An Examination of the Medicinal Potential of Pittosporum phylliraeoides: Toxicity, Antibacterial and Antifungal Activities INTRODUCTION

Abstract

Introduction: Pittosporum phylliraeoides is an endemic Australian plant historically used as a medicinal
agent by indigenous Australians. P. phylliraeoides solvent extracts were tested for antibacterial and antifungal
activities and toxicity in vitro. Results: All extracts displayed antibacterial activity in the disc diffusion assay. The
methanol and hexane extracts demonstrated the broadest specificity, inhibiting the growth of 4 of the 14 bacteria
tested (28.6%). The water, ethyl acetate, and chloroform extracts inhibited the growth of 2 (14.3%), 3 (21.4%), and
3 (21.4%) of the 14 bacteria tested respectively. P. phylliraeoides methanolic extract was also effective as an antifungal
agent, inhibiting the growth of a nystatin resistant strain of Aspergillus niger. It did not affect the growth of Candida
albicans. All extracts were more effective at inhibiting the growth of Gram-negative bacteria than Gram-positive
bacteria. Indeed, only the methanol and hexane extracts were capable of inhibiting the growth of any of the Grampositive
bacteria, inhibiting the growth of only 1 of the 4 (25%) Gram-positive bacteria tested each. All P. phylliraeoides
extracts displayed low toxicity in the Artemia franciscana bioassay. The only significant increase in mortality above
that of the control was seen for the ethyl acetate, chloroform and hexane extracts, although even these extracts
displayed low toxicity, inducing less than 50% mortality at 72 h. Conclusions: The low toxicity of the P. phylliraeoides
extracts and their inhibitory bioactivity against bacteria and fungi validate Australian Aboriginal usage of
P. phylliraeoides and indicates its medicinal potential.

Full text

Correspondence:
DOI: 10.5530/pc.2011.2.1
8 (c) Copyright 2011 EManuscript Publishing Services, India
Research Article
Pharmacognosy Communications www.phcogcommn.org
Volume 1 | Issue 2 | Oct-Dec 2011
*Correspondence:
Dr. I. E. Cock: E-mail: I.Cock@griffith.edu.au
DOI: 10.5530/pc.2011.2.3
An Examination of the Medicinal Potential
of Pittosporum phylliraeoides: Toxicity, Antibacterial
and Antifungal Activities
J. Vesoula,c I. E. Cocka,b*,
aBiomolecular and Physical Sciences, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Queensland 4111, Australia.
bEnvironmental Futures Centre, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Queensland 4111, Australia. cEcole Supérieure
d’Ingénieurs en Développement Agroalimentaire Intégré, Université de la Réunion, Parc Technologique, 2 rue Joseph Wetzell, 27490 Sainte
Clotilde, Ile de La Réunion
INTRODUCTION
Pittosporum phylliraeoides (Family Pittosporaceae) (commonly
known as weeping Pittosporum, cattlebush, Gumbi Gumbi,
Gumby Gumby, Cumbi Cumbi and native apricot) is an
endemic Australian tree/shrub that grows to approximately
8 m tall and is distributed throughout the drier areas of
the Australian continent. It has pendulous branchlets with
alternate, narrow tapered leaves, often with a hooked point.
In the winter and spring, the tree develops small yellow
owers which may either be solitary or occur as clusters
in leaf forks. The owers develop to small (6-20 mm) oval
yellow skinned fruit which ripen in late summer/early
autumn, opening into two valves with several seeds inside.
Australian Aborigines used P. phylliraeoides as a medical plant
to treat a variety of conditions.[1-5] An infusion of the leaves,
seeds, fruit pulp or wood was used to treat bruises, muscle
ache, sprains and cramps. P. phylliraeoides infusions were
drunk to treat coughs and colds as well as to induce lactation.
A decoction of fruit was used both externally and by
ingestion to treat eczema and pruritus.
Despite its range of traditional medicinal uses, the
phytochemistry and therapeutic potential of P. phylliraeoides
has not been extensively studied. One study examined 40
different Australian plants for antiviral bioactivities.[6] This
study found that P. phylliraeoides leaf extracts were capable
of inhibiting greater than 25% of Ross River virus (RRV)
induced cytopathicity. This demonstrated the antiviral
potential of P. phylliraeoides and provided support for the
ABSTRACT: Introduction: Pittosporum phylliraeoides is an endemic Australian plant historically used as a medicinal
agent by indigenous Australians. P. phylliraeoides solvent extracts were tested for antibacterial and antifungal
activities and toxicity in vitro. Results: All extracts displayed antibacterial activity in the disc diffusion assay. The
methanol and hexane extracts demonstrated the broadest specicity, inhibiting the growth of 4 of the 14 bacteria
tested (28.6%). The water, ethyl acetate, and chloroform extracts inhibited the growth of 2 (14.3%), 3 (21.4%), and
3 (21.4%) of the 14 bacteria tested respectively. P. phylliraeoides methanolic extract was also effective as an antifungal
agent, inhibiting the growth of a nystatin resistant strain of Aspergillus niger. It did not affect the growth of Candida
albicans. All extracts were more effective at inhibiting the growth of Gram-negative bacteria than Gram-positive
bacteria. Indeed, only the methanol and hexane extracts were capable of inhibiting the growth of any of the Gram-
positive bacteria, inhibiting the growth of only 1 of the 4 (25%) Gram-positive bacteria tested each. All P. phylliraeoides
extracts displayed low toxicity in the Artemia franciscana bioassay. The only signicant increase in mortality above
that of the control was seen for the ethyl acetate, chloroform and hexane extracts, although even these extracts
displayed low toxicity, inducing less than 50% mortality at 72 h. Conclusions: The low toxicity of the P. phylliraeoides
extracts and their inhibitory bioactivity against bacteria and fungi validate Australian Aboriginal usage of
P. phylliraeoides and indicates its medicinal potential.
KEYWORDS: Pittosporum phylliraeoides, Gumbi Gumbi, Australian plants, antibacterial, medicinal plants

Research Article
9
Vesoul, et. al.: An Examination of the Medicinal Potential of Pittosporum phylliraeoides: Toxicity, Antibacterial and Antifungal Activities
antifungal properties of P. phylliraeoides extracts as well as
examining their toxicity to determine their potential as
antibiotic agents.
MATERIALS AND METHODS
Plant collection and extraction
P. phylliraeoides plant material was provided by Philip Higson
of the Queensland Bush Foods Association as pre-dried
and coarse milled whole plant material. The material was
stored at -30 °C until use.
1 g of plant material was weighed into each of 5 tubes and
5 different extracts were prepared by adding 50 ml of
methanol, water, ethyl acetate, chloroform, or hexane
respectively. All solvents were obtained from Ajax and were
AR grade. Leaf material was extracted in each solvent for
24 h at 4 °C with gentle shaking. The extracts were ltered
through lter paper (Whatman No. 54) under vacuum
followed by drying by rotary evaporation in an Eppendorf
concentrator 5301. The resultant dry extract was weighed
and redissolved in 10 ml deionised water.
Qualitative phytochemical studies
Phytochemical analysis of P. phylliraeoides extracts were
conducted by modied versions of previously described
assays.[25,36,37] The modied assays are briey outlined below.
Saponins
1 ml of pure extract was added to 1 ml deionised water
and shaken vigorously for 30s. The tubes were allowed to
stand for 15 min and the presence or absence of persistent
frothing was noted. Persistent frothing indicated the
presence of saponins.
Phenolic compounds
Phenolic compounds were detected using a modied version
of the Folin-Ciocalteu procedure.[37] 200 μl of crude extract
was added to 2 ml of 3% aqueous sodium carbonate,
followed by the addition of 200 μl Folin-Ciocalteu reagent.
The mixture was allowed to stand for 30 min at room
temperature. The formation of blue/gray colour indicated
the presence of phenolic groups.
Water soluble phenol test
2 drops of 1% ferric chloride were added to 500 µl of each
extract. A red colour change indicated presence of water
soluble phenols.
Water insoluble phenol test
500 µl of dichloromethane, 3 drops of 1% ferric chloride
and 1 drop of pyridine were added to 500 µl of each extract
and mixed. The presence of insoluble phenols was indicated
by a colour change.
traditional Aboriginal use of P. phylliraeoides infusions to
treat viral diseases including colds and coughs. The same
study found P. phylliraeoides leaf extracts inaffective against
poliovirus or cytomegalovirus (CMV) induced cytopathicity,
although different cell lines were used for all three assays.
The authors questioned whether the lack of antiviral activity
towards poliovirus and RRV in their study was due to
variations in the way the extract components behaved in
the different cell lines, or whether it suggested that plant
components interfered with a step in the replication cycle
which is specic to RRV.
P. phylliraeoides also had uses in the treatment of various
cancers by Aborigines.[1-5] This ethnopharmacological
knowledge was traditionally passed on by word of mouth,
instead of by written record, and unfortunately much of
our understanding of Aboriginal medicine has been lost
with Aboriginal society merging into mainstream Australian
society. Recent anecdotal accounts have also credited P.
phylliraeoides with anticancer activity,[7] although these have
yet to be veried by rigorous scientic examination. A
recent scientic study examined the anticancer potential
of P. phylliraeoides leaf extracts.[8] The extracts were shown
to have moderate cytotoxic activity towards A427 lung
cancer cells, however, as these were preliminary studies
only the efcacy of these extracts requires verication.
Anticancer activity has also been detected in other related
Pittosporum species from Madagascar,[9] South Africa,[10] New
Caledonia[11] and Asia.[12]
P. phylliraeoides is reported to contain a number of pentacyclic
triterpenoid saponogenins.[13] In particular, phillyregenin
(a dihydroxylactone), R1-barrigenol, 27 - desoxyphillyrigenin
(3 - hydroxytaraxastan - 28, 20 - olide), 23 -
hydroxyphillyrigenin (3, 23, 27- trihydroxytaraxastan - 28,
20 - olide), dihydropriverogenin A, 16 - desoxybarringtogenol
C and barringtogenol C, were isolated from P. phylliraeoides
and identied in this study. Similar pentacyclic triterpenoids
isolated from Alchornea latifolia have been linked with
cytotoxic activity towards Hep-G2 and A-431 human cancer
cell lines and are potent inhibitors of topoisomerase II.[14]
Pentacyclic triterpenoids have also been associated with
antitumour,[15] anti-HIV[16] and antioxidant bioactivities.[17]
Additionally, pentacyclic triterpenoids from Laggera pterodonta
have shown antiviral activity against herpes viruses.[18] Studies
have also demonstrated the antibacterial activity of
pentacyclic triterpenoids from a variety of plants.[19-21]
Surprisingly, the antiseptic properties of P. phylliraeoides
remain largely unstudied. The antibacterial and antifungal
properties of other Australian plants are well known. For
instance, species belonging to the genera Eucalyptus,[22-25]
Callistemon,[26] Leptospermum,[14,27-29] Melaleuca[30,31] and
Syzygium[27,32-35] are known to have antimicrobial activities.
The current study reports on the antibacterial, and

10
Vesoul, et. al.: An Examination of the Medicinal Potential of Pittosporum phylliraeoides: Toxicity, Antibacterial and Antifungal Activities
Alkaloids
Two methods were used to test for the presence of alkaloids:
Mayers reagent test
200 μl of pure extract was treated with a few drops of
aqueous solution of hydrochloric acid and 500 μl Mayer’s
reagent. Formation of a white precipitate indicated the
presence of alkaloids.
Mayer’s reagent: Mercuric chloride (1.358 g) was dissolved
in 60 ml deionised water. Potassium Iodide (5.0 g) was
dissolved in 10 ml deionised water. The mercuric chloride
and potassium iodide solutions were mixed and made up
to 100 ml with deionised water.
Wagners reagent test
200 μl of each extract was treated with a few drops of an
aqueous solution of hydrochloric acid and 500 μl Wagner’s
reagent. A reddish-brown occulent precipitate indicated
the presence of alkaloid.
Wagner’s reagent: 1.27 g iodine and 2 g potassium iodide
were dissolved in 5 ml deionised water and made up to
nal volume 100 ml with deionised water.
Antimicrobial screening
Test microorganisms
All microbial strains were obtained from Michelle Mendell
and Tarita Morais, Grifth University, Australia. Stock
cultures of Aeromonas hydrophila, Alcaligenes feacalis, Bacillus
cereus, Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae,
Proteus mirabilis, Pseudomonas uorescens, Salmonella newport,
Serratia marcescens, Shigella sonnei, Staphylococcus aureus,
Staphylococcus epidermidis and Streptococcus pyogenes were
subcultured and maintained in nutrient broth at 4 °C. Stock
cultures of Aspergillus niger and Candida albicans were
subcultured and maintained in Sabouraud media at 4 °C.
Evaluation of antimicrobial activity
Antimicrobial activity of all plant extracts was determined
using a modied Kirby-Bauer disc diffusion method.[38]
Briey, 100 µl of the test bacteria/fungi were grown in 10
ml of the appropriate fresh broth until they reached a
count of approximately 108 cells/ml of bacteria or 105
cells/ml for fungi (as determined by direct microscopic
determination). One hundred microliters of microbial
suspension was spread onto agar plates corresponding to
the broth in which they were maintained.
The extracts were tested using 5 mm sterilised lter paper
discs. Discs were impregnated with 10 µl of the test sample,
allowed to dry and placed onto inoculated plates. The
plates were allowed to stand at 4 °C for 2 h before incubation
with the test microbial agents. Plates inoculated with
A. feacalis, A. hydrophilia, B. cereus, C. freundii, K. pneumoniae,
Flavonoids
Flavonoids were detected using a modied Kumar test.[36]
100 μl of aqueous sodium hydroxide was added to 1 ml
of each extract. The development of an intense yellow
colour indicated the presence of avonoids. 100 μl of
concentrated HCl was added to the solution. Reversion to
the original colour conrmed the presence of avonoids.
Polysteroids
Polysteroids were detected using a modied version of
the Leiberman-Buchard test.[36] Three drops of acetic
anhydride was added to 500 μl of crude extract followed
by the addition of a few drops concentrated sulphuric
acid. The solution was allowed to sit at room temperature
for 5 min. Formation of a blue/green colour indicated
the presence of polysteroids.
Triterpenoids
Triterpenoids were detected using a modied version of
the Salkowski test.[25] 1 ml of extract was slowly added to
400 µl chloroform, followed by careful addition of 400 µl
concentrated sulphuric acid. Formation of a red/brown/
purple colour at the interface indicated the presence of
triterpenoids.
Cardiac glycosides
Cardiac glycosides were detected using a modied version
of the Keller Kiliani test.[37] 500 μl of extract was added
to 500 μl glacial acetic acid. A few drops of 1% aqueous
iron chloride and concentrated sulphuric acid were then
carefully added. The presence of a red/brown ring of the
interface or the formation of a green/blue colour
throughout the solution indicated the presence of cardiac
glycosides.
Anthraquinones
Anthraquinones were detected using modied versions of
the Kumar and Ajaiyeoba tests.[36,37] The modied Kumar
test involved addition of a few drops of concentrated
sulphuric acid to 500 μl pure extract, followed by the careful
addition of 500 μl of ammonia. A rose pink colour indicated
the presence of free anthraquinones. For the Ajaiyeoba
test, 450 μl of crude extract was added to 50 μl concentrated
HCl and allowed to stand at room temperature for several
minutes. 500 μl chloroform was then carefully added. The
formation of a rose pink colour indicated the presence of
combined anthraquinones.
Tannins
Tannins were detected using a modied version of the
Ferric chloride test.[36] Two drops of 1% aqueous ferric
chloride reagent were added to 500 μl of crude extract.
The mixture was observed for the formation of blue, blue-
black, green or green-black colouration which indicated
the presence of tannins.

11
Vesoul, et. al.: An Examination of the Medicinal Potential of Pittosporum phylliraeoides: Toxicity, Antibacterial and Antifungal Activities
from the shells and remaining cysts and were concentrated
to a suitable density by placing an articial light at one end
of their incubation vessel and the nauplii rich water closest
to the light was removed for biological assays. 400 µl of
seawater containing approximately 42 (mean 41.6, n = 150,
SD 17.8) nauplii were added to wells of a 48 well plate and
immediately used for bioassay. The plant extracts were
diluted to 4 mg/ml in seawater for toxicity testing, resulting
in a 2 mg/ml concentration in the bioassay (except where
specied). 400 µl of diluted plant extract and the reference
toxins were transferred to the wells and incubated at
25 ± 1 °C under articial light (1000 Lux). A negative
control (400 µl seawater) was run in at least triplicate for
each plate. All treatments were performed in at least
triplicate. The wells were checked at regular intervals and
the number of dead counted. The nauplii were considered
moribund if no movement of the appendages was observed
within 10 s. After 72 h all nauplii were sacriced and counted
to determine the total number per well. The LC50 with 95%
condence limits for each treatment was calculated using
probit analysis.[40]
Statistical analysis
Data are expressed as the mean ± SD of at least three
independent experiments. The Paired T-Test was used to
calculate statistical signicance between control and treated
groups with a P value < 0.05 considered to statistically
signicant.
RESULTS
Liquid extraction yields and qualitative
phytochemical screening
Extraction of 1 g of dried plant material with various
solvents yielded dried plant extracts ranging from 20.6 mg
to 120.1 mg (Table 1). Deionised water and chloroform
both gave relatively high yields of dried extracted material
(111.2 and 120.1 mg respectively) whilst ethyl acetate
extracted the lowest mass (20.6 mg). The dried extracts
were resuspended in 10 ml of deionised water resulting in
the extract concentrations shown in Table 1.
Qualitative phytochemical studies (Table 2) show that
methanol and water extracted the widest range of
phytochemicals. Both showed moderate to high levels of
phenolics (both water soluble and insoluble phenolics),
saponins, triterpenoids and avanoids. The only difference
detected between the methanol and water extracts was the
presence of low levels of tannins in the water extract. The
ethyl acetate and chloroform extracts had low levels of
avanoids whilst the ethyl acetate extract also had low levels
of saponins. However, only a low response was seen for each
of these solvents in these tests. None of the classes of
phytochemicals tested for were detected in the hexane extract.
P. mirabilis, P. uorescens, S. marcescens, and C. albicans were
incubated at 30 °C for 24 h, the diameters of the inhibition
zones were then measured in mm. Plates inoculated with
E. coli, S. newport, S. sonnei, S. aureus, S. epidermidis and
S. pyogenes were incubated at 37 °C for 24 h, he diameters
of the inhibition zones were then measured. A. niger
inoculated plates were incubated at 25 °C for 48 h, the
zones of inhibition were then measured. All measurements
were to the closest whole mm. Each antimicrobial
assay was performed in at least triplicate. Mean values
are reported in this study. Standard discs of ampicillin
(2 µg), chloramphenicol (10 µg) and nystatin (100 µg) were
obtained from Oxoid Ltd. and served as positive controls
for antimicrobial activity. Filter discs impregnated with
10 µl of distilled water or 10 µl of 10% methanol were
used as negative controls.
Minimum inhibitory concentration (MIC)
determination
The minimum inhibitory concentration (MIC) of the
P. phylliraeoides extracts were determined by the disc diffusion
method across a range of doses. The plant extracts were
diluted in deionised water across a concentration range of
5 mg/ml to 0.1 mg/ml. Discs were impregnated with 10
µl of the test dilutions, allowed to dry and placed onto
inoculated plates. The assay was performed as outlined
above and graphs of the zone of inhibition versus
concentration were plotted for each extract. Linear regression
was used to calculate the MIC values.
Toxicity screening
Reference toxins for biological screening
Potassium dichromate (K2Cr2O7) (AR grade, Chem-Supply,
Australia) was prepared as a 1.6 mg/ml solution in distilled
water and was serially diluted in synthetic seawater for use
in the Artemia franciscana nauplii bioassay. Mevinphos
(2-methoxycarbonyl-1-methylvinyl dimethyl phosphate)
was obtained from Sigma-Aldrich as a mixture of cis (76.6%)
and trans (23.0%) isomers and prepared as a 4 mg/ml stock
in distilled water. The stock was serially diluted in articial
seawater for use in the bioassay.
Artemia franciscana nauplii toxicity screening
Toxicity was tested using the A. franciscana nauplii lethality
assay developed for the screening of active plant constituents
with the following modications.[39] A. franciscana cysts were
obtained from North American Brine Shrimp, LLC, USA
(harvested from the Great Salt Lake, Utah). Synthetic
seawater was prepared using Reef Salt, AZOO Co., USA.
Seawater solutions at 34 g/l distilled water were prepared
prior to use. 2 g of A. franciscana cysts were incubated in 1
l synthetic seawater under articial light at 25 °C, 2000 Lux
with continuous aeration. Hatching commenced within
16-18 h of incubation. Newly hatched A. franciscana (nauplii)
were used within 10 h of hatching. Nauplii were separated

12
Vesoul, et. al.: An Examination of the Medicinal Potential of Pittosporum phylliraeoides: Toxicity, Antibacterial and Antifungal Activities
the water extract displayed potent antibacterial activity
against P. mirabilis as determined from the zone of inhibition
(16.3 ± 0.3 mm).
P. phylliraeoides methanol extract also demonstrated limited
antifungal activity. The extract inhibited the growth of a
nystatin resistant strain of A. niger but was ineffective against
C. albicans. Although the inhibition of A. niger was not
particularly strong (as determined by the zone of inhibition),
this does identify the P. phylliraeoides methanol extract as a
possible antifungal agent to combat otherwise resistant
strains of A. niger. No other extract inhibited the growth
of either of the fungi tested.
The relative level of antibacterial activity was further
evaluated by determining the MIC values for each extract
against the bacterial species which were determined to be
susceptible. MIC values were evaluated in the current studies
by disc diffusion across a range of concentrations. This
has previously been determined to be a valid method of
MIC determination as MIC values determined by disc
diffusion correlate well with those determined by broth
dilution assays.[41]
The methanol extract was particularly effective at inhibiting
the growth of S. pyogenes, with growth inhibition seen
at concentrations as low as approximately 8 µg/ml. The
methanolic extracts MIC values were also below 1000 µg/ ml
for mirabilis and S. marcenscens. Furthermore, the methanol
extract was the only sample tested that displayed antifungal
activity, inhibiting the growth of A. niger at concentrations
as low as approximately 40 µg/ml. This is a signicant
Antimicrobial activity
10 µl of each extract was tested in the disc diffusion assay
against 14 bacteria (Table 3). All extracts displayed
antibacterial activity, as evidenced by the inhibition of
growth for 2-4 species of the tested bacterial panel. The
methanolic and hexane extracts displayed the broadest
antibiotic specicity, inhibiting the growth of 4 of the 14
bacteria tested (28.6%). The methanolic extract was
particularly potent against P. mirabilis as determined from
the zone of inhibition (16.3 ± 0.3 mm). Indeed, the methanol
extract was more effective than the ampicillin and
chloramphenicol controls in inhibiting P. mirabilis growth.
The methanol and hexane extracts were the only extracts
capable of inhibiting the growth of any Gram-positive
bacteria, each inhibiting 1 of the 4 bacteria tested (25%).
P. phylliraeoides ethyl acetate and chloroform extracts each
inhibited the growth of 3 of the 14 bacteria tested (21.4%).
The water extract had the narrowest antibacterial specicity,
inhibiting the growth of only 2 of the 14 bacteria tested
(14.3%). Whilst displaying a narrow range of specicity,
Table 1: The mass of dried material extracted
with the various solvents and the concentration
after resuspension in deionised water
Solvent Mass of Dried
Extract (mg)
Resuspended Extract
Concentration (mg/ml)
Methanol 64.0 6.4
Deionised Water 111.2 11.1
Ethyl Acetate 20.6 2.1
Chloroform 120.1 12.0
Hexane 70.0 7.0
Table 2: Qualitative phytochemical screenings of solvent extractions
Phenols
Cardiac Glycosides
Saponins
Triterpenes
Phytosteroids
Alkaloids
Flavanoids
Tannins
Anthraquinones
Extract
Total Phenolics
Water Soluble
Water Insoluble
Keller-Kiliani Test
Froth Persistence
Salkowski Test
Acetic Anhydride Test
Meyers Test
Wagners Test
Shinoda Test
Kumar test
Ferric Chloride Test
Free
Combined
Methanol ++ + + – ++ ++ – – – ++ ++ – – –
Water +++ + +++ – +++ ++ – – – +++ +++ + – –
Ethyl Acetate – – – – + – – – – + + – – –
Chloroform – – – – – – – – – + + – – –
Hexane – – – – – – – – – – – – – –
+++ indicates a large response; ++ indicates a moderate response; + indicates a minor response; - indicates no response in the assay.

13
Vesoul, et. al.: An Examination of the Medicinal Potential of Pittosporum phylliraeoides: Toxicity, Antibacterial and Antifungal Activities
with MIC values less than 1000 µg/ml for all the bacterial
species whose growth it inhibited. However, the potent
growth inhibition of the chloroform extract towards S.
sonnei (32.2 µg/ml) is noteworthy. The water extract inhibited
the growth of only two bacterial species (P. mirabilis and
S. marcenscens), although MIC values below 1000 µg/ml
were seen for only a single species (P. mirabilis).
result as the strain of A. niger tested was a resistant strain
which was not inhibited by the nystatin control. The hexane
extract also displayed potent antibacterial activity with MIC
values below 1000 µg/ml for two bacteria (K. pneumonia
and S. marcenscens). The ethyl acetate and chloroform extracts
both inhibited the growth of three bacteria, although the
ethyl acetate extract exhibited the more potent bioactivity,
Table 4: Minimum inhibitory concentrations (µg/ml) of P. phylliraeoides extracts against susceptible microbes
MIC (µg/ml)
A. hydrophilia
E. coli
K.pneumoniae
P. mirabilis
S. marcenscens
S. sonnei
S. aureus
S. pyogenes
A. niger
Methanol extract – 1369.2 – 473.4 737.3 – – 7.8 40.4
Water extract – – – 770.4 1544.5 – – – –
Ethyl acetate
extract
506.7 – – 665.2 216.1 – – – –
Chloroform extract – 2518.7 – – 1314.5 32.2 – – –
Hexane extract – – 307.9 1203.1 784.1 – 5557.6 – –
Numbers indicate the mean MIC values of at least triplicate determinations.
– indicates no growth inhibition.
Table 3: Antimicrobial activity of P. phylliraeoides solvent extracts measured as zones of inhibition (mm)
Methanol
extract
Water
extract
Ethyl
acetate
extract
Chloroform
extract
Hexane
extract Ampicillin Chloramphenicol Nystatin
Gram negative rods
A. faecalis – – – – – 9.8 ± 0.8 6.2 ± 0.6 NT
A. hydrophilia – – 7.3 ± 0.6 – – 9.5 ± 1.4 18.0 ± 0.8 NT
C. freundi – – – – – 9.8 ± 0.8 25.3 ± 1.5 NT
E. coli 6.5 ± 0.0 – – 6.0 ± 0.0 – 10.7 ± 1.8 20.0 ± 0.8 NT
K. pneumoniae – – – – 9.0 ± 0.0 9.8 ± 1.2 24.0 ± 0.7 NT
P. mirabilis 16.3 ± 0.3 14.7 ± 0.3 9.3 ± 0.6 – 9.2 ± 0.6 14.6 ± 0.7 8.2 ± 0.6 NT
P. fluorescens – – – – – 13.7 ± 1.8 16.8 ± 1.5 NT
S. newport – – – – – 9.5 ± 1.4 15.2 ± 1.2 NT
S. marcenscens 7.5 ± 0.5 8.7 ± 0.4 8.0 ± 0.5 7.7 ± 0.3 8.3 ± 0.3 – 14.9 ± 1.6 NT
S. sonnei – – – 7.7 ± 0.3 – 12.4 ± 0.8 18.3 ± 1.3 NT
Gram positive rods
B. cereus – – – – – 16.8 ± 1.2 12.8 ± 0.9 NT
Gram positive cocci
S. aureus – – – – 7.3 ± 0.6 14.3 ± 1.5 18.8 ± 0.6 NT
S. epidermidis – – – – – 32.2 ± 1.3 13.0 ± 1.0 NT
S. pyogenes 6.7 ± 0.3 – – – – 14.0 ± 1.3 10.3 ± 0.7 NT
Fungi
A. niger 6.6 ± 0.3 – – – – NT NT –
C. albicans – – – – – NT NT 25.0 ± 1.8
Numbers indicate the mean diameters (mm) of inhibition of at least triplicate experiments ± standard deviation. – indicates no growth inhibition. NT indicates not tested.
Ampicillin (2 µg), chloramphenicol (10 µg) and nystatin (100 µg) were used as positive controls. Deionised water was included as a negative control.

14
Vesoul, et. al.: An Examination of the Medicinal Potential of Pittosporum phylliraeoides: Toxicity, Antibacterial and Antifungal Activities
2000µg/ ml in articial seawater for toxicity testing, resulting
in 1000 µg/ml concentrations in the Artemia nauplii lethality
bioassay. For comparison, the reference toxins potassium
dichromate (1000 µg/ml) and Mevinphos (2000 µg/ml)
were also tested in the bioassay. Figure 1 shows the %
mortality induced by each extract and by the controls at
Quantification of toxicity
P. phylliraeoides methanol, water, chloroform and hexane
extracts were diluted to a concentration of 4000 µg/ml in
articial seawater for toxicity testing, resulting in 2000 µg/ ml
concentrations in the Artemia nauplii lethality bioassay. The
less concentrated ethyl acetate extract was diluted to
Figure 1: Brine shrimp lethality of (a) P. phylliraeoides methanol extract (2000 µg/ml), (b) P. phylliraeoides water extract (2000 µg/ml),
(c) P. phylliraeoides ethyl acetate extract (1000 µg/ml), (d) P. phylliraeoides chloroform extract (2000 µg/ml), (e) P. phylliraeoides hexane extract
(2000 µg/ml), (f) artificial seawater negative control, (c) potassium dichromate (1000 µg/ml), (d) Mevinphos (2000 µg/ml). All bioassays were
performed in at least triplicate and are expressed as mean ± standard deviation.

15
Vesoul, et. al.: An Examination of the Medicinal Potential of Pittosporum phylliraeoides: Toxicity, Antibacterial and Antifungal Activities
various times. The potassium dichromate (Figure 1g) and
Mevinphos (Figure 1h) reference toxins were rapid in their
onset of mortality. Both reference toxins induced mortality
within the rst 3 hours of exposure and 100% mortality
was evident following 4-5 hours. In contrast, all of the
P. phylliraeoides extracts (Figures 1a-e) displayed mortality
rates only slightly elevated above those of the articial
seawater negative control (Figure 1f) at 24, 48 and 72 h.
At no time point tested did the mortality induction by any
P. phylliraeoides extract reach 50%, even at the relatively high
doses tested (2000 µg/ml for methanol, water, chloroform
and hexane extracts; 1000 µg/ml for ethyl acetate extract)
so it was not possible to accurately determine an LC50 for
any extract. Furthermore, as toxicity has previously been
dened for plant extracts as the induction of ≥ 50%
mortality at concentrations ≤ 1000 µg/ml,[39] all P. phylliraeoides
extracts are considered to be of low toxicity.
DISCUSSION
The current study reports on the antibacterial and antifungal
activities of various P. phylliraeoides extracts, and on their
toxicity. The ability of P. phylliraeoides extracts to inhibit the
growth of Gram-negative, and to a lesser extent Gram-
positive bacteria, is in agreement with previous reports of
the antibacterial activity of other Australian native plants
that have a history of medicinal usage by Australian
Aborigines. The antiseptic properties of Eucalyptus,[22-25]
Leptospermum,[14,27-29] and Melaleuca[30,31] species have been
extensively studied and shown to inhibit the growth of a
wide variety of both Gram-positive and Gram-negative
bacteria.
The current study shows Gram-negative bacteria to be
more susceptible to P. phylliraeoides extracts than Gram-
positive bacteria, although this may be due to the small
sample of Gram-positive bacteria tested. Indeed, only the
methanolic and hexane extracts were capable of inhibiting
any Gram-positive bacteria, each inhibiting the growth of
a single bacterium of the 4 Gram-positive bacteria tested
(25%). The greater susceptibility of Gram-negative bacteria
observed in this study is in contrast to previous studies
which have reported a greater susceptibility of Gram-
positive bacteria towards solvent extracts for South
American,[42] African[43,44] and Australian[45] plant extracts.
Results within this laboratory have also conrmed the
greater susceptibility of Gram-positive bacteria towards
other many other Australian plant extracts,[27] although
examples of nonpolar extracts from Australian plants having
a greater effect on Gram-negative bacteria have also been
reported.[46]
Individual P. phylliraeoides extract components responsible
for the antibacterial potential of the solvent extracts were
not identied in the current study. Previous reports have
identied various bioactive components of other Australian
medicinal plants (Eucalyptus,[47] Leptospermum,[48] Melaleuca[49]).
These plants all contain terpenes including 1, 8-cineole,
terpinen-4-ol, α-pinene and β-pinene. Both 1, 8-cineole
and terpinen-4-ol have antimicrobial activity.[50,51] Recent
studies have also reported on the antibacterial activities of
Callistemon[26] and Syzygium[20,32-34] species. It has been
postulated that terpene components may also be responsible
for the antiseptic properties of these plants.[52] P. phylliraeoides
leaves contain a number of pentacyclic triterpenoid
saponogenins including phillyregenin (a dihydroxylactone),
R1-barrigenol, 27 - desoxyphillyrigenin (3 –
hydroxytaraxastan - 28, 20 - olide), 23 - hydroxyphillyrigenin
(3, 23, 27- trihydroxytaraxastan - 28, 20 - olide),
dihydropriverogenin A, 16 - desoxybarringtogenol C and
barringtogenol C.[13] Whilst the phytochemistry of the P.
phylliraeoides extracts investigated in the current study was
not extensively examined, phenolic compounds,
triterpenoids, saponins, avanoids and tannins were detected
by qualitative assays. Further studies are required to
determine which of these classes of phytochemical is
responsible for the recorded bioactivities and to further
identify the individual bioactive compounds.
The ndings reported here also demonstrate that none of
the P. phylliraeoides extracts displayed signicant toxicity
towards A. franciscana. None of the extracts tested induced
mortality above 50%. Whilst the ethyl acetate, chloroform
and hexane extracts induced mortality approaching 50%,
this was not until 72 h. As most toxicity studies using
Artemia nauplii usually report 24 h and/or 48 h LC50 values,
the reporting of mortality at 72 h may not be required as
it does not provide data for cross study comparisons.
Furthermore, even at 72 h the mortality induction by all
extracts was below 50%. The extracts in the current study
were all tested at concentrations of 2000 µg/ml in the
bioassay (with the exception of the ethyl acetate extract
which was tested at 1000 µg/ml). Previously, compounds
with an LC50 of greater than 1000 µg/ml towards Artemia
nauplii have been dened as being non-toxic.[39] It was
therefore determined that all P. phylliraeoides extracts were
non-toxic towards Artemia nauplii.
In conclusion, the results of this study partially validate
the traditional Australian Aboriginal usage of P. phylliraeoides
to treat bacterial and fungal diseases and indicate that
P. phylliraeoides is worthy of further study. Further evaluation
of the antibacterial and antifungal properties of these
extracts against a more extensive panel of microbial agents
is warranted. Likewise, purication and identication of
the bioactive components is needed to examine the
mechanisms of action of these agents. Whilst the extracts
examined in this report are promising as antibacterial and
antifungal agents, caution is needed before these

16
Vesoul, et. al.: An Examination of the Medicinal Potential of Pittosporum phylliraeoides: Toxicity, Antibacterial and Antifungal Activities
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ACKNOWLEDGEMENTS
The P. phylliraeoides plant material used in these studies was
provided by Philip Higson of the Queensland Bush Foods
Association. Financial support for this work was provided
by School of Biomolecular and Physical Sciences, Grifth
University.
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