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Journal of Ethnopharmacology 132 (2010) 340–343

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

thnopharmacological communication

valuation of the anti-inflammatory properties of Dodonaea polyandra, a Kaanjuraditional medicine

radley Simpsona, David Claudieb, Nick Smitha, Jiping Wanga, Ross McKinnona, Susan Semplea,∗

School of Pharmacy and Medical Sciences, University of South Australia, Adelaide 5000, South Australia, AustraliaChuulangun Aboriginal Corporation, PMB 30, Cairns Mail Centre, Cairns 4870, Queensland, Australia

r t i c l e i n f o

rticle history:eceived 19 March 2010eceived in revised form 18 June 2010ccepted 6 July 2010vailable online 13 July 2010

eywords:odonaeaouse ear oedema

nti-inflammatoryustralian indigenous medicine

a b s t r a c t

Ethnopharmacological relevance: Extracts of the medicinal plant species Dodonaea polyandra were investi-gated as part of a collegial research partnership between Northern Kaanju traditional owners representedby Chuulangun Aboriginal Corporation (centred on the Wenlock and Pascoe Rivers, Cape York Peninsula,Queensland, Australia) and university-based researchers. D. polyandra, known as “Uncha” in Kaanju lan-guage, is used in Northern Kaanju Traditional Medicine for relief from pain associated with toothacheand related ailments. The species has a restricted distribution in Cape York Peninsula and there has beenno previous Western scientific investigation of its pharmacology or chemistry.Aim of the study: The current study investigates the anti-inflammatory effects of several extracts from D.polyandra.Materials and methods: Phytochemical screening was conducted using TLC. Anti-inflammatory effectsof leaf extracts were determined using an acute mouse ear oedema model induced by croton oil and12-o-tetradecanoylphorbol-13-acetate (TPA) chemical irritants.

Results: Flavonoid and terpenoid secondary compounds were detected in leaf extracts of D. polyandra.Non-polar hexane and methylene chloride/methanol extracts showed potent inhibition of inflammationin TPA-induced mouse ear oedema by 72.12 and 79.81%, respectively, after 24 h at 0.4 mg/ear.Conclusion: In a mouse model of acute inflammation, this study revealed that leaf extracts of D. polyan-dra possess significant anti-inflammatory potential. These results contribute to a Western scientificunderstanding of the ethnopharmacological use of the plant in Northern Kaanju Medicine for reducing tooth-related pain.

. Introduction

The research reported here is part of a collegial project initiatedy traditional owners of the Northern Kaanju homelands (Kuuku’yu) centred on the Wenlock and Pascoe Rivers in Cape York Penin-ula in the state of Queensland, Australia. Traditional owners wereeen to examine opportunities for development of their naturalesources such as medicinal plants in a way that could contribute toconomic development on the homelands. A research partnershipas formed with university-based researchers to share traditional

nd Western scientific perspectives on the pharmacological prop-

rties of traditionally used native plants.

Collaborative field work on Northern Kaanju homelandsetween Kaanju and Western researchers identified Dodonaeaolyandra (Sapindaceae), traditionally known as “Uncha”, as a

∗ Corresponding author at: University of South Australia, GPO Box 2471, Adelaide001, South Australia, Australia. Tel.: +61 8 83022395; fax: +61 8 83021087.

E-mail address: susan.semple@unisa.edu.au (S. Semple).

378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.07.012

© 2010 Elsevier Ireland Ltd. All rights reserved.

strong candidate for further study. The species is a small shrubwhich grows in a localized area along the North and East coastsof Cape York Peninsula. In Kaanju traditional medicine the plantis a favored medicine for relief of pain associated with toothacheand removal of rotten teeth. Kaanju researchers in the project usethe plant by breaking off one of the terminal branchlets with a leafattached. The plant material is applied directly to the mouth orinserted into the hole left after the removal of a rotten tooth. Par-ticular Kuuku I’yu (Northern Kaanju) people are entitled to use theirknowledge about this plant. In Kuuku I’yu traditional law the par-ticular knowledge of this medicinal plant and its usage is passedthrough the male (patrilineal) bloodline.

Previous studies have reported anti-inflammatory effects fromextracts of a related species Dodonaea viscosa using carrageenan-induced rat paw oedema models (Alagarsamy et al., 2007).

However, the active anti-inflammatory constituents were not iden-tified. No previous report of the pharmacological properties for D.polyandra has been published in the scientific literature.

The aim of the study was therefore to evaluate the anti-inflammatory properties of several extracts prepared from the

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eaves of D. polyandra and to examine the classes of compoundshich may be responsible for the activity. This article presents

esults for the anti-inflammatory effects in a mouse ear oedemaodel using croton oil and 12-o-tetradecanoylphorbol-13-acetate

TPA) as inflammation inducers.

. Materials and methods

.1. Collaborative research agreement

At the commencement of the project a written collabora-ive agreement was established between Chuulangun Aboriginalorporation and University of South Australia to include equal shar-

ng of project intellectual property and decision-making for howroject intellectual property is used. The agreement also ensuresrotection of indigenous cultural and intellectual property forraditional owners. The agreement was approved by the Humanesearch Ethics Committee of the University of South Australia.

.2. Plant material

Plant material was collected by Kaanju researchers at Chu-la outstation (Kaanju homelands), Central Cape York Peninsula,ueensland, in collaboration with ethnobotanist (author N. Smith)ho confirmed Western scientific species name. The leaves of D.

olyandra (collection number NMS5293) were collected from aopulation of 30 plant samples from both male and female typesjuveniles ≤2 m) on Chuula Homeland in December 2007. Voucherpecimens and details of plant location (13◦07′14′′, 142◦59′45′′)ere recorded and lodged at Brisbane Herbarium, Queensland

voucher specimen number AQ 749703). Plant material wasllowed to air dry in the shade and packed into paper bags forransportation. The leaves were stored at −20 ◦C until extraction.

.3. Preparation of extracts

Leaf material (50 g) was extracted with 80% (v/v) aqueousthanol (LE) using a solvent to dry plant material ratio of 5:1 at5 ◦C with agitation for 24 ± 1 h. After 24 h the ethanolic extractas decanted and filtered in vacuo through Whatman No.1 filteraper (Whatman, UK). A second equivalent amount of solvent wasdded and allowed to extract for a further 24 h before removal andltration. Leaves (50 g) were also sequentially extracted with n-exane, followed by methylene chloride/methanol (1:1) and lastly0% (v/v) aqueous ethanol. The filtered extracts were concen-rated using Buchi Rotavap at 40–42 ◦C to remove solvent andubsequently freeze-dried (Christ Alpha 2-4 LD) (if required) toemove residual water. The yield of LE extract was 34.4% (17.7 g).

hile for the sequential method they were 1.34% (670 mg), 15.4%7.69 g) and 14.1% (7.03 g) for the n-hexane (SeLH), methylene chlo-ide/methanol (1:1) (SeMM) and 80% (v/v) aqueous ethanol (SeLE)xtracts, respectively. Long-term storage of dried extracts was at20 ◦C.

.4. Reagents

Croton oil, TPA, hydrocortisone 21-hemisuccinate sodium saltnd p-anisaldehyde were obtained from Sigma Chemical Co. (St.ouis, MO, USA). Solvents were of analytical grade and purchasedrom Merck (Darmstadt, Germany). TLC plates were silica gel 60254, aluminum backed from Merck (Darmstadt, Germany).

.5. Phytochemical screening

Crude extracts from D. polyandra leaves were tested on TLCor the presence of flavonoids and terpenoids. Natural products

rmacology 132 (2010) 340–343 341

(NP)–polyethylene glycol (PEG) 4000 and anisaldehyde reagents(Wagner and Bladt, 1996), respectively, were sprayed onto devel-oped TLC plates (normal phase, methylene chloride/methanol(90:10)) and used to visualize these types of secondary metabo-lites. NP–PEG4000 reagent was composed of solution A (1%diphenyl boric acid-�-ethyl amino ester) followed by application ofsolution B (5% ethanolic PEG), viewed under UV 365 nm. Anisalde-hyde was composed of 95 mL ethanol, 2.5 mL acetic acid, 2.5 mLsulphuric acid and 0.5 mL anisaldehyde, viewed under naturallight.

2.6. Animals

Male Balb/C mice 7–9 weeks old and weighing 20–25 g obtainedfrom Animal Care Facility, University of Adelaide, were used. Micewere housed in cages at constant room temperature (20 ± 2 ◦C)with access to food (standard rat/mouse pellet) and water ad libi-tum. A 12 h day/night cycle was maintained in the animal holdingfacility (Reid Building Animal House, University of South Australia)and all experiments were carried out during the day phase. Exper-iments were conducted in accordance with ethical standards asoutlined in the Australian Code of Practice for the Care and Useof Animals for Scientific Purposes (9th ed.) and approved by TheInstitute of Medical and Veterinary Science Animal Ethics Commit-tee.

2.7. Mouse ear oedema model

The experiment was conducted based on previously describedmethods (Bralley et al., 2008). Baseline measurements of earthickness were measured using a digital micrometer (±0.001 mm,Mitutoyo, Japan) prior to the experiment. Croton oil (0.1 mg/ear)or 12-o-tetradecanoylphorbol 13-acetate (TPA) (2.5 �g/ear) (dis-solved in acetone) was then applied in a volume of 20 �L to theinner surface of the right ear and 20 �L of acetone to the left earas control. Thirty minutes later the test samples (dissolved in 80%ethanol) were applied to the inner surface of the right ear and80% ethanol on the left ear to the respective treatment groups.Hydrocortisone 21-hemisuccinate sodium salt (6 or 2 mg/ear) wasused as a positive control based upon doses used in a similarstudy (Iskander et al., 2002). The extract doses tested were 0.004,0.04 and 0.4 mg/ear based on preliminary experiments that indi-cated that a dose of 4 mg/ear or above gave poor extract solubilityand no additional anti-inflammatory activity. At 2, 4, 6, 8, 24 and48 h after the application, the ear thicknesses were measured. Anon-treated control group to which only croton oil or TPA withsolvent controls was applied was used as a measure of max-imum inflammation. Percent inhibition of inflammation of testsample was calculated relative to this group. Maximum inflam-mation is achieved at 6–8 h post-application of sample and is acommonly used endpoint for this particular model (Iskander et al.,2002; Medeiros et al., 2007). Therefore, results after the 6-h timepoint were used as the primary endpoint for comparison of resultsbetween the extracts. Following completion of the experiment micewere euthanized by inhalation of isoflurane followed by cervicaldislocation.

2.8. Statistics

Results are presented as mean ± S.E.M. and statistical signif-icance between groups was determined by one-way analysisof variance (ANOVA) with Dunnett t-test post hoc analysis. P-values < 0.05 were considered significant.

342B.Sim

psonet

al./JournalofEthnopharmacology

132 (2010) 340–343

Fig.1.TLC

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.polyandra

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Table 1Effect of leaf extracts on croton oil and TPA-induced mouse ear oedema.

)

4 h 6 h 8 h 24 h 48 h

0.095 ± 0.007 0.121 ± 0.012 0.153 ± 0.027 0.103 ± 0.024 0.036 ± 0.0030.054 ± 0.019 (43.1%)* 0.093 ± 0.011 (23.1%) 0.124 ± 0.024 (18.8%) 0.072 ± 0.011 (30.1% 0.042 ± 0.010 (−16.7%)0.019 ± 0.005 (80.0%)* 0.051 ± 0.008 (58.8%)* 0.106 ± 0.013 (30.7%) 0.044 ± 0.021 (57.3%) 0.017 ± 0.024 (52.8%)0.031 ± 0.002 (67.4%)* 0.047 ± 0.011 (61.2%)* 0.101 ± 0.037 (34.0%) 0.045 ± 0.035 (56.3%) 0.012 ± 0.011 (66.7%)0.024 ± 0.001 (74.7%)* 0.015 ± 0.003 (87.6%)* 0.022 ± 0.007 (85.6%)* 0.020 ± 0.013 (80.6%)* −0.003 ± 0.018 (110.0%)*

0.122 ± 0.038 0.243 ± 0.004 0.244 ± 0.024 0.208 ± 0.039 0.064 ± 0.0080.040 ± 0.011 (67.2%)* 0.096 ± 0.009 (60.5%)* 0.137 ± 0.007 (43.9%)* 0.048 ± 0.013 (76.9%)* 0.028 ± 0.004 (56.2%)*

0.055 ± 0.015 (54.9%) 0.106 ± 0.016 (56.4%)* 0.131 ± 0.015 (46.3%)* 0.058 ± 0.009 (72.1%)* 0.040 ± 0.010 (37.5%)0.107 ± 0.014 (12.3%) 0.145 ± 0.007 (40.3%)* 0.137 ± 0.005 (43.8%)* 0.042 ± 0.007 (79.8%)* 0.017 ± 0.011 (73.4%)*

0.124 ± 0.020 (−1.6%) 0.152 ± 0.009 (37.4%)* 0.180 ± 0.010 (26.23%)* 0.077 ± 0.023 (63.0%)* 0.037 ± 0.006 (42.2%)0.047 ± 0.019 (61.5%) 0.083 ± 0.001 (65.8%)* 0.169 ± 0.003 (30.7%)* 0.095 ± 0.047 (54.3%)* 0.042 ± 0.001 (34.4%)

reated). Asterisks denote statistical significance of treated groups compared to control. LE, 80% aqueous ethanol extract of Dodonaea polyandra leaves;tion inducer) in acetone.

Group Dose (mg/ear) Ear thickness difference (mm

2 h

Control (CO) 0.1 0.018 ± 0.006LE 0.004 0.024 ± 0.007 (−33.3%)

0.04 0.014 ± 0.005 (22.2%)0.4 0.008 ± 0.004 (55.5%)

Hydrocortisone 6.0 0.015 ± 0.004 (16.7%)

Control (TPA) 0.0025 0.037 ± 0.013LE 0.4 0.016 ± 0.002 (56.7%)*

SeLH 0.4 0.005 ± 0.004 (86.5%)*

SeMM 0.4 0.021 ± 0.001 (43.2%)SeLE 0.4 0.039 ± 0.005 (−5.4%)Hydrocortisone 2.0 0.024 ± 0.010 (35.1%)

Results are expressed as mean ± S.E.M. (n = 3 for CO treated and n = 4 for TPA tCO and TPA, croton oil and 12-o-tetradecanoylphorbol-13-acetate (inflamma

* p < 0.05.

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pounds from Dodonaea viscosa. Planta Medica 62, 154–159.Sánchez, T., Moreno, J.J., 1999. Role of leukocyte influx in tissue prostaglandin

B. Simpson et al. / Journal of Eth

. Discussion

To our knowledge there has been no previously published studyeporting the pharmacological activity of D. polyandra. This studyas demonstrated that D. polyandra leaf extracts possess significantnti-inflammatory activity using the mouse ear oedema model ofcute inflammation induced by croton oil and TPA. The greatestffects were observed from the extracts which were extracted withntermediate to non-polar solvents (methylene chloride/methanolnd n-hexane). However, as indicated in the TLC profile, there wasome overlap of components in extracts. Inhibitory values of up to0% were measured for SeLH and SeMM indicating that the activeubstance(s) are non-polar in nature.

In the mouse ear model, TPA (and croton oil from which TPAay be extracted) as an inflammation inducer is responsible for

he local influx of neutrophils and leukocytes and an increase in thexpression of the inducible COX-2 enzyme at the site of inflamma-ion (Sánchez and Moreno, 1999). The significant inhibitory effectsf the extracts tested suggest that the prostaglandin pathway ofhe inflammation process may be a potential target of the activeomponent(s).

A few discrete species of the genus Dodonaea have been widelynvestigated both chemically and pharmacologically (Payne andefferies, 1973; Rojas et al., 1996; Amabeoku et al., 2001; Moshia etl., 2005). The most recognized species of Dodonaea in the literatures D. viscosa, which is closely related to D. polyandra. The most abun-ant secondary compounds identified and isolated from Dodonaeare flavonoids and diterpenoids (Ghisalberti, 1998). Phytochemicalcreening of D. polyandra conducted during this study suggests thispecies is also rich in these classes of secondary metabolites. Bothf these classes of compounds are well known for their biologicalctivities, although none of those which have been isolated fromther Dodonaea species have been attributed to anti-inflammatoryctivity. It may be speculated that the compounds responsible forhe prominent spots observed from TLC screening here are at leastn some part contributing to the anti-inflammatory activities of D.olyandra leaf extracts. Further analysis is required to characterizehe active compounds, the mechanism of their anti-inflammatoryctions and toxicity.

Demonstrating the anti-inflammatory activity of D. polyandra issignificant outcome for Kaanju people as this provides Western

cientific evidence which may be used in conjunction with tra-itional use evidence to support the development of plant-basededicinal products. It is envisaged that this will support the fur-

her research and development of sustainable products based on

his species. Chuulangun Aboriginal Corporation is planning whathey call “whole picture development” of the homeland’s naturalesources. The Corporation is currently examining internationalodels for conservation and sustainable development based on

raditional knowledge systems (Karst, 2010).

rmacology 132 (2010) 340–343 343

This study has not explored other possible reasons as to whytoothache is relieved by this plant. However, it provides a workingbasis to investigate other possibilities. Additionally, further studyneeds to examine the administration of plant material in a way thatmore closely reflects the traditional usage methods – where plantmaterial is used as a direct application to the affected area.

In conclusion, this study has provided the first report of pharma-cological activity from D. polyandra. Leaf extracts of this plant arepotent inhibitors of inflammation in the croton oil and TPA-inducedmouse ear oedema model. The findings of the study contribute toa Western scientific understanding of the ethnopharmacologicaluse of the plant in Kaanju Medicine. Further work is under wayto determine the chemistry of the active component(s) and themechanisms by which they act.

Acknowledgments

This work was funded by an Australian Research Council LinkageProject grant. The authors are grateful for the involvement of Kaanjupeople and their enthusiasm towards the Kaanju Medicinal Plantsproject.

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