1 | P a g e
Research Proposal
Title: Antimicrobial activities of Selected Essential oils from Traditional Medicinal
Plants (TMPs) found in the South Pacific
Principal Investigators: Mr Ravneel Chand1, Assoc. Prof Alison Ung
2 and Prof Shawkat
Ali1
1School of Science and Technology, University of Fiji, Lautoka, Fiji
2School of Mathematics and Physical Sciences, University of Technology Sydney, Broadway NSW 2007,
Australia
Research facilitator: Douglas Pharmaceutical, Nadi (Microbiological Analysis)
Objective:
This study aims to assess the potential antibacterial and antifungal activities of essential oils
from 10-19 traditional medicinal plants commonly used in Fiji and the South Pacific. As part
of this study, the best technique for extraction of essential oils will be determined. The
essential oils will also be analysed using GC-MS analysis to identify compounds and their
relative percentage compositions.
1. Brief Background:
Essential oils are considered as antimicrobial agents that can fight viral, bacteria and fungi
infections (Chand et al., 2016b; Chandra et al., 2017; Cowan, 1999; Hintz et al., 2015;
Pandey & Kumar, 2013). The safest and best methods of eliminating microorganism are
essential oils (Negi, 2012). Essential oils are favoured over synthetic chemicals mainly due to
consumer safety in relation to chemical preservatives (Chand et al., 2016b; Fernández et al.,
2015; Hassoun & Emir Çoban, 2017; Lucera et al., 2011). The concerns associated with
synthetic chemicals involve teratogenic and carcinogenic attributes, microbial resistance and
remaining contaminants (Chand et al., 2016b; Moreira et al., 2005; Raybaudi‐Massilia et al.,
2009).
The active essential oil constituents are often found in the edible herbs and spices. Extensive
toxicity studies of commonly occurring natural products found in essential oils were shown to
have no toxicity or carcinogenic potential.
2 | P a g e
1.1 Essential oils
The effectiveness of essential oils is mainly resulting from presence of different active
chemical compounds such as phenolics, fatty esters, aldehydes and terpenes. These natural
products are secondary metabolites that are produced by plants for their own defences. The
complex mixture of these compounds has a synergistic activity against multiple targets of the
microorganisms. The essential oils are known for their ability to penetrate pathogenic
microorganisms’ cell membranes allowing the active components to work effectively against
the intercellular targets. Essential oils have also been reported to directly interacting with
cytoplasmic membrance of both Gram (+) bacteria and Gram (-) bacteria, resulting in the
leakage of low molecular weight of cytoplasmic constituents and ions, leading to interruption
in cell respiration and enymatic activities (Akthar et al., 2014; Johnston et al., 2003). This
mode of actions are effective in killing the organism, and more importantly, there are fewer
chances of pathogens developing resistance (Akthar et al., 2014; Faleiro, 2011). This
approach would, therefore, may help circumvent the antimicrobial multidrug-resistance and
reduce the toxicity.
1.2 Fijian Traditional Medicinal Plants (TMPs)
Fijian traditional medicinal plants are still widely used by both indigenous Fijians and Indian
Fijians-Indians (Ayurveda) as an alternative to Western-style medicine (Singh, 1986). The
traditional uses of the plants are for herbal remedies or for treatments of various diseases and
ailments (Cambie & Ash, 1994). The medicinal plants are either endemic, indigenous or
introductions. The introduced species were brought to Fiji for their medicinal properties by
the Indians, Chinese and Europeans.
2. Methodology
2.1 Collection and identification of Plant materials
The selection criteria for the plants to be studied will be based on their ethnopharmacology,
chemotaxonomy, those that may contain chemical scaffolds of interest and their availabilities
(Table 1 – Potential Traditional Medicinal Plants to be analysed).
3 | P a g e
Table 1: Potential essential oil containing plants that are commonly used in the South Pacific
for Traditional medicines.
Scientific
Names
Family Common English
name
Plant part used *Traditional Uses in
the South Pacific
(Treatment)
Ocimum
tenuiflorum L.,
Ocimum
basilicum L.,
Ocimum
sanctum L.
Lamiaceae
Holy or sacred basil
Essential oils from leaves Earache, sore, hair lice,
stomach ache, filariasis,
throat, gastric
Ulcer, fevers, nasal
infections, colds and
cough.
Ocimum
basilicum Linn.
var. pilosum
(willd)-Benth
Leaf extracts
Ocimum
tenuiflorum
Adenanthera
pavonina
L.
Mimosaceae Red bead tree Seed Extract Leprosy
Ageratum
conyzoides L.
Asteraceae Goat weed Canopy of plant species
(above ground plant parts)
Sore eyes, Infective
hepatitis, eczyma,
dysentery, headaches,
intestinal worms,
filariasis, vomiting,
epilepsy, dizziness,
Nausea wounds and
cuts.
Crude hexane extract of
aerial parts of A. conyzoides
Crude petroleum ether
extract aerial parts of A.
conyzoides
Aloe vera L.
Agavaceae Aloe, aloe vera Leaf extract Treat wounds and
burns, sun burns,
rashes, x-ray burns and
stomach ache.
Acetone, ethyl
acetate, water, and ethanol
extracts
Leaf extract
Annona
muricata L.
Annonaceae Soursop, custard
apple
Crude ethanoic seed extract Treating stomach
ailments.
Fruit (pericarp) extract
Ethanoic seed extract
Azadirachta
indica A. Juss.
Meliaceae Margosa, neem,
Indian Lilac.
Seed water extract For diabetes, skin
diseases, asthma,
syphilis and used as
insecticide. Neem oil from seeds
Crude ethanol extracts of
leaves
4 | P a g e
Cananga
odorata (Lam.)
Hook. F. &
Thoms.
Annonaceae Ylang-ylang,
Kenanga
Essential oil Extracts from
flowers.
Earaches, toothaches,
headaches, stomach
aches, boils, skin
irritation, coughs and
dizziness.
EOs extracts of leaves.
EOs extracts of leaves.
Capsicum
frutescens L.
Solanaceae Chili pepper, red
pepper, paprika.
Methanol extract of fruits
and leaves
Skin tuberculosis, mild
conjunctivitis and
jaundice, boils and
cough. Powdered fruits
Ethanolic extract of fruit
Carica papaya
L.
Caricacea Papaya, Pawpaw Hexanic, acetonic and
methanolic
extracts of seed
Sores, high blood
pressure and treat
diarrhoea.
Leaf extract
Chloroform seed extract
Cassia alata L
(Senna alata)
Fabaceae
(Caesalpiniaceae)
Ringworm bush,
roman candle tree
Ethanoic extracts of
leaves
Skin diarrhoea, worms,
purifies blood and
scabies. Solvent extract of fruits
Leaf and stem extract
Centella
asiatica (L.)
Urban
Apiaceae Indian pennywort,
Asiatic pennywort
Leaves extract Dysentery, fever,
headache, diarrhoea,
pimples, rashes, itchy
lumps, Fractures,
migraines and boils.
Leave extracts
Leaf extract (hexane,
diethyl ether,
dichloromethane, and
methanol)
Citrus
aurantium
L
Rutaceae Seville or sour
orang
Fruit extracts Headache, abdominal
pain and urinary tract
infections. Leaf extracts
Shoot extracts
Citrus sinensis
(L.) Osbeck
Rutaceae orange, sweet
orange
Essential oils from fruits Sickness, abdominal
pains and remedies for
internal ailments. Peels from fresh oranges.
Essential oils from the seeds
Curcuma longa
L.
Zingiberaceae Turmeric Leaf essential oils Painful skin, sores and
rashes in infant, sprains,
bruises, eye diseases
and open wounds,
Colds and runny nose,
dysentery and infected
puncture wounds.
Turmeric rhizome oils
Leaves
Rhizomes
Erythrina
variegata
L.
Fabaceae Coral tree Ethanoic extracts from root
and bark
Filariasis, stomach ache
and fever.
Leaf extract using solvents
5 | P a g e
Methanoic leaf extracts
Momordica
charantia L.
Cucurbitaceae Bitter gourd;
balsam pear;
balsam apple
Leaf extracts Hypertension, stomach
worms, malignant
ulcers, hypertension,
diabetes, Leprosy and
dysentery. Acetone, n-hexane, and
methanol extract of leaves
Methanoic fruit extracts
Passiflora
foetida (L.) var.
hispida (DC.)
Killip
Passifloraceae Wild passion fruit Leaves and the stem Improve fertility in
women.
Psilotum
nudum (L.) P.
Beauv.
Psilotaceae Psilotum Aerial extract Pain relief and remedy
for thrush and the spore.
Vitex trifolia L.
Verbenaceae Vitex Leaf extracts Stomach pains and
mouth infections. Hexanic and
dichloromethanic (DCM)
extracts of leaves and stems
Leaves and stem bark
extracts * reference to (World Health Organization, 1998)
Please note the plants that are listed above may be substituted with other plants depending
on the availability.
The plant samples will be collected from the local communities around Viti Levu, Fiji
islands. In addition, this research will be carried out in consultation with University of the
South Pacific (USP) Herbarium to ensure correct protocol of Botanic identification of plant
materials are used in comparison to Herbarium voucher number identity.
2.2. Extraction of essential oils from the Traditional Medicinal plants
2.2.1 Solvent extraction and steam distillation
Organic solvent extraction method is mostly used in industrial process to obtain very pure
essential oils. The technique uses organic solvents to extract out organic compounds.
Organic solvent is removed under reduced pressure to yield crude extracts which often
require further washing with presence of alcohol to obtain pure essential oils. The use of this
technique is very costly, highly flammable and has detrimental effect on the environment and
hence it is not preferred (Chaichana, 2009).
The common and simplest method is steam distillation. However, this method requires a high
temperature of heating which can lead to decomposition and low yielding of the essential
oils.
6 | P a g e
Current method that meets our criteria for this proposal is hydrodistillation (HD). This
method is feasible regarding logistics and infrastructure at SOST.
2.2.2 Hydrodistillation (HD)
The Clevenger apparatus (Figure 1) will be used for extraction of essential oils (mostly
leaves) from Traditional Medicinal Plants as similarly reported by Chand et al. (2016a).
2.3 Essential oils Analysis
Gas Chromatography-Mass Spectrometry (GC-MS) is used for analysing and identifying
individual components of essential oils. Chemical analysis of essential oils are qualitatively
and quantitatively analysed using Gas Chromatography-Mass Spectrometry (GC-MS)
technique. Identification of the principal components are usually carried by the comparison of
Gas Chromatography retention times with Mass Spectrometry data of reference standards.
Chemical structures of active constituents will be determined using 1D & 2D NMR Infrared
spectroscopy (ATR-FTIR, Agilent Cary 630), and High-resolution mass spectrometry
techniques. The samples will be run using an Agilent 500MHz NMR spectrometry for 1H and
13C NMR, with 2D NMR. High-resolution mass spectra will be obtained using Agilent 6510
7 | P a g e
Q-TOF Mass Spectrometer fitted with an electron ionisation source (ESI). The analyses will
be carried out in collaboration with the University of Technology Sydney.
2.4 Antimicrobial Activities of Essential oils
Broad Biological screening of the extracted essential oils for antibacterial and antifungal
activities will be conducted in the standard microbiology laboratory at Douglas
Pharmaceutical, Nadi, Fiji.
2.4.1 Sample preparation
The essential oils are poorly soluble in water and this can lead to difficulty in determining the
MIC and biological activity of the oils. DMSO is the common solvent used in sample
preparation; however, the oils could still result in a bad solubility. Emulsifier detergents such
as Tween 80, Tween 20 and Triton X100 and noble agar will be used to encourage the
solubility as well as the stability of the oils.
2.4.2 Antibacterial activity screening
The different extracts of essential oils are screened for antimicrobial activities using agar cup
diffision method (Deans & Ritchie, 1987) with a suitable solvent (DSMO, hexane or ethanol)
(Del-Vechio-Vieira et al., 2009; Mimica-Dukic et al., 2004; Okoh et al., 2010). All analyses
are carried out in triplicate. A standard antibiotic such as gentamicin will be used as a control
for respective bacterial species (Okoh et al., 2010).
2.4.2.1 Screening of MIC (Minimum Inhibitory Concentration) and MBC (Minimum Inhibitory
Concentration) activities
Determination of the essential oils bactericidal nature will first be evaluated by the
compounds MIC. From the MIC values, the minimum bactericidal concentrations (MBC) can
be determined to identify the concentration of essential oils that killed the pathogen
completely, indicative of bactericidal properties. To ensure the comparable, reproducible and
standardisation of the results, the uniform procedures outlined in the Clinical and Laboratory
Standards [CLSI (Matthew et al. 20060)] will be used. Determining of the MIC of the
essential oil constitution will be done using a standard colorimetric broth micro titre
technique. Measuring the absorbance with micro plate reader will determine the percentage
inhibition of bacterial growth. This same process will be done in triplicate for each
concentration. A standard antibiotic will be used as a control for different bacterial species.
The MBC is an essential piece of information when testing for a potential antibiotic. The
MIC above indicates whether the concentration of the compound used is capable of only
inhibiting cell growth (a reversible process). The MBC, however, will determine whether the
compound kill the pathogen completely, indicative of bactericidal properties.
2.5 Plans for Data Processing and Analysis
8 | P a g e
The microbiological data will be statistically analysed using SPSS software to compare the
mean difference of activities recorded for different essential oils and microorganisms. The
type of analysis will depend on the recorded data. Professor Shawkat Ali will provide full
guidance with the usage of different data analysis for the recorded data. Besides, for chemical
constituents - the GC-MS instrument will provide relevant data.
3. Project plan
Task Starting Date Duration (days) Ending Date
Literature Review 2nd
Dec 2017 7 months 2nd
Aug 2018
Sample collection
and Extraction
Jan 3 2018 1 month 3 Feb 2018
Microbiological
Studies
Availability/dependency of external parties (approx – 3 months)
Analysis of essential
oils – GC-MS
Up to 7 months
Gantt chart
Budget
Allocation Line Items Allocated Budget (FJ$)
Plant samples
Plant sample collection -
Traditional Medicinal Plants
Purchase/collection of samples
from different sites around the
Viti Levu
800.00
Chemical Analysis
Chemical Analysis - Analysis to be carried out at
2/12/17 21/1/18 12/3/18 1/5/18 20/6/18 9/8/18 28/9/18 17/11/18
Literature Review
Sample collection and extraction
Microbiological studies
Analysis of essential oils - GC-MS
Length (days)
9 | P a g e
Chromatography Analysis UTS, Sydney 5,800.00
Microbiological Analysis -
antibacterial and antifungal
activities
3,500.00
Chemicals
Tween 20
100.00
Anhydrous sodium sulphate
80.00
Motor Mileage ($0.50 x 600km) For visiting sites and collection
of samples from west via Bus
and taxi.
200.00
Total contingency
750.00
Total
11,230.00
Justification of Budget
The proposed budget for this research study is approximately 11, 230 FJ dollars. Almost half
of budget money (5800 FJD) will be used to carry out chemical analysis at the University of
Technology Sydney. None of the laboratories in Fiji have access to the instrument - Gas-
Chromatography combined with Mass Spectrometry and only for this reason the samples will
be sent abroad for analysis. Likewise, the antimicrobial test analysis will be carried out at the
Douglas Pharmaceuticals in Nadi due to lack of proper facilities at the University of Fiji. The
cost of the analysis is expected to be 3,500 FJ dollars. The team has also proposed to have
contingency amount of 750 FJ dollars in their budget for any unforeseen circumstances.
Please note: The amount proposed in the budget is just an estimate, and the team is very
much sure that it will not exceed the proposed amount.
Research Team Bio-data
1. Mr Ravneel Rajneel Chand is an Assistant Lecturer in Biology at the University of Fiji.
Prior to joining Unifiji, he has worked as a Researcher for the Faculty of Science, technology
and Environment at the University of the South Pacific. He has a Master of Science degree in
Biology from the University of the South Pacific. He also has few ranked publications with
reference to the Australian Research Council (ARC) in Toxicological, repellent and
antimicrobial potential of Traditional Medicinal Plants (TMPs). Recently, he was awarded
with Excellency in Research and Publications at University of the South Pacific during the 6th
Research Excellence and Innovation Award Evening held on 10th
November, 2017. His
10 | P a g e
current research focuses on the biological control of termites and its impact in the South
Pacific.
2. Associate Professor Alison Ung is an accomplished medicinal chemist, organic synthetic
and natural products chemist. She has been actively working in the areas of drug design and
synthesis for more than 20 years. For 14 years, she has worked for Johnson & Johnson
Research Pharmaceutical Research Laboratory (JJRPRL), housed in University of
Wollongong (UOW). As a Research Fellow (1994-1996), she was instrumental in developing
immune modulators that could arrest diabetes in nude mice and suppress the immune system
in allograph transplantation in the animal. This breakthrough led to the filing of an
international patent (WO 9746543 A1 19971211). She has played a crucial role in driving the
research to produce a large number of bioactive molecules derived from natural alkaloids.
The success of these works led to the expanding of research at JJRPRL in 2003 with an
increase in research funding ($1.7 M per year for five years).
Reference
Akthar, M. S., Degaga, B., & Azam, T. (2014). Antimicrobial activity of essential oils
extracted from medicinal plants against the pathogenic microorganisms: a review.
Issues in Biological Sciences and Pharmaceutical Research, 2, 1-7.
Cambie, R. C., & Ash, J. (1994). Fijian Medicinal Plants. East Melbourne, Victoria,
Australia: CSIRO Publications.
Chaichana, J. (2009). Chemical constituents and biological activities of Thai aromatic plants.
(Master of Science in Pharmaceutical Sciences), Chiang Mai: Graduate School,
Chiang Mai University, 2009, Thailand
11 | P a g e
Chand, R. R., Jokhan, A. D., & Gopalan, R. D. (2016a). Bioactivity of selected essential oil
from medicinal plants found in Fiji against the Spiralling whiteflies. Advances in
Horticultural Science, 30, 165-174.
Chand, R. R., Jokhan, A. D., & Gopalan, R. D. (2016b). Whitefly control and anti-
microbiological activities of essential oils from medicinal plants found in Fiji Islands.
. (Master of Science), University of the South Pacific, Suva, Fiji. Retrieved from
http://digilib.library.usp.ac.fj/gsdl/collect/usplibr1/index/assoc/HASH01fd/f6510e8d.d
ir/doc.pdf
Chandra, H., Bishnoi, P., Yadav, A., Patni, B., Mishra, A. P., & Nautiyal, A. R. (2017).
Antimicrobial Resistance and the Alternative Resources with Special Emphasis on
Plant-Based Antimicrobials—A Review. Plants, 6, 16.
Cowan, M. M. (1999). Plant Products as Antimicrobial Agents. Clinical Microbiology
Reviews, 12, 564-582.
Deans, S. G., & Ritchie, G. (1987). Antibacterial properties of plant essential oils.
International Journal of Food Microbiology, 5, 165-180.
Del-Vechio-Vieira, G., Sousa, O. V., Yamamoto, C. H., & Kaplan, M. A. (2009). Chemical
composition and antimicrobial activity of the essential oils of Ageratum fastigiatum
(Asteraceae). Records of Natural Products, 3, 52.
Faleiro, L. (2011). The mode of antibacterial action of essential oils. Science Against
Microbial Pathogens: Communicating Current Research And Technological
Advances, 2, 1143-1156.
Fernández, M., Hudson, J., Korpela, R., & de Los Reyes-Gavilán, C. (2015). Impact on
Human Health of Microorganisms Present in Fermented Dairy Products: An
Overview. BioMed Research International, 2015, 1-13.
Hassoun, A., & Emir Çoban, Ö. (2017). Essential oils for antimicrobial and antioxidant
applications in fish and other seafood products. Trends in Food Science &
Technology, 68, 26-36.
Hintz, T., Matthews, K. K., & Di, R. (2015). The Use of Plant Antimicrobial Compounds for
Food Preservation. BioMed Research International, 2015, 1-12.
Johnston, M. D., Hanlon, G. W., Denyer, S. P., & Lambert, R. J. W. (2003). Membrane
damage to bacteria caused by single and combined biocides. Journal of Applied
Microbiology, 94, 1015-1023.
Lucera, A., Costa, C., Conte, A., & Del Nobile, M. (2011). Food applications of natural
antimicrobial compounds. Frontiers in Microbiology, 3, 287-287.
Mimica-Dukic, N., Bozin, B., Sokovic, M., & Simin, N. (2004). Antimicrobial and
antioxidant activities of Melissa officinalis L.(Lamiaceae) essential oil. Journal of
agricultural and food chemistry, 52, 2485-2489.
12 | P a g e
Moreira, M. R., Ponce, A. G., del Valle, C. E., & Roura, S. I. (2005). Inhibitory parameters of
essential oils to reduce a foodborne pathogen. Lebensmittel-Wissenschaft &
Technologie - Food Science and Technology, 38, 565-570.
Negi, P. S. (2012). Plant extracts for the control of bacterial growth: Efficacy, stability and
safety issues for food application. International Journal of Food Microbiology, 156,
7-17.
Okoh, O. O., Sadimenko, A. P., & Afolayan, A. J. (2010). Comparative evaluation of the
antibacterial activities of the essential oils of Rosmarinus officinalis L. obtained by
hydrodistillation and solvent free microwave extraction methods. Food Chemistry,
120, 308-312.
Pandey, A., & Kumar, S. (2013). Perspective on plant products as antimicrobial agents: a
review. Pharmacologia, 4, 469-480.
Raybaudi‐Massilia, R. M., Mosqueda‐Melgar, J., Soliva‐Fortuny, R., & Martín‐Belloso, O.
(2009). Control of Pathogenic and Spoilage Microorganisms in Fresh‐cut Fruits and
Fruit Juices by Traditional and Alternative Natural Antimicrobials. Comprehensive
Reviews In Food Science And Food Safety, 8, 157-180.
Singh, Y. N. (1986). Traditional medicine in Fiji: some herbal folk cures used by Fiji Indians.
Journal of Ethnopharmacology, 15, 57-88.
World Health Organization. (1998). Medicinal plants in the South Pacific. Western Pacific
Series, 19, 1-151.