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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.

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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).

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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

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

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

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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 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.

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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).

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

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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

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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)

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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

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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).

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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

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