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Antimicrobial activity of customary medicinal plants of the Yaegl Aboriginal

community of northern New South Wales, Australia: A preliminary study

Article  in  BMC Research Notes · June 2015

DOI: 10.1186/s13104-015-1258-x · Source: PubMed

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Packer et al. BMC Res Notes (2015) 8:276
DOI 10.1186/s13104-015-1258-x

RESEARCH ARTICLE Open Access

Antimicrobial activity of customary

medicinal plants of the Yaegl Aboriginal
community of northern New South Wales,
Australia: a preliminary study
Joanne Packer1, Tarannum Naz1, Yaegl Community Elders, David Harrington1, Joanne F Jamie1 and
Subramanyam R Vemulpad1*

Abstract 
Background:  This study is a collaboration between Macquarie University researchers and the Yaegl Aboriginal Com-
munity of northern NSW, Australia to investigate the antimicrobial potential of plants used in the topical treatment of
wounds, sores and skin infections. Based on previously documented medicinal applications, aqueous and aqueous
ethanolic extracts of Alocasia brisbanensis, Canavalia rosea, Corymbia intermedia, Hibbertia scandens, Ipomoea brasilien-
sis, Lophostemon suaveolens and Syncarpia glomulifera and the aqueous extracts of Smilax australis and Smilax glyci-
phylla were tested against common wound pathogens, including antibiotic resistant bacterial strains.
Methods:  Plant material was prepared as aqueous extractions modelled on customary preparations and using 80%
aqueous ethanol. Extracts were assayed against a selection of clinically relevant Gram positive (Streptococcus pyogenes
and sensitive and resistant strains of Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa, Escherichia
coli and Salmonella typhimurium) bacteria and a fungus (Candida albicans) using disc diffusion and MTT microdilution
methods. Viability of treated microorganisms was determined by subculturing from microdilution assays.
Results:  The extracts of Corymbia intermedia, Lophostemon suaveolens and Syncarpia glomulifera had promising levels
of antimicrobial activity (MIC 31–1,000 µg/mL) against both antibiotic sensitive and resistant Staphylococcus aureus as
well as the fungus Candida albicans (clinical isolate).
Conclusion:  Aqueous and 80% aqueous ethanolic extracts of Lophostemon suaveolens, Corymbia intermedia and Syn-
carpia glomulifera exhibited promising levels of antimicrobial activity against a range of both antibiotic sensitive and
resistant strains of microorganisms. This is the first report of antimicrobial activities for C. intermedia and L. suaveolens
and the leaves of S. glomulifera. This study demonstrates the value of customary knowledge in the identification of
new sources of antimicrobial treatments.
Keywords:  Antimicrobials, Multidrug resistant, Wound healing, Traditional medicine, Ethnobotany

Background healing agents and other resources. Studies of custom-

Australian Aboriginal people have over 40,000  years ary (traditional and contemporary) medicinal plant
of knowledge of flora and fauna as sources of food, preparations, especially in recent years, have revealed
interesting medicinal properties and valuable biologically
active compounds [1]. Australian Aboriginal medici-
*Correspondence: [email protected] nal flora have had limited biological screening studies
1
Indigenous Bioresources Research Group, Faculty of Science aligned with their medicinal uses. Furthermore, the
and Engineering, Macquarie University, North Ryde, Sydney 2109,
Australia
knowledge of their medicinal uses is quickly disappearing
Full list of author information is available at the end of the article [2], particularly in some regions of the country, such as

© 2015 Packer et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Packer et al. BMC Res Notes (2015) 8:276 Page 2 of 7

the southern states of Australia. Assessment of the bioac- holders of the Yaegl community. Plant identification was
tive potential of these plants with long historical use may confirmed by and voucher specimens deposited at the
support their wider application and also provide a source Herbarium of the Royal Botanic Gardens of NSW or the
for safe, accessible alternative medicines and leads for the Macquarie University Herbarium [registered with the
discovery of new drug-like molecules [3]. Index Herbariorum, New York Botanic Gardens (http://
The ongoing crisis of antimicrobial resistance [4] calls sciweb.nybg.org/science2/IndexHerbariorum.asp)], with
for the investigation of novel sources of antimicrobi- the prefix MQU.
als. Working with Indigenous communities who have For all plants the material used was leaves, except for
been using natural remedies over the centuries can pro- I. brasiliensis where the stem and leaves were used. Plant
vide novel sources of antimicrobial therapies and/or lead material was weighed and washed twice with water to
compounds for the development of new medicines [3]. remove surface contaminants and extraneous mate-
Discussions with Elders of the Yaegl Aboriginal com- rial. Approximately 50 g of fresh plant material for each
munity of New South Wales, Australia on their custom- extraction method was collected (except for S. australis
ary (traditional and contemporary medicines) identified and S. glyciphylla, where 10 g was collected for the aque-
plants used in the topical treatment of conditions of a ous extraction only, due to inadequate availability of the
microbial aetiology [5]. These included Alocasia brisban- plant material in the area).
ensis for burns and boils, cuts, sores and open wounds;
Lophostemon suaveolens, Smilax australis, Smilax glyci- Chemicals
phylla and Syncarpia glomulifera for antiseptic purposes; Unless otherwise indicated, all chemicals were sourced
Canavalia rosea for boils and sores, Corymbia interme- from Sigma Aldrich, St Louis, USA. All media listed
dia for the treatment of wounds; Hibbertia scandens for below were sourced from Bacto Laboratories Pty Ltd,
sores and rashes; and Ipomoea brasiliensis as a poultice Australia and prepared as per the manufacturer’s
for boils. instructions.
There has been little investigation into these plants
identified by the Yaegl community for their antimicrobial Extraction of plant material
potential. This study looks into the antimicrobial activity Aqueous extraction of samples for antimicrobial screening
of these plants against eight pathogenic organisms com- Plant material was, where possible, prepared in a way
monly implicated in wounds, sores and skin infections. consistent with the preparation methods described by the
Yaegl Elders [5] within 48  h of collection. In the case of
Methods pink bloodwood (C. intermedia) and apple gum (L. sua-
Ethics protocol veolens or S. glomulifera), sap or latex while customarily
This research was approved by the Human Ethics Com- used. Due to the risk of considerable damage to the tree,
mittee at Macquarie University, (HE27 JUL2007-R05356 sap or latex was not collected and the Elders requested
and 5201200763). It was conducted under the frame- the leaves be collected and tested as an accessible and
work of best ethical practice, working in partnerships sustainable option. For C. rosea and H. scandens, the spe-
with Indigenous people [6, 7] and was governed by a cifics of the preparation method could not be recalled by
cooperative research agreement with the Yaegl commu- the knowledge holders.
nity [8]. The choice of plants and the biological testing Aqueous extractions were prepared as either a decoc-
to be undertaken were determined in consultation with tion or an infusion as described below.
the Yaegl community through the Yaegl Local Aboriginal Decoction (D): Plant material was pounded and boiled
Land Council as the appropriate authorising body. The in 10× (w/v) tap water for 10  min, before being left to
proposed methods and workflow were discussed before steep and cool (covered, for approximately 60 min). The
work commenced, and the results of the screening were decoction was filtered and the filtrate collected.
presented back to the Yaegl community in the form of Hot infusion (I): Freshly boiled water was poured over
regular reports and presentations. The regular contact roughly chopped or torn plant material (10× w/v). The
with the community during all phases of this research infusion was covered and allowed to steep for 2  h. The
project ensured that the progress of the project remained infusion was filtered and the filtrate collected.
in accordance with the requirements of the community Cold infusion (CI): Plant material was roughly chopped
and academics. or torn and submerged in 10× (w/v) water at room tem-
perature. The infusion was shaken gently for 24  h, the
Plant collection and identification mixture was vacuum filtered and the solid was shaken
Plants were collected in March 2011 after being located with fresh water (10× w/v) for an additional 24 h and fil-
and identified with the assistance of the knowledge tered. The filtrates were pooled.
Packer et al. BMC Res Notes (2015) 8:276 Page 3 of 7

All crude aqueous extracts were dried by rotary evapo- Disc diffusion assay
ration (Büchi, Germany) at reduced pressure at approxi- Dried plant material was dissolved in DMSO at 50  mg/
mately 50°C, followed by freeze drying (Labconco freeze mL. Antibiotic controls were prepared in broth at a con-
dryer, USA). Extracts were stored at −20°C. centration of 0.1  mg/mL. Sterile paper discs (∅ 6  mm,
Whatman, Maidstone, UK) were impregnated with
Aqueous ethanolic extraction of samples for antimicrobial 2 × 10 µL of plant extract (1 mg/disc) or antibiotic con-
screening trol (2 µg/disc) and air-dried. 20 µL DMSO impregnated
Fresh plant material was ground to a coarse powder using discs were included as a negative control. Discs were
a coffee grinder (IKA® A11 Basic, Selangor, Malaysia) placed on appropriate agar plates (horse blood agar for
and shaken in 10× w/v of 80% v/v aqueous ethanol at S. pyogenes, potato dextrose agar for C. albicans or MH
room temperature [9] for approximately 48  h. The mix- agar for other microorganisms), inoculated with broth
ture was filtered and the solid shaken with fresh 80% v/v cultures at A600  =  0.08 (bacteria: 107–108  cfu/mL, C.
aqueous ethanol (10× w/v) for an additional 48  h, and albicans: 105 cfu/mL) and incubated at 37°C for 18–24 h
the filtrates pooled. The ethanolic portion was removed (30°C for 48  h for C. albicans). The antimicrobial activ-
by rotary evaporation under reduced pressure at a low ity was measured as the zone of inhibition from the edge
heat (<40°C), leaving the water portion, which was freeze of the disc. Each sample and microbial combination was
dried and stored at −20°C. tested as at least four replicates.

Microbial strains MTT microdilution assay

All extracts were initially screened against one Gram The assay was performed as outlined by Steenkamp
positive (Staphylococcus aureus ATCC 29213) and two et al., with minor modifications [12]. Plant extracts were
Gram negative (Escherichia coli β-lactamase nega- prepared at 10  mg/mL in MH broth/20% DMSO (or
tive ATCC 25922 and Pseudomonas aeruginosa ATCC SAB/20% DMSO for C. albicans) and serially diluted
27853) bacterial strains, representing common patho- to give 20  μL final plant sample concentrations of
gens associated with wounds, sores and skin infections 2–1,000  µg/mL (0.02–10  µg/mL of antibiotic control)
[10, 11]. Selected extracts were also assayed against a in 96-well clear bottom microtitre plates. Test sam-
broader range of microorganisms including: Streptococ- ples were inoculated with 175  µL of microbial culture
cus pyogenes—Group A, clinical isolate; Salmonella typh- (A600 = 0.08 diluted 1/100 in MH broth); a sterile broth
imurium—Group B, clinical isolate; resistant S. aureus control was included. The plates were incubated at 37°C
strains (ATCC BAA 1026 community acquired methicil- for 18  h (30°C for C. albicans), 5  µL of a sterile metha-
lin resistant (CMRSA); wild multidrug resistant MRSA nolic solution (5 mg/mL) of MTT was added to each well
clinical isolate) and a yeast (Candida albicans—clinical and incubated at 37°C for 60 min. The MIC was the low-
isolate). est concentration of test material in which no growth (as
All microbial strains were kindly provided by Dr measured by no blue colour) was observed [13].
John Merlino (Department of Microbiology, Con-
cord Hospital, Sydney) and the work was approved Microbial viability determination
by the Macquarie University Biosafety Committee Plant extracts with antimicrobial activity (Table  1),
(Approval Reference 08/06/LAB, JOP250512BHA, TAN- as determined by the MTT microdilution assay, were
180512BHA). Müller Hinton II (MH) broth was used as assessed for their microbicidal/static nature by subcul-
the liquid medium for all bacteria; MH agar was used for turing onto fresh agar plates after the assay [14]. Sub-
the growth of E. coli, S. aureus, S. typhimurium and P. cultures not producing colonies were deemed to have
aeruginosa strains; horse blood agar (Edwards Instru- been affected by the plant extract by a microbicidal
ments, Australia), was used for S. pyogenes. Potato dex- mechanism. The minimum microbicidal concentration
trose agar (PDA, Oxoid, Basingstoke, UK) and Difco was defined as the concentration of the plant extract in
Sabouraud dextrose broth (SAB) were used for C. albi- the last well showing no further microbial growth on
cans. Gentamycin was used as the antibiotic control for subculture.
the sensitive strains of S. aureus, E. coli, S. typhimurium
and P. aeruginosa, vancomycin for the resistant strains of Results
S. aureus and penicillin G for S. pyogenes (all antibiot- Aqueous or 80% aqueous ethanolic extracts from nine
ics were supplied by Amresco, Ohio, USA). Fluconazole plants used by the Yaegl community were initially tested
(MP Biomedicals, Ohio, USA) or miconazole (MP Bio- against the Gram positive and Gram negative antibi-
medicals, LLC, France) was used as the antifungal con- otic sensitive bacteria, S. aureus (ATCC 29213), E. coli
trol for the assays involving C. albicans. (β-lactamase negative, ATCC 25922) and P. aeruginosa
Packer et al. BMC Res Notes (2015) 8:276 Page 4 of 7

Table 1  Initial screening for antimicrobial activity of extracts of plants used customarily to treat skin conditions
Planta (voucher number) Extractb Pseudomonas aeruginosa Staphylococcus aureus
(ATCC 27853) (ATCC 29213)

Disc diffusionc MTTd Disc diffusionc MTTd

Average (range) MIC median Average (range) MIC median
(range) (range)

Apple gum
 Lophostemon suaveolens (Sol. ex Gaertn.) Peter G. H2O (I) na na 2 (1–3) 188 (125–250)
Wilson & J.T. Waterh (MQ 73008908) EtOH na na 3 (2.5–3.5) 93 (63–125)
 Syncarpia glomulifera H2O (I) 5.5 (0–10) na 6.5 (5–10) 300 (250–500)
subsp. glomulifera (Sm.) Nied. (MQ 73009066) EtOH na na 3 (3–6) 63 (31–63)
Beach morning glory
 Ipomoea brasiliensis (L.) Sweet (MQ 73007958)A H2O (D) na na na na
EtOH na na na na
Bloodwood
 Corymbia intermedia (R.T. Baker) K.D. Hill & L.A.S. John- H2O (I) 1 (1) na 2 (1–3.5) 250 (250–500)
son (NSW 792670) EtOH na na 2.5 (2–4) 299 (174–500)
Coastal jackbean
 Canavalia rosea (Sw.) DC. (MQ 73008909) H2O (D) na na na na
EtOH na na na na
Cunjevoi
 Alocasia brisbanensis Domin (MQ 73008737) H2O (D) na na na na
EtOH na na na na
Sarsaparilla
 Smilax australis R.Br. (wide leaf ) (NSW 792674) H2O (I) na na na na
 Smilax glyciphylla Sm. (narrow leaf ) (NSW 792380) H2O (I) na na na na
Yellowvine
 Hibbertia scandens (Willd.) Gilg (MQ 73008905) H2O (D) na na na na
EtOH na na na na
 Antibiotic control (Gentamycin) 8 (8) 1.25 8 (8) 0.5
na no activity detected.
a
  Leaves of plants used in all cases except where indicated.
b
  Extraction method used: H2O—based on traditional preparation (D, decoction; I, infusion; CI, cold infusion), EtOH—80% aqueous ethanol.
c
  Values given are inhibition in mm from the edge of the disc; average (range) of 4 replicates.
d
  MIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of four replicates.
A
  Leaves and stems.

(ATCC 27853) using the disc diffusion and MTT assays. microorganisms viz. Candida albicans, Streptococcus
The plant extracts showed activity only against S. aureus pyogenes, Salmonella typhimurium and resistant strains
(in both disc diffusion and MTT assays) and P. aerugi- of S. aureus—multidrug resistant (MDRSA) and commu-
nosa (in disc diffusion assay only). L. suaveolens, C. inter- nity acquired methicillin resistant (CMRSA). Table 2 (and
media and S. glomulifera extracts had the highest levels Additional file 1) presents the activities of these extracts.
of antimicrobial activity (Table 1). Results for S. typhimurium are not included as no activ-
Antimicrobial activity for mixtures or crude plant ity was observed with any of the three plants. The 80%
extracts of between 0.1 and 1  mg/mL have been sug- aqueous ethanolic extract of S. glomulifera was the most
gested as being appropriate for further investigation active of the extracts, based on low MIC values (31  µg/
[15]. L. suaveolens, C. intermedia and S. glomulifera mL) against the tested bacteria as well as C. albicans. The
crude plant extracts were therefore further investigated 80% aqueous ethanolic extracts of both L. suaveolens and
as they had MIC values ≤500  µg/mL (Table  1). These S. glomulifera were microbicidal in their activity against
extracts were tested against a broader spectrum of the susceptible microorganisms.
Table 2  Broad spectrum antimicrobial screening of extracts of selected plants
MicrobeA: CA CMRSA MDRSA SP
Packer et al. BMC Res Notes (2015) 8:276

Plant ExtB Disc diffusionC MTT MICD Disc diffusionC MTT MICD Disc diffusionC MTT MICD Disc diffusionC MTT MICD
Average Median Average Median Average Median Average Median
(range) (range) (range) (range) (range) (range) (range) (range)

Corymbia intermedia H2O 6.5 (6–8) 500 (250–500) 3 (2.5–3) 500 (250–500) 3 (2–4) 500 na na
(R.T. Baker) K.D. Hill & EtOH 2 (0–6.5) 500 (250–500) 2.5 (2–5.5) 375 (250–500) 2.5 (1.5–4) 250 (250–500) na 250 (125–>1,000)
L.A.S. Johnson (NSW
792670)
Lophostemon suaveolens H2O 6 (5.5–6) 1,000 (500–1,000) 2.5 (2–3) 1,000 (500–1,000) 2.5 (2–3) 1,000 (250–1,000) na na
(Sol. ex Gaertn.) Peter G. EtOH 6.5 (6–7) 125 (31–125) 3.5 (3–4.5) 63 (63–125) 3.5 (3–5) 125 (63–250) na 125 (16–>1,000)
Wilson & J.T. Waterh
(MQ 73008908)
Syncarpia glomulifera H2O 6.5 (5.5–7.5) 500 (250–1,000) 3 (3–4) 375 (250–500) 2.5 (2–3) 250 (31–500) na na
subsp. glomulifera (Sm.) EtOH 5.5 (5–6) 31 (16–1,000) 5 (2.5–5) 31 (16–1,000) 3.5 (2–5) 31 (16–>1,000) 3 (2–4.5) 31 (31–>1,000)
Nied. (MQ 73009066)
Antibiotic control 17a 2.8b 3c 1.5c 2c 1.6c 8d 0.02d

Antibiotic controls: a fluconazole, bmiconazole ,cvancomycin, dpenicillin.

na no activity detected.
A
 CA: Candida albicans (clinical isolate); CMRSA: S. aureus (ATCC BAA 1026); MDRSA: S. aureus (multidrug resistant clinical isolate); SP: Streptococcus pyogenes (Group A—clinical isolate).
B
  Extraction method used: H2O, traditional prep; EtOH, 80% aqueous ethanol.
C
  Values given are inhibition in mm from the edge of the disc; average of four replicates (plant extracts tested at 1 mg/disc and antibiotic controls tested at 2 µg/disc).
D
  MIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of 4 replicates.
Page 5 of 7
Packer et al. BMC Res Notes (2015) 8:276 Page 6 of 7

Discussion gum (Lophostemon suaveolens or Syncarpia glomulifera)

Ethnobotanical studies conducted by us with Yaegl Abo- have been used as antiseptics by the Yaegl community
riginal Elders of northern New South Wales, Australia, [5]. These two plants are sometimes interchangeably used
described the use of Alocasia brisbanensis, Canavalia due to their similar morphology.
rosea, Corymbia intermedia, Hibbertia scandens, Ipomoea No antimicrobial activity studies have been reported
brasiliensis, Lophostemon suaveolens, Smilax australis, for L. suaveolens or the leaves of S. glomulifera. How-
Smilax glyciphylla and Syncarpia glomulifera for the treat- ever, both plants have been found to contain the follow-
ment of wounds, sores and skin infections [5]. None of ing compounds that have been independently reported
these plants have been investigated for their antimicrobial for antimicrobial activity [18–22]. Leaves of L. suaveolens
activity, with the exception of C. rosea [16]. The success have been found to contain α-pinene, β-caryophyllene,
of finding antimicrobial activity and bioactive compounds aromadendrene, globulol and spathulenol [23], which
of medicinal value from traditional/customary medicines have been independently reported for antimicrobial activ-
[3], prompted the investigation of these plants. ity. The leaves of S. glomulifera also contain α-pinene,
The plants were extracted with water to mimic the aromadendrene and globulol [24]. Eucalyptin, which has
common customary practice of the community and/or been reported to be antibacterial, has been found in the
with 80% aqueous ethanol, as a standard natural products leaf wax of S. glomulifera [25]. Additionally, S. glomulifera
research method [9]. The plant parts extracted were the bark has been reported to be antibacterial and to con-
same as those used by the Yaegl community, except for tain the antibacterial compounds betulinic acid, oleanolic
bloodwood (C. intermedia) and apple gum (L. suaveolens acid-3-acetate and ursolic acid-3-acetate [26].
or S. glomulifera), where customarily the sap or latex was This is the first report of antimicrobial activities for C.
used in the treatment of wounds. However, as sap or latex intermedia and L. suaveolens and the leaves of S. glomu-
were not readily accessible, we tested the leaves at the lifera. These results warrant further investigation of the
request of the Yaegl Elders. active ingredients of these medicinal plants.
The microorganisms selected for the assays are com-
monly implicated in wounds, sores and skin infections Conclusion
[11]. The two assay methods were chosen as they comple- 80% aqueous ethanolic extracts of Lophostemon sua-
ment each other in that the disc diffusion assay is simple veolens and Syncarpia glomulifera showed microbicidal
but not always appropriate for non-polar and large mol- activity at concentrations of <200 µg/mL against C. albi-
ecules [17] and the more sensitive, versatile and quantita- cans and both sensitive and resistant strains of S. aureus.
tive MTT microdilution assay can have interference with Aqueous extracts of L. suaveolens and S. glomulifera
the colour and redox activity of plant extracts. and aqueous and ethanolic extracts of Corymbia inter-
The extracts of C. intermedia, L. suaveolens and S. glomu- media exhibited microbicidal activity against a range of
lifera had reasonable levels of antimicrobial activity (MIC both antibiotic sensitive and resistant strains of micro-
0.1–1  mg/mL; [15]) against antibiotic sensitive and resist- organisms at levels below 1,000  µg/mL. This is the first
ant Staphylococcus aureus as well as the fungus Candida report of antimicrobial activities for C. intermedia and
albicans (clinical isolate). Although antibacterial activity of L. suaveolens and the leaves of S. glomulifera. This study
the ethanolic extract of C. rosea leaves has been previously demonstrates the value of customary knowledge in the
reported against S. aureus, albeit at a low level [16], no anti- identification of new sources of antimicrobial treatments.
bacterial activity was seen in this study, possibly due to the
different bacterial strains and assay conditions used. Additional file
The 80% aqueous ethanolic extracts of C. intermedia,
L. suaveolens and S. glomulifera were substantially more Additional file 1.  Picture showing typical results of the disc diffusion
assay.
active than the aqueous extracts. This indicates that the
antimicrobial components in these plants were more
easily extractable in 80% aqueous ethanol. S. glomulifera Authors’ contribution
was the most active of the extracts (Table  2). The 80% JP and TN carried out the extractions and biological assays, wrote the paper
and evaluated the data. YCE provided the valuable customary knowledge and
aqueous ethanolic extracts of both L. suaveolens and S. input to the research directions. DH collected the plant samples and contrib-
glomulifera were microbicidal in their activity against the uted to the literature search. JJ and SV initiated and directed the research. All
susceptible microorganisms. authors read and approved the final manuscript.
Sap from bloodwood (C. intermedia) has been used by Author details
the Yaegl community in the treatment of wounds [5]. No 1
Indigenous Bioresources Research Group, Faculty of Science and Engineer-
chemical or biological studies have been reported for C. ing, Macquarie University, North Ryde, Sydney 2109, Australia. 2 Yaegl Local
Aboriginal Land Council, Jubilee St, Hillcrest, Maclean, NSW 2463, Australia.
intermedia. Milky sap and ash from the bark of the apple
 Packer et al. BMC Res Notes (2015) 8:276 Page 7 of 7

Acknowledgements 9. Houghton PJ, Raman A (1998) Laboratory handbook for the fractionation
The authors thank the Yaegl Elders for sharing their knowledge on behalf of of natural extracts. Chapman & Hall, London
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