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EVALUATION OF ANTIBACTERIAL, ANTIFUNGAL AND ANTIOXIDANT POTENTIAL OF ESSENTIAL OIL FROM
AMYRIS BALSAMIFERA AGAINST MULTI DRUG RESISTANT CLINICAL ISOLATES
PRAVEEN DAHIYA1*, AKSHI MANGLIK1
Amity Institute of Biotechnology, Amity University- Noida, Uttar Pradesh, India
Received: 30 July 2013, Revised and Accepted: 24 August 2013
Objective: To investigate the phytochemical constituents, TLC bioauto graphy and antioxidants of Amyris balsamifera e ssential oil. The antimicr obial
potential was also determined against various multi drug resistant clinical isolates.
Methods: Preliminary phytochemical analysis was performed. The antimicrobial potential of essential oil from Amyris was evaluated by agar well
diffusion method against multi drug resistant clinical isolates. T he antibacterial effect was investigated using the TLC -bioautographic method. The
antioxidants analyzed include catalase, peroxidase, superoxide dismutase, glutathione-S-transferase and glutathione reductase.
Results: Phytoconstituents analysis demonstrated the presence of few phytochemicals present including saponins, terpenoids and phlobatanins.
Amyris balsamifera essential oil was further investigated for its antimicrobial activity against twelve Multi drug resistant pathogenic bacteri a and
three fungi respectively. The oil showed broad antimicrobial activity against MDR Gram-positive bacteria and Gram-negative bacteria and fungal
isolates such as Staphylococcus aureus, Salmonella paratyphi, Escherichia coli, Klebsiella pneumoniae and Candida albicans. The highest in vitro
inhibitory activity was observed for Klebsiella pneu moniae with wide inhibition zone diameters (20±0.11 mm) followed by Staphylococcus aureus
(18±0.15) mm. Among fungal isolates, the growth of only Candida albicans was inhibited. Thin layer chromatogra phy bioautography assay
demonstrated one large growth inhibition zone observed at Rf values of 0.63 against Klebsiella pneumoniae and Staphylococcus aureus 1. Amyris
balsamifera essential oil was found to be rich in antioxidants such as superoxide dismutase, glutathione -S-transferase and glutathione reductase.
Conclusions: It can be concluded that, Amyris essential oil with good antimicrobial activity against several multi drug resistant clinical isolates and
possessing antioxidant activity, thus can be used in the treatment of various microbial infections.
Keywords: Antibacterial activity; Antifungal activity; Antioxidant potential; Amyris balsamifera; TLC bioautography
The spread of antibiotic-resistant strains of bacteria is one of the
most serious threats to successful treatment of microbial diseases as
it may render the current antimicrobial agents insuffici ent to control
the diseases. The continuous emergence of multi drug resistant
organisms poses serious threat to the treatment of infectious
diseases. Down the ages essential oils and other extracts of plants
have evoked interest as sources of natural products. They have been
screened for their potential uses as alternative remedies for the
treatment of many infectious diseases . World Health
Organization (WHO) noted that majority of the world's population
depends on traditional Medicine for primary healthcare. Various
infectious diseases have been known to be treated with herbal
remedies for the betterment of mankind throughout the globe. Thus,
scientists are increasingly turning their attention to natural
products, either as pure compounds or as standardized plant
extracts, looking for new leads to develop better drugs against
microbial infections .
Essential oils (also called volatile oils) are the concentrated,
hydrophobic liquids containing volatile aromatic compounds from
plants. They are naturally synthesized by plants for different reasons
according to t heir needs. They are called “essential” - because they
carry the very essence, and undoubtedly the most important part of
the plant. They can be obtained by expression, fermentation or
extraction but the method of steam distillation is most commonly
used for commercial production. Essential oils are a rich source of
biologically active compounds . Their potential antimicrobial
traits are due to compounds synthesized in the secondary
metabolism of plants.
Amyris balsamifera known as torchwood belongs to the family
Rutaceae. A. balsamifera is a small evergreen tree grows in the
Caribbean area and along the Gulf of Mexico. It is also found in
tropical Asia especially India, Sri Lanka, Malaysia, Indonesia and
Taiwan. Amyris oil or West Indian sandal wood oil is obtained by
steam distillation of the wood from this tree which is pleasantly
woody with a balsamic to uch. It is much cheaper than sandalwood
oil and is used as a fixative in perfumes. Amyris oil is rich in
sesquiterpene alcohols, six sesquiterpenes were isolated a nd
identified to be 10-epi-γ-eudesmol, α-agarofuran, 4-
hydroxydihydroagarofuran, valerianol, β-eudesmol and elemol .
Sandalwood oil has been used to treat skin eruptions and
inflammatory diseases in India for centuries. Amyris oil has
historically been associated wi th antiseptics, wound cleaners,
childbirth recovery, diarrhea and influenza. It is also reportedly used
in Chinese medicine for the relief of stomach ache, vomiting and
gonorrhoea. In Europe it was also used for the treatment of pains,
fevers and ‘strengthening the heart’ .
The present study aimed at evaluating the phytochemical screening,
in vitro antimicrobial activity and antioxidant potentials of Amyris
balsamifera essential oil against multi drug resistant clinical isolates.
MATERIALS AND METHODS
Acquisition of Amyris balsamifera essential oil
Commercial brands of Amyris balsamifera oil (Amyris) was
purchased from Delhi, India. As per manufacturer’s information, it
was prepared by steam distillation. The oil was further distilled by
rotary evaporator. The essential oil was dissolved in methanol (0.3
ml oil/ 2 ml methanol). The oil was transferred into sterile vials and
stored at 4oC till further analysis.
Microbial cultures and Growth conditions
The microbial cultures included multi-drug resistant isolates of
Enterobacter sp, Salmonella paratyphi, Salmonella typhi,
Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa and Acinetobacter sp. The cultures of
bacteria were maintained in their appropriate agar slants at 4°C
throughout the study and sub-cultured on to nutrient broth for 24 h
prior to testing. Three fungal isolates studied includes Candida
albicans, Aspergillus niger and Rhizopus nigricans. The cultures were
Vol 6, Suppl 5, 2013 ISSN - 097 4-2441
Dahiya et al.
Asian J Pharm Clin Res, Vol 6 Suppl 5, 2013, 57-60
maintained on potato dextrose agar at 4°C. These microbial isolates
served as test pathogens for antimicrobial activity assay.
The oil dissolved in methanol (0.3 ml oil/ 2 ml methanol) was
subjected to phytochemical screening for the presence of saponins,
tannins, steroids, phlobatanins, anthraquinones, cardiac glycosides,
alkaloids, reducing sugars and flavonoids by using wet reactions [6-
Antimicrobial activity assay
The agar well diffusion method was employed with slight
modifications to determine the antibacterial activities of Amyris oil
in methanol . About 25 ml of nutrient agar and potato dextrose
agar was poured into each petri plate. Once the agar solidified, the
cultures were inoculated on the surface of the plates (1 × 108
cfu/ml). Subsequently, the surface of the a gar was punched with a 6
mm diameter wells. Each well was filled with 50 μl of oil in
methanol. The concentration of the extracts employed was 20
mg/ml. Control wells containing the same volume of methanol were
made. After 24 h incubation at respective temperatures, the plates
were observed for zones of growth inhibition, and the diameter of
these zones was measured in millimeters. All tests were performed
in triplicate and t he antimicrobial activity was expressed as the
mean of inhibition.
TLC bioautography assay
Amyris essential oil exhibiting significant antimicrobial potential
against Klebsiella pneumoniae and Staphylococcus aureus 1 as
determined by agar well diffusion method (Table 2) was analyzed
using TLC bioautography assay. About 10 μl of oil in methanol was
applied on glass coated silica gel plates. The plates were developed
with toluene and ethyl acetate (93:7 v/v). The TLC plates were run
in duplicate. One of the strips was visualized under UV light to see if
the separated spots were UV active after which it was sprayed with
2% vanillin sulphuric acid reagent, the second strip was used for
bioautography assay. Individual Rf for each spot was measured. TLC
bioautography was carried out using the selected strains of bacteria.
The developed TLC plates were thinly overlaid with molten nutrient
agar inoculated with an overnight culture of bacteria. The plates
were incubated in a dark and humid chamber overnight at 37°C.
Subsequently, the bioautogram was sprayed with an aqueous
solution of 2, 3, 5 triphenyl tetrazolium chloride and further
incubated for at 37°C for 4 h. Microbial growth inhibition appeared
as clear zones against a pink background. The Rf values of the spots
showing inhibition were determined.
Antioxidant activity of essential oil
Amyris oil was evaluated for the presence of various antioxidants
including superoxide dismutase (SOD), catalase (CAT), glutathione-
s- transferase (GST), reduced glutathione (GSH) and lipid
peroxidation. Superoxide dismutase activity was measured by the
NBT reduction . Catalase activity was estimated by measuring the
rate of decomposition of hydrogen peroxi de at 240nm . Assay of
glutathione-s- transferase was done according to Habig et al. .
Lipid peroxidation was measured by using the TBA method as per
Ohkawa et al. . Glutathione activity (GSH) was assayed based on
the reaction with DTNB . Data was expressed as mean± standard
Preliminary phytochemical analysis of Amyris essential oil
demonstrated the presence of few phtyochemicals including,
saponins, terpenoids and phlobatanins. Most of the phytochemicals
tested including tannins, glycosides, steroids, anthraquinones and
reducing sugars were not observed in Amyris oil (Table 1).
Table 1: Phytochemical analysis of Amyris balsamifera oil
a) +: Positive, b) −: Negative
Table 2: Antibacterial and antifungal activity of Amyris
essential oil determined by agar well diffusion assay
Zone of Inhibition (in mm)
Escherichia coli 1
Escherichia coli 2
Staphylococcus aureus 1
Staphylococcus aureus 2
Zone of inhibition is expressed as mean± standard deviation, -: no
Amyris balsamifera essential oil was further investigated for its
antimicrobial activity against twelve Multi drug resistant pathogenic
bacteria and three fungi r espectively (Table 2). The oil showed
broad antimicrobial activity against MDR Gram-positive bacteria and
Gram-negative bacteria and fungal isolates such as Staphylococcus
aureus, Salmonella paratyphi, Escherichia coli, Klebsiella pneumoniae
and Candida albicans. The highest in vitro inhibitory activity was
observed for Klebsiella pneumoniae with wide inhibition zone
diameters (20±0.11 mm) followed by S. aureus 1 (18±0.15) mm and
Acinetobacter sp (13±0.22). The oil showed poor antifungal activity
and inhibited the growth of only Candida albicans. No inhibitory
activity was observed against the fungal isolates Aspergillus niger
and Rhizopus nigricans.
TLC Bioautographic assay are usually used to screen the
antimicrobial activity by separating components onto the surface of
chromatographic plates and overlaying the TLC plate with molten
bacterial agar. TLC analysis revealed the presence of saponins in the
essential oil tested ( data not show n). TLC bioautography was
performed for Amyris essential oil against Klebsiella pneumoniae and
Staphylococcus aureus 1 isolates. One large inhibitory zone with Rf
value 0.63 was observed against the growth of isolates Klebsiella
pneumoniae and Staphylococcus aureus 1 on the TLC plates B and C
as white spot on pink b ackground when sprayed with aqueous
solution of 2, 3, 5 triphenyl tetrazolium chloride (Figure 1).
Antioxidants namely SOD, CAT, GSH, GST and lipid peroxidation in
Amyris balsamifera oil were analyzed as re ported in Table 3. Amyris
was found to possess high amount of GSH ( 19.89±0.091µg/mg) and
GST (14.2±0.132µg/mg) followed by SOD (10.2±0.035 U/mg). Small
amount of CAT and lipid peroxidation was observed.
Dahiya et al.
Asian J Pharm Clin Res, Vol 6 Suppl 5, 2013, 57-60
Table 3: Level of enzymatic and non-enzymatic antioxidants in Am yris balsamifera essential oil
Plate A) (Plate B) (Plate C)
Fig. 1: Chromatogram for (Plate A) and Bioautograms (Plates B
and C) for Amyris essential oil against Klebsiella pneumoniae
and Staphylococcus aureus 1
Plate A: arrow indicates spot visualized when sprayed with 2%
vanillin sulphuric acid reagent. Zones of inhibition (Plates B and C)
are observed as clear spots against pink background. Mobile phase:
Toluene/Ethyl acetate (93:7 v/v)
Plant essential oils have immense potential to be used as
antimicrobial compounds. Thus, they can be used in the treatment of
infectious diseases caused by the numerous resistant
microorganisms present. Technically, essential oils are not true oils
as they contain no lipid content. The essential oil is made up of a
variety of complex, volatile compounds so they are a rich source of
biologically active compounds. Therefore, it is reasonable to expect a
variety of plant compounds in these oils with specific as well as
general antimicrobial activity and antibiotic potential . An
estimated 3000 essential oils are known, of which 300 are
commercially important in fragrance market . These are also
used in food preservative, aromatherapy, as muscle relaxant a nd as
stimulant. Essential oils have been shown to possess antibacterial,
antifungal, antiviral insecticidal and antioxidant properties [16-17].
Some oils have been used in cancer treatment . In the present
study, medicinally important Amyris balsamifera essential oils was
screened for the presence of phytoconstituents, antimicrobial
potential, separation and analysis of bioactive compounds by TLC
bioautography and anti oxidant enzyme analysis.
Phytoconstituents anaysis of Amyris oil showed that the oil contains
few of the phytoconstituents including saponins, t erpenoids and
phlobatanins. Beek et al.  reported that the oil consisted of
17.5% sesquiterpene hydrocarbons and 82.5% oxygenated
sesquiterpenes. Terpenoids encompass a diversity of structures and
have many functional roles in nature, including protection against
pest arthropods. Naturally occurring sesquiterpenes contained in
Amyris oils are significantly repellent to a spectrum of arthropod
pests and ticks [20-21].
In vitro antimicrobial activity by agar well diffusion method of
Amyris essential oil was quantitatively assessed on the basis of zone
of inhibition. Amyris oil in methanol exhibited varying degree of
inhibitory effect against the selected multi drug resistant bacterial
and fungal clinical isolates. The results are consistent with the
reports of previous investigators. The antimicrobial activity of
Amyris oil against the yeast Candida albicans, the Gram-positive
bacterium Staphylococcus aureus and the Gram-negative
bacteria Pseudomonas aeruginosa, Escherichia coli and Klebsiella
pneumonia has al so been reported by Jirovetz et al. . Kloucek et
al.  and Setzer et al.  reported antibacterial activity against of
Amyris oil against S. aureus.
TLC was carried out in order to separate the bioactive components
present. TLC is an easy and cost-efficient technique used in the
separation of components of complex mixtures, commonly used for
natural products. The main benefits of this technique include low
cost analysis, high-throughput screening of samples, and minimal
sample preparation . An additional benefit is that the
chromatograms can be screened for antimicrobial activity. The
bioactive components were separated on TLC followed by TLC
bioautography of Amyris essential oil against clinical isolates K.
Pneumoniae and Staphylococcus aureus 1. One large inhibitory zone
with Rf value 0.63 was observed against the growth of both K.
Pneumoniae a nd Staphylococcus aureus 1 on the TLC plates B and C
as white spot on pink b ackground when sprayed with aqueous
solution of 2, 3, 5 triphenyl tetrazolium chloride. It is possible that
the observed inhibition was likely due to one or more active
compounds which overlap possibly due to the solvent system used
for screening. Synergism might play a major role in extracts that
were active when the MIC of the mixture was determined, while the
separated components showed no antimicrobial activity.
Amyris balsamifera oil was analyzed for the presence of various
antioxidants. Amyris was found to possess high level of GST, GSH
followed by SOD and small amount of CAT and lipid peroxidation.
Catalase is regarded as one of the most significant antioxidant
enzyme that can protect plants by scavenging fr ee radicals and H 2O2.
Low level of catalase was observed in Amyris balsamifera whereas
significant amount of SOD was recorded. SOD prevents the
formation of .OH and provides essential defence against the
potential toxicity of oxygen. High level of GST and GSH are observed
in Amyris essential oil. GST offers protection against LPO by the
conjugation of toxic effect with GSH .
The results of the present study support partially the use of the
selected essential oil in traditional medicine notably in the treatment
of microbial infections. A good antimicrobial compound coupled
with high antioxidant activity is a very interesting lead compound to
fight with the present scenario of multidrug resistance. It would
have a dual role to inhibit the growth of MRSA and reduce the
inflammation triggered by this pathogen by its high antioxidant
activity. However further elucidation of the chemical structures of
the biologically active compounds will be required along with
studies in animal model to generate a potent drug.
The authors are thankful to Amity Institute of Biotechnology, Amity
University, Noida, U.P, India for providing infrastructural facilities to
carry out this study.
Conflict of interest statement
We declare that we have no conflict of interest.
Dahiya et al.
Asian J Pharm Clin Res, Vol 6 Suppl 5, 2013, 57-60
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