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Research Proposal
Objective:
This study aims to evaluate the antimicrobial 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 known for their antimicrobial activities since ancient times, which were not
scientifically proven till the 20th
century (Chand et al., 2016b; Dagli et al., 2015; George et
al., 2014). 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). The main reason for favouring essential oils over synthetic
chemicals is due to consumer concern towards chemical preservatives (Chand et al., 2016b;
Fernández et al., 2015; Hassoun & Emir Çoban, 2017; Lucera et al., 2011). These concerns
mostly involve carcinogenic and teratogenic attributes, residual toxicity and microbial
resistance (Chand et al., 2016b; Moreira et al., 2005; Raybaudi‐Massilia et al., 2009).
The active constituents of the essential oils 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.
1.1 Essential oils
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The effectiveness of essential oils is mainly due to the presence of many different active
natural products such as phenolics, terpenes derivative, fatty esters, aldehydes and other
antimicrobial compounds. 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. Esseential oils
have also been reported to directly interacting with cytoplasmic membrance of both Gram-
negative and Gram-positive bacteria, resulting in the leakage of low molecular weight of
cytoplasmic constituents and ions, leading to the disruption of the 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).
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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
var. CIM AYU
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
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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
diarrhea.
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
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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 alcohol to remove wax and finally resulting in the pure essential
oils. However, this method is not preferred because it is very costly, highly flammable and
harmful to the environment (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.
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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).
Figure 1: Set-up for hydro-distillation.
2.3 Analysis of chemical components of Essential oils
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 GC/MS. Identification of the principal components is
carried out by the comparing of both the GC retention times and MS data against those of the
reference standards or the available reference library of compounds. 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
Condenser
3- Neck round bottom
flask
Clamp stand with the
holder
Collecting tube
Heating base
- H2O in
- H2O out
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NMR.High-resolution mass spectra will be obtained using Agilent 6510 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 test bacteria can be screened for susceptibility to the differently extracted essential oils
using the standard agar cup well diffusion method (Deans & Ritchie, 1987) at a cut-off
screening concentration of 10 mg/mL of the oils in 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 two gram-positive and two gram-negative bacterial species (Okoh et
al., 2010).
2.4.2.1 Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration (MBC)
Determination of the essential oils bactericidal nature will first be evaluated by the
compounds minimum inhibitory concentration (MIC). The MIC results determine the
concentration required of each essential oil (g/mL) to inhibit the growth (95-100%) of the
microorganisms. 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 determine the MIC and MBC.
Determining of the MIC of the essential oil constitution will be done using a standard
colorimetric broth micro titre technique. The % inhibition of bacterial growth is observed by
measuring the absorbance at 595 nm with a micro plate reader. This same process will be
done in triplicate for each concentration. A standard antibiotic will be used as a control for
respective two gram-positive and two gram-negative 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
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inhibiting cell growth (a reversible process). The MBC, however, will determine whether the
compound kill the pathogen completely, indicative of bactericidal properties. The MBC is
estimated as the least concentration of the extract where no visible growth is observed (Okoh
et al., 2010). This same process is tested in triplicate for each concentration.
2.5 Plans for Data Processing and Analysis
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 in the form of a Work Schedule
Task Start Date Duration (days) End 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
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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 -
Chromatography Analysis
Analysis to be carried out at
UTS, Sydney
5,800.00
Microbiological Analysis -
antibacterial and antifungal
activities
3,500.00
Chemicals including Freight charges
Tween 20 or polysorbate 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
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)
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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
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.
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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).
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