1993). The temperature was initially programmed at 50 C (hold for
activity is compared with known antioxidant standards ascor- 1 min) and finally at 300 C (hold for 5 min) at a rate of
bic acid, butylated hydroxylanisole and a-tocopherol. 80 C/min while the trial temperature was 37.25 C. The col-
umn heater was set at 250 C and was at split less mode while
2. Materials and methods the pressure was 10.153 psi with an average velocity of
66.45 cm/s and a hold-up time of 0.75245 min was recorded.
2.1. Materials Mass spectrometry was run in the electron impact mode (EI)
at 70 eV. The percentage compositions were obtained from
2.1.1. Plant materials electronic integration measurements using flame ionization
Fresh whole plant samples of L. aestuans were collected in detector (FID), set at 250 C. The peak numbers and relative
June 2010 at the Botanical Gardens, University of Ibadan. percentages of the characterized components are given in
Specimens were identified by Mr. E. Donatus at the Botany Table 1.
and Microbiology Department, University of Ibadan, Oyo
State, Nigeria. The volatile oil was immediately collected from 2.2.2.2. Gas chromatography–mass spectrometry. The essential
the fresh plant material by hydro-distillation using an all-glass oils were analysed by GC–MS on an Agilent Technologies
scavenger apparatus. 7890A GC system coupled to a 5975C VLMSD mass spectrom-
eter with an injector 7683B series device. An Agilent (9091)-
2.1.2. Reagents 413:325 C HP-5 column (30 m · 320 lm · 0.25 lm) was used
Hexane and methanol (BDH chemicals), butylated hydroxyan- with helium as the carrier gas at a flow rate of 3.3245 ml/min.
isole (BHA), a-tocopherol and 2,2-diphenyl-1-picrylhydrazyl GC oven temperature and conditions were as described above.
radical (DPPH) were obtained from Sigma Chemical Co. The injector temperature was at 250 C. Mass spectra were re-
(Germany). corded at 70 eV. Mass range was from m/z 30 to 500.

2.1.2.1. Test organisms. Escherichia coli, Staphylococcus aureus, 2.2.2.3. Identification of components. The individual constitu-
Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumo- ents of the oil were identified on the basis of their retention
niae, Salmonella typhi, Candida albicans, Rhizopus stolon, indices determined with a reference to a homologous series
Aspergillus niger and Penicillium nonatum (micro-organisms of n-alkanes and by comparison of their mass spectral
were collected from the stock of the Department of Pharmaceu- fragmentation patterns (NIST 08.L database/chemstation data
tical Microbiology, Faculty of Pharmacy of University of Iba- system) with data previously reported in literature (Adams,
dan). The test organisms were maintained on nutrient agar 1989; Joulain and Konig, 1998; Mclafferty and Stauffer, 1989).
S842 G.K. Oloyede

Table 1 Chemical composition of essential oil of L. aestuans from GC–MS analysis.

S/N RT (min) Chemical composition T.P. (%)
1 9.261 N-2-propynyl-2-Propyn-1amine 1.17
2 10.159 P-cymenene 1.27
3 10.554 Linalool 3.26
4 11.201 Fenchol 10.59
5 11.555 Trans-Rose oxide 2.65
6 11.796 2,4,6-Cycloheptatriene-1-one 2.53
7 13.198 Lavandulol 0.41
8 13.329 1,2-Cyclohexanedione dioxime 9.40
9 13.449 (E,E)-1,5-Cyclododecadiene 1.23
10 14.273 Methyl salicylate 54.50
11 14.451 Myrtenol 2.66
12 16.608 (E)-2-Octen-1-ol 0.88
13 14.748 1,4-Octadiene 8.86
14 15.678 p-Benzoquinone 0.59
Total 100.00
RT = Retention time, T.P. = Total percentage.

2.2.3. Brine shrimp lethality test bial testing. Inoculation of the test organisms on nutrient agar-
The toxicity level of the extracts was conducted according to prepared plates was achieved by flaming a wire loop on a spirit
Falope et al. (1993) and Oloyede et al. (2010). The brine shrimp lamp, cooling the wire loop (air cooling) and fetching the test
lethality test (BST) was used to predict the presence, in the oils, organisms. The discs were prepared using a Grade No. 1
of toxicity activity (Meyer et al., 1982). The shrimp’s eggs were Whatman filter paper. One hundred discs were obtained by
hatched in sea water for 48 h at room temperature. The nauplii punching and putting in vial bottles and sterilizing in an oven
(harvested shrimps) were attracted to one side of the vials with at 150 C for 15 min. Thereafter the cups (9 mm diameter)
a light source. Solutions of the extracts were made in DMSO, were aseptically bored into the solid nutrient agar using a ster-
at varying concentrations (10,000, 1000 and 100 ppm) and ile cork-borer. A sterile cork-borer was used to create wells (or
incubated in triplicate vials with the brine shrimp larvae. Ten holes) inside the set plates. The test solutions of oils (50 ll) at a
brine shrimp larvae were placed in each of the triplicate vials. concentration of 40 g/ml were then introduced into each of the
Control brine shrimp larvae were placed in a mixture of sea designated cups on each plate ensuring that no spillage oc-
water and DMSO only. After 24 h the vials were examined curred. The same amount of the standard antimicrobial agent
against a lighted background and the average number of larvae and solvents was introduced using syringes into the remaining
that survived in each vial was determined. The concentration cups on each plate to act as positive and negative controls,
for killing fifty percent of the larvae (LC50) was determined respectively. The plates were left at room temperature for
using the Finney computer programme. 2 h, allowed to diffuse into the medium, turned upside-down
and thereafter incubated at 37 C for 24 h in an incubator.
2.2.4. Antimicrobial analysis Clear zones of inhibition were observed. Activity or
Antimicrobial activities of the essential oils of L. aestuans were inactivity of the oil was tested in triplicate and the diameters
carried out using the agar well diffusion method. The 0.2 ml of of zones of inhibition were measured in millimetre (mm) using
an overnight broth culture of test micro-organisms was added a transparent well-calibrated ruler. The positive control for
to 20 ml of cooled molten agar. bacteria is gentamicin at the concentration of 5 mg/ml (Rojas
et al., 2003; Cushine and Lamb, 2005; Duraipandiyan et al.,
2.2.5. Preparation of essential oil sample for antimicrobial 2006).
analysis
A stock solution of the oil sample (100%) was prepared in hex-
ane, 1 ml was taken to prepare 50% of the sample’s concentra- 2.2.5.2. Agar diffusion: pour plate method for fungi. Molten
tion and this was done until 3.125% concentration was sterile Sabouraud dextrose agar (SDA) was poured aseptically
obtained. into the sterile plates and allowed to cool down for 45 min. The
0.2 ml of 1:100 dilutions of the organisms C. albicans,
2.2.5.1. Agar diffusion: pour plate method for bacteria. An over- R. stolon, A. niger and P. nonatum was spread on the surface
night culture of each organism S. aureus, E. coli, B. subtilis, P. using a sterile spreader. Then, a sterile cork-borer was used
aeruginosa, S. typhi, and K. pneumoniae was prepared. The to create wells inside the plates. The same procedure described
0.1 ml of each of the organism was taken into 9.9 ml of sterile for the above anti-bacterial activity was followed from this
distilled water (SDW) to give 10 ml of 1:100 (102) dilution. The stage. The positive control for the fungi is 70% tioconazole.
0.2 ml was taken into the prepared molten nutrient agar (NA) All the plates for the fungi were incubated at 28 C for 48 h
at 45 C and this was aseptically poured into the sterile plates unlike bacteria that was incubated at 37 C for 24 h. The clear
and allowed to set on the bench for about 45 min. The stock zones of inhibition were observed and recorded using the same
was maintained on nutrient agar slant and sub-cultured in method as described in the case of bacteria (Hadecek and
nutrient broth for incubation at 37 C prior to each antimicro- Greger, 2000; Bayer et al., 1986).
Toxicity, antimicrobial and antioxidant activities of methyl salicylate dominated essential oils S843

the total essential oils were identified in the plant. The result
Table 2 Brine shrimp lethality test of L. aestuans essential
of the analysis is presented in Table 1.
oilsa.
The following compounds N-2-propynyl-2-propyn-1amine,
1000 Concentration 100 Control P-cymenene, linalool, fenchol, trans-rose oxide, 2,4,6-cyclo-
ppm 1000 ppm ppm heptatriene-1-one, lavandulol, 1,2-cyclohexanedionedioxime,
Survivor 0 0 0 30 (E,E)-1,5-cyclododecadiene, methyl salicylate, myrtenol, (E)-
Dead 30 30 28 0 2-octen-1-ol, 1,4-octadiene, and p-benzoquinone, were ob-
LC50 (lg/ml) 367.1805 tained in order of the retention time in the hydro distilled oil.
a
LC50 < 1000 = Toxic, LC50 > 1000 = Not toxic, upper con-
fidence limit 1339.4570, lower confidence limit 21.1602.

2.2.6. Free radical scavenging activity

2.2.6.1. Scavenging effect on DPPH. A 0.5 mM of 2,2-diphe-
nyl-1-picrylhydrazyl radical (DPPH) solution in methanol
was prepared and 3 ml of this solution was mixed with the
oil sample in methanol. The free radical scavenging activity
of the oil at various concentrations 0.1 and 0.2 mg/ml was then The major constituents in the oil were methyl salicylate
determined. The decrease in absorption at 517 nm of DPPH (54.50%), fenchol (10.59%), 1,2-cyclohexanedione dioxime
was measured after 10 min of incubation. The actual decrease (9.40%), 1,4-octadiene (8.86%) and linalool (3.26%). The
in absorption was measured against that of the control and the presence of methyl salicylate in this plant confirms the ethno
percentage inhibition was also calculated. The same experi- medicinal importance of L. aestuans. Methyl salicylate is used
ment was carried out on ascorbic acid, butylatedhydroxylani- in perfumery and as a flavouring material. Fenchol or 1,3,3-tri-
sole (BHA) and a-tocopherol which are known antioxidant methyl-2-norbornanol is a terpene and an isomer of borneol
agents. All tests and analyses were run in triplicates and the and is used also in perfumery.
result obtained was averaged (Koleva et al., 2002; Oloyede
and Farombi, 2010). The activities were determined as a func-
tion of their % inhibition which was calculated using the 3.2. Brine shrimp toxicity test
formula:
The toxicity result of the essential oils of L. aestuans showed
Acontrol  Asample that it is toxic to brine shrimp larvae (Table 2). The essential
%I ¼  100
Acontrol oil with LC50 of 367.1805 lg/ml corroborated the presence in
the oil of medicinally active compounds as it has been observed
3. Results and discussion that medicinally active natural products are at most times toxic
to Artemia silina nauplii (Oloyede et al., 2010; Onocha and Ali,
2010). The high toxicity of the oil can be beneficial in the ther-
3.1. GC–MS analysis of the essential oils
apy of some ailments involving cell or tumour growth.
Essential oils from 300 g of freshly collected whole plant
samples of L. aestuans, obtained by means of hydro-distilla- 3.3. Antimicrobial screening
tion gave 0.60% (w/w). The essential oils, colourless with char-
acteristic smell were analysed by GC and GC/MS systems The essential oils showed promising inhibitory potential espe-
using a polar column. 14 constituents representing 100% of cially against Klebsiella pneumoniae, Salmonella typhi, Candida

Table 3 Antimicrobial screening of the essential oils of L. aestuans.

Concentration Zones of inhibition (mm)
S.a E.coli B.sub Ps.a Kleb Sal. C.a Rhiz A.n Pen
1 12 10 10 10 14 16 14 16 16 12
2 10 – – – 12 14 12 14 14 10
3 – – – – 10 12 10 12 12 –
4 – – – – – – – 10 10 –
5 – – – – – – – – – –
6 – – – – – – – – – –
ve – – – – – – – – – –
+ve 36 34 36 34 36 36 24 24 26 26
Integers 1–6 represent essential oil at various concentrations viz: (1) 200 mg/ml, (2) 100 mg/ml, (3) 50 mg/ml, (4) 25 mg/ml, (5) 12.5 mg/ml, (6)
6.25 mg/ml. ve = negative control (hexane),+ve = positive control {gentamicin at 5 mg/ml for bacteria or tioconazole (70%) for fungi} = no
inhibition, S.a = Staphylococcus aureus, E. coli = Escherichia coli, B. sub = Bacillus subtilis, Ps.a = Pseudomonas aeruginosa, Kleb = Kleb-
siella pneumoniae, Sal. = Salmonella typhi, C.a = Candida albicans, Phiz = Rhizopus stolon, A.n = Aspergillus niger, Pen = Penicillium
nonatum.
S844 G.K. Oloyede

Table 4 Scavenging effect of L. aestuans essential oils on DPPH.

Conc. (mg/ml) Oil sample Ascorbic acid BHA a-Tocopherol
0.1 0.15355 ± 0.07 0.0843 ± 0.010 0.0370 ± 0.006 0.6800 ± 0.029
0.2 0.12975 ± 0.01 0.2893 ± 0.128 0.0460 ± 0.008 0.7040 ± 0.003
Absorbance of essential oil, ascorbic acid, BHA and a-tocopherol at 517 nm.

the oil showed very good activity as a radical scavenger in

the experiment involving 2, 2-diphenyl-1-picrylhydrazyl radi-
cal (DPPH).

Acknowledgement

The author would like to thank Mr. Festus of the Department

of Pharmaceutical Microbiology, University of Ibadan, Nige-
ria, for assisting in carrying out the antimicrobial analysis.
This project is sponsored fully by the author as part of her sole
authorship research activity. No financial/commercial conflict
of interest is therefore associated with this work.
Figure 1 % Inhibition of DPPH free radical scavenging activity
of essential oils of L. aestuans*. *% Inhibition of oil and reference
standards. References
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