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Industrial Crops and Products 64 (2015) 167–174

Contents lists available at ScienceDirect

Industrial Crops and Products

jo ur nal home p age: www.elsev ier .com/ locate / indcrop

hysicochemical, antioxidant and anti-cancer activity of a Eucalyptusobusta (Sm.) leaf aqueous extract

uan V. Vuonga,b, Sathira Hiruna,b, Tiffany L.K. Chuena,b, Chloe D. Goldsmitha,b,enjamin Munrob, Michael C. Bowyera,b, Anita C. Chalmersb, Jennette A. Sakoff c,hoebe A. Phillipsd, Christopher J. Scarletta,b,e,∗

Pancreatic Cancer Research, Nutrition Food & Health Research Group, Newcastle, NSW, AustraliaSchool of Environmental and Life Sciences, University of Newcastle, Ourimbah, NSW, AustraliaDepartment of Medical Oncology, Calvary Mater Newcastle Hospital, Waratah, NSW, AustraliaPancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, Prince of Wales Clinical School, Faculty of Medicine, The University of Newouth Wales, Sydney, AustraliaCancer Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia

r t i c l e i n f o

rticle history:eceived 17 July 2014eceived in revised form 22 October 2014ccepted 27 October 2014vailable online 13 November 2014

eywords:ucalyptus robusta (Sm.)olyphenolsntioxidantancreatic cancer

a b s t r a c t

Eucalyptus robusta (Sm.) (ER) is a widely distributed tree native to the east coast of Australia, which hasalso been established in numerous other countries. ER leaves contain high levels of essential oils and arerich in total phenolic compounds (TPC), which have been linked with health benefits; however, there islimited information on the bioactivity of ER leaf extracts. This study aimed to optimise water extractionconditions for TPC, prepare a spray-dried powdered extract and test its physicochemical, antioxidantand anti-proliferative properties. The results showed that optimal water extraction conditions for TPCwere 85 ◦C, 15 min and a water-to-leaf ratio of 20:1 mL/g. Under these conditions, spray-dried powderedextract was prepared with a recovery yield of 85%. The extract was water-soluble and had a TPC level of407 mg GAE/g. It also possessed potent antioxidant capacity, comparable to pure ascorbic acid, but higherthan pure �-tocopherol. In addition, the powdered extract demonstrated significant activity against a

panel of cancer cell lines, which included cancers of the pancreas, breast, lung, brain, skin, colon andovary. Of note, the ER extract exerted a more significant toxic effect on pancreatic cancer (PC) cellscompared to gemcitabine, the first line chemotherapeutic agent for PC. We suggest that future studiesshould purify individual bioactive compounds from ER for further investigation of its potential healthpromoting and anti-cancer activity.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

The natural distribution of Eucalyptus robusta (Sm.) (ER), alsonown as swamp mahogany, is in a narrow belt along the east coastf Australia (Boland et al., 2006). However, it has been well estab-ished in other countries and reportedly occupies a total area of.3 million hectares worldwide (Rejmanek and Richardson, 2011).

he eucalyptus leaf contains high levels of essential oils and islso rich in total phenolic compounds (TPC), with several studiesxtracting phenolics from the eucalyptus leaf using either organic

∗ Corresponding author at: University of Newcastle, School of Environmental andife Sciences, Head, Pancreatic Cancer Research, Brush Rd, Ourimbah 2258, Australia.el.: +61 2 4348 4680; fax: +61 2 4348 4145.

E-mail address: c.scarlett@newcastle.edu.au (C.J. Scarlett).

ttp://dx.doi.org/10.1016/j.indcrop.2014.10.061926-6690/© 2014 Elsevier B.V. All rights reserved.

solvents or a mixture of organic solvents and water (Amakura et al.,2002; Bachir and Benali, 2012; Bhagat et al., 2012; Rejmanek andRichardson, 2011; Takasaki et al., 2000). Numerous methods havebeen employed for the extraction of phenolic compounds, withmany procedures often associated with high-energy costs and theproduction of excessive solvent waste, increasing hazard poten-tial as well as increased expenses associated with its disposal. Assuch, there is a need for the development of “green” extractionprocedures with water being the ideal solvent of choice. Impor-tantly water is a safe, inexpensive, and environmentally friendlysolvent and several studies have extracted polyphenols from euca-lyptus leaf under aqueous conditions (Chapuis-Lardy et al., 2002;

Hasegawa et al., 2008); however, no previous studies have opti-mised conditions for water extraction to yield maximal quantitiesof phenolic compounds from eucalyptus leaf, nor assessed theactivity of aqueous extracts as potential anti-cancer agents.

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Recent studies have shown potential anti-cancer activity ofucalyptus extracts against some cancer cells, such as cancers ofhe colon, lung, prostate, ovary, cervix, liver and neuroblastoma (Al-ayed et al., 2012; Bhagat et al., 2012; Islam et al., 2012). Thus, thequeous ER extracts may hold potent anti-cancer agents againstancers, including pancreatic cancer, a disease with very limitedherapeutic options (Scarlett et al., 2011). To date, activity againstancreatic cancer has yet to be investigated.

To obtain the powdered extract for utilisation in the food orharmaceutical industries, plant extracts have been prepared usingifferent drying methods such as evaporation, freeze drying andpray drying; of which spray drying is the most popular method ast is rapid and can be completed in a very short time (Walzel anduruta, 2011). To date, the preparation of spray-dried extracts fromucalyptus leaves has not been reported. As such, this study aimedo optimise water extraction conditions for high phenolic com-ound yield from ER leaves; to prepare a crude powdered ER extractsing a spray drier and to test its physicochemical, antioxidant andnti-cancer properties.

. Materials and methods

.1. Eucalyptus robusta leaf collection and preparation

The Eucalyptus robusta leaves (immature and mature) werearvested in March, 2013 from cultivated plants located at Ourim-ah, Central Coast, NSW, Australia (latitude of 33.4◦S, longitudef 151.4◦E). The plants were authenticated by one of the authorsA.C.C.) and a voucher specimen deposited at the Don McNairerbarium (accession number 10492), the University of New-astle, NSW, Australia. The materials were immediately dried at0 ◦C to constant weight. Using a blender (John Morris Scientific,hatswood, NSW, Australia) the dried leaves were ground, thenieved (≤1 mm) using a 1 mm EFL 2000 stainless steel mesh sieveEndecotts Ltd., London, England), then packed in a sealed containernd stored at 5 ◦C until required.

.2. Experimental design

Based on a preliminary study (data not shown), three impor-ant parameters and their optimal ranges were selected for theptimisation of extraction conditions for increased total pheno-ic compound (TPC) yield. These included temperature (70–90 ◦C),ime (5–25 min) and water-to-leaf ratio (10:1–100:1 mL/g). Extrac-ion was conducted using a shaking water bath (Ratek Instruments,oronia, VIC, Australia). Response surface methodology (RSM) with

Box–Behnken factorial design with 3 center points was used forhe experimental design (Table 1) and optimisation. The experi-

ental data obtained for the fifteen experimental runs were fittedo the following second-order polynomial model (EQ1):

+ ˇ0 +k∑

i=1

ˇiXi +k−1∑

i = 1

i < j

ˇijXiXj +k∑

i=1

ˇiiX2i (1)

here various Xi values are independent variables affecting theesponses Y; ˇ0, ˇi, ˇii, and ˇij are the regression coefficients forntercept, linear, quadratic, and interaction terms, respectively; and

is the number of variables.To prepare a spray-dried ER powdered extract for further anal-

sis of its phytochemical, antioxidant and anti-cancer properties,

round ER leaves were extracted in water under optimal condi-ions (85 ◦C, 15 min, water-to-leaf ratio of 20:1 mL/g). The extractas filtered using filter paper (2.5 micron; Lomb Scientific, Taren

oint, NSW, Australia) to remove solids. The filtrate was then

d Products 64 (2015) 167–174

concentrated using a rotary evaporator (Buchi Rotavapor B-480,Buchi Australia, Noble Park, Vic., Australia) at 55 ◦C with reducedpressure to one third of the initial volume. The concentrated extractwas then dried using a Buchi mini spray drier (Model B-480, BuchiAustralia, Noble Park, Vic., Australia) under optimal conditions(Vuong et al., 2013a) with inlet temperature at 180 ± 1 ◦C, outlettemperature at 115 ± 1 ◦C, aspiration rate at 100% and the com-pressed air flow at 301 L/h. The extracted powder was then storedat −18 ◦C until required.

2.3. Determination of physical properties

Physical properties of ER leaves and ER powdered extract includ-ing moisture content, dry weight, water activities, bulk density,solubility, pH and colour were determined as described in previousstudies (S ahin Nadeem et al., 2011; Vuong et al., 2012).

2.4. Determination of total phenolic compounds, flavonoids andproanthocyanidins of the ER powdered extract

Total phenolic content (TPC), flavonoids and proanthocyaninswere determined using colorimetric assays and measured using aUV Vis Spectrophotometer (Varian Australia Pty. Ltd., VIC Australia)as described by Vuong et al. (2013b). Each analytical procedurewas performed in triplicate. For the solution extract, the extractwas diluted 20 times with water prior to analysis. For the pow-dered extract, 4 mg of powder was diluted in 10 mL of water forthe analysis. Gallic acid in the ranges of 6.25–100 �g/mL was usedas the standard and the results were expressed as mg of gallic acidequivalents per g of sample (mg GAE/g).

The flavonoid content of ER powdered extract was measuredusing a method described by Zhishen et al. (1999). 4 mg of pow-der was diluted in 10 mL of water for analysis. Catechin in theranges 6.25–100 �g/mL was used as the standard and the resultswere expressed as mg of catechin equivalents per gram of sample(mg CE/g).

Proanthocyanidin content of the ER powdered extract wasdetermined using a method described by Li et al. (2006). 4 mg ofpowder was diluted in 10 mL of water for analysis. Catechin in theranges 6.25–100 �g/mL was used as the reference standard, and theresults expressed as mg of catechin equivalents per gram of sample(mg CE/g).

2.5. Determination of antioxidant activity of the ER powderedextract

To determine the antioxidant properties of ER powdered extractand to compare with those of �-tocopherol (90% purity) and ascor-bic acid (95% purity), the powdered extract, �-tocopherol andascorbic acid were diluted at the same concentration of 100 �g/mL.Butylated hydroxytoluene (BHT) in the ranges 6.25-100 �g/mL wasused as the standard and the results were expressed as mg of BHTequivalents per gram of sample (mg BHT/g). To further test thedependence of antioxidant capacity on the dose of the powderedextract applied; the powdered extract was diluted in a series ofconcentrations (25, 50, 100 and 200 �g/mL) for analysis.

Six different antioxidant assays were employed to testantioxidant properties of ER powdered extract. The SSA (0.6 Msulfuric acid, 28 mM sodium phosphate and 4 mM ammo-nium molybdate) assay was conducted as described by Prietoet al. (1999). The ABTS (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) assay was performed as described by Thaipong et al.

(2006). The DPPH (1,1-diphenyl-2-picrylhydrazyl) assay describedby Vuong et al. (2013b) and hydrogen peroxide (H2O2) assayreported by Kannan et al. (2013) were employed for measuringantioxidant capacity of ER powdered extract. Finally, the CUPRAC

Q.V. Vuong et al. / Industrial Crops and Products 64 (2015) 167–174 169

Table 1Variables, experimental design and observed response.

Runs X1 temperature (◦C) X2 time (min) X3 water-to-leaf ratio (mL/g) Extraction yield (mg GAE/g)

1 90 12.5 10:1 128.112 70 12.5 10:1 101.003 70 5 55:1 105.644 90 5 55:1 115.715 80 12.5 55:1 127.636 80 20 100:1 142.327 80 12.5 55:1 127.878 70 20 55:1 103.359 80 12.5 55:1 127.61

10 80 5 10:1 108.5711 90 12.5 100:1 146.1812 80 5 100:1 136.10

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15 90 20

cupric ion reducing antioxidant capacity) assay of Apak et al.2004) and the FRAP (ferric reducing antioxidant power) assay ofhaipong et al. (2006) were also applied for determining antioxi-ant properties of ER powdered extract.

.6. HPLC analysis of bioactive components in the crude ERxtract

To determine the major bioactive components, including galliccid, syringic acid, catechin, epicatechin, quercetin and apigenin inhe ER powdered extract. 0.2 g of the powdered extract was dilutedhoroughly in 10 mL of water and then filtered through a 0.45 �mellulose syringe filter for HPLC analysis. Analysis was conductedsing a Shimadzu HPLC system (Shimadzu Australia, Rydalmere,SW, Australia) using UV detection at 210 nm and 280 nm, on a50 × 4.6 mm Synergi 4 mm Fusion-RP 80A reversed-phase col-mn (Phenomenex Australia Pty. Ltd, Lane Cove, NSW, Australia)aintained at a temperature of 35 ◦C. The mobile phases consisted

f three solvent systems; Solvent A—0.2% (v/v) orthophosphoriccid:acetonitrile:tetrahydro-furan, 95.5:3.5:1.5% (v:v:v), Solvent—0.2% (v/v) orthophosphoric acid:acetonitrile:tetrahydrofuran,3.5:30:1.5 (v:v:v); and Solvent C—100% acetonitrile, with the gra-ient elution schedule as described in our previous study (Vuongt al., 2014a). Compounds in the ER extract were identified by com-aring their retention times against known reference standardsnder identical chromatographic conditions.

.7. Assessment of growth inhibition of ER powdered extract onancer cell lines

Human cancer cell lines were obtained from the Ameri-an Type Culture Collection (ATCC, Manassas, VA, USA). Theytotoxicity of the crude ER extracts was screened using the-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideMTT) assay to detect cell growth inhibition across a panel ofancer cell lines (Deane et al., 2013). The following cell linesere assessed: HT29 (colon); U87, SJ-G2, SMA (glioblastoma);CF-7 (breast); A2780 (ovarian); H460 (lung); A431 (skin); Du145

prostate); BE2-C (neuroblastoma); and MiaPaCa-2 (pancreas)ogether with the one non-tumour derived normal breast celline (MCF10A). Briefly, all cancer cell lines were cultured inulbecco’s Modified Eagle Medium (DMEM) supplemented with0% foetal bovine serum, 50IU/mL penicillin, 50ug/mL strepto-ycin and 2 mM l-glutamine. The MCF10A cells were cultured

n DMEM:F12 (1:1) cell culture media, 5% heat inactivated horseerum, supplemented with penicillin (50 IU/mL), streptomycin50 �g/mL), 20 mM Hepes, l-glutamine (2 mM), epidermal growthactor (20 ng/mL), hydrocortisone (500 ng/mL), cholera toxin

100:1 130.2610:1 117.3555:1 132.98

(100 ng/mL), and insulin (10ug/mL). Cells were plated in triplicatein DMEM (100 �L) on a 96 well plate, at a density of 2500–4000cells per well. When cells were at logarithmic growth after 24 h,medium without (control) and with crude ER extract (100 �L)was added to each well to give a final at concentrations of 100and 200 �g/mL (day 0). The MTT assay was employed where theabsorbance was read at 540 nm to determine growth inhibitionafter 72 h of incubation based on the difference between the opticaldensity values on day 0 and those at the end of drug exposure.Cell growth inhibition as a percentage was determined where avalue of 100% is indicative of total growth inhibition, while a valuegreater than 100% is indicative of growth inhibition and cell death.An eight-point dose response curve (200–0.5 �g/mL) was alsoproduced, from which a GI50 value was obtained representing theER concentration that induced 50% growth inhibition.

2.8. Determination of pancreatic cancer cell viability

2.8.1. Cell cultureHuman pancreatic cancer cells (MiaPaCa2, ASPC-1 and HPAF-II)

were cultured at 37 ◦C, 5% CO2. Dulbecco’s Modified Eagle’s Medium(DMEM) supplemented with 10% fetal bovine serum (FBS), 2.5%horse serum and l-glutamine (100 �g/mL) was used for MiaPaCa-2 cells. 10% FBS in RPMI media was used for ASPC-1 cells, whileEagle’s Minimum Essential Medium (EMEM) supplemented with10% FBS was used for HPAF-II cells.

2.8.2. Cell viabilityCell viability was determined using the Dojindo Cell Count-

ing Kit-8 (CCK-8: Dojindo Molecular Technologies, INC., Maryland,USA). Cells were seeded into a 96 well plate at 5 × 103 cells perwell and allowed to adhere for 24 h. The cells were then treatedwith 100 �g/mL of crude ER extract, gemcitabine (IC50—50 nM), orvehicle control and after 72 h 10 �L of CCK-8 solution was addedand incubated at 37 ◦C for 90 min. The absorbance was measured at450 nm and cell viability was determined as a percentage of control.All experiments were performed in triplicate.

2.9. Statistical analysis

RSM analysis within JMP software (Version 10) was used toestablish the model equation, to graph the 3-D plot, 2-D contourof the response and to predict the optimum values for the three

response variables. The Student t-test was used when there wereonly two treatments to compare. The one-way ANOVA and theLSD post-hoc test were conducted using the SPSS statistical soft-ware (Version 20). Differences between the mean levels of the

170 Q.V. Vuong et al. / Industrial Crops and Products 64 (2015) 167–174

Table 2Statistical analysis of regression equation results.

Source Degree of freedom F-ratio Probability > F

Model 9 323.7411 <0.0001**

X1 (temperature) 1 895.1099 <0.0001**

X2 (time) 1 117.6055 0.0001**

X3 (water-to-leaf ratio) 1 1303.5060 <0.0001**

X1 X2 1 100.0737 0.0002**

X1 X3 1 32.6931 0.0023**

X2 X3 1 1.7278 0.2458X1

2 1 162.7144 <0.0001**

X22 1 178.2590 <0.0001**

X32 1 103.3138 0.0002**

R2 0.9987

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solution being moderately acidic (pH = 5). Such physicochemi-cal properties favour utilisation in the food or pharmaceuticalindustries.

Table 3Physicochemical properties of spray dried powdered extract.

Properties Values*

General physical propertiesDried leaf from fresh leaf (%) 43.45 ± 1.05Moisture content in dried leaf (%) 5.20 ± 0.33Dry weight (%) 16.43 ± 0.96Yield (%) 84.7 ± 2.21Moisture content 3.54 ± 0.03Water activity (aw) 0.65 ± 0.02Bulk density (g/cm3) 0.37 ± 0.01Solubility (%) 95.75 ± 0.15pH of reconstituted solution 5.0 ± 0.01Colour of crude extract

Lightness 39.65 ± 0.23Chroma 18.31 ± 0.08Hue angle 178.44 ± 0.01

Total phenolic compositionTotal phenolic compounds (mg GAE/g) 407.54 ± 23.99Flavonoids (mg CAE/g) 144.50 ± 18.33Proanthocyanidins (mg CAE/g) 14.42 ± 2.40Quantified Phenolic compoundsGallic acid (mg/g) 2.97 ± 0.05Syringic acid (mg/g) 10.08 ± 0.41Catechin (mg/g) 4.49 ± 0.09

Significant (P < 0.05).** Extremely significant (P < 0.01).

omponents in the different experiments were taken to be statis-ically significant at p < 0.05.

. Results and discussion

.1. Optimisation of water extraction conditions for TPC

To examine the model fitting and the true response surface suit-bility, analysis of the variance was conducted. The results (Table 2)howed that the regression model had low dispersion (R2 = 0.9987),evealing that the estimation of regression equations presented aood adjustment to the sample data, with 99% of predicted valuesatching with actual values. The p value of the model (assuming

confidence interval of 95%) was less than 0.01, suggesting thathe model could be a very good predictor (Mota et al., 2012). Usinghe coefficients determined, the predicted model (Y) for extractionield of TPC was:

TPC = 127.70 + 10.34X1 + 3.42X2 + 12.15X3 + 4.89X1X2

− 2.79X1X3 − 1.29X2X3 − 6.17X21 − 7.12X2

2 + 4.85X23 (2)

Table 2 also showed that temperature, extraction time andater-to-leaf ratio significantly affected the extraction efficiency of

PC (p < 0.01). There was a significant effect between temperaturend time or temperature and water-to-leaf ratio on the extractionfficiency of TPC, but there was no interaction between time andater-to-leaf ratio on the extraction efficiency of TPC.

The effect of temperature, time and water-to-leaf ratio onxtraction efficiency of TPC is shown in Fig. 1. Higher TPC wasbserved when higher temperature, longer extraction time andigher volumes of water were applied for ER leaf extraction. Theighest level of TPC (146.9 mg GAE/g) was obtained when extract-

ng the leaf at 85 ◦C for 15 min. at a water-to-leaf ratio of 100:1 mL/g.owever, at this ratio, a very high volume of water is needed forxtraction, requiring greater energy input to heat the water duringxtraction and to subsequently remove it during drying and pow-ering. Reducing water volume fivefold during extraction resulted

n approximately 86% of maximum TPC (125.8 mg GAE/g) beingxtracted. Therefore, the conditions of 85 ◦C, 15 min. extraction andater-to-leaf ratio of 20:1 mL/g were chosen as optimal conditions.

To validate the predicted extraction value at these optimal con-itions, ER leaf was extracted in triplicate and our results show

hat TPC levels of 124.9 ± 2.46 mg GAE/g were obtained. This levelas not found to be significantly different from the predicted value

125.8 ± 1.42 mg GAE/g; p > 0.05), and therefore the predicted andxperimental values were satisfactorily matched.

3.2. Preparation of spray-dried powdered extract anddetermination of physicochemical properties

Table 3 shows that 43.5% dried leaf could be obtained from freshleaf, revealing that approximately 2.3 kg of fresh ER leaves couldproduce 1 kg of dried leaves, which in turn produced 0.14 kg ofpowdered extract. The production recovery yield of the powderedextract was 85%, with the mass loss of 15% potentially explained bypartial loss of the powdered extract due to adherence to the insidesof the piping and the collection cylinder of the spray dryer (Vuonget al., 2012). Therefore, using the current method approximately62 g of the powdered extract could be obtained from 1 kg of freshleaves.

The powdered extract had low water activity and low mois-ture content (0.65 and 4%, respectively), indicating that it can bestored for a long period of time with minimum loss of quality(Sinija et al., 2007; Vuong et al., 2013a). The extract also had lowbulk density (∼0.37 g/cm3), meaning that storage transport costsare minimised. In addition, it exhibited high water solubility (96%dissolution upon reconstitution in water), with the reconstituted

Epicatechin (mg/g) 10.06 ± 0.06Quercetin (mg/g) 10.29 ± 0.52Apigenin (mg/g) 5.23 ± 0.59

* Values are mean ± SD for triplicate experiments.

Q.V. Vuong et al. / Industrial Crops and Products 64 (2015) 167–174 171

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ig. 1. Effect of extraction conditions on total phenolic compounds and the 3D respoime and water-to-leaf ratio.

The powdered extract had approximately 407 mg GAE/g of TPC,ccounted for 40% of the powdered extract (Table 3). Levels of TPCn this extract was higher than TPC of the extracts prepared fromhe leaves of Eucalyptus camaldulensis (Singab et al., 2011), the barkf Eucalyptus globulus (159 mg GAE/g) (Santos et al., 2012b), andhe bark of Eucalyptus grandis and Eucalyptus urograndis (385 and47 mg GAE/g, respectively) (Santos et al., 2012a). These findings

ndicated that this powdered extract is an excellent lead materialor further purification to obtain high purity TPC.

The powdered extract was found to contain approximately44 mg CAE/g of flavonoids, which correlates to approximately5% of total TPC (Table 3). This level was significantly higher

han aqueous extracts from the leaves of Eucalyptus camaldulen-is (53 mg QE/g) (Singab et al., 2011), and the leaves of other plantpecies such as laurel, oregano, olive, hypericum and hawthorn0.08, 0.257, 0.382, 1.479 and 0.245 mg/g, respectively) (Skerget

rface and 2D contour plots of total polyphenols affected by extraction temperature,

et al., 2005). Proanthocyanidins in the ER powdered extractaccounted for approximately 3.4% of TPC. This level was lowerthan that found in the extract of the leaves of laurel, hypericumand hawthorn (29.9, 48.2 and 40.6 mg/g, respectively), but wassignificantly higher than those in the extract of the leaves oforegano and olive (2.5 and 1.2 mg/g, respectively) (Skerget et al.,2005).

To further identify the individual phenolic compounds in thepowdered extract, Table 3 shows that the crude ER extract con-tained high levels of epicatechin, quercetin and syringic acid withapproximately 10 mg/g of the extract. Lower levels of other pheno-lic compounds such as gallic acid, catechin and apigenin were also

found in the powdered extract (3, 4.5, and 5 mg/g, respectively).These individual phenolic compounds have been found to link withanti-cancer activity both in vitro and in vivo (Visioli et al., 2011;Vuong et al., 2014b). Of note, other major compounds were also

172 Q.V. Vuong et al. / Industrial Crops and Products 64 (2015) 167–174

Table 4Antioxidant properties of ER extract in comparison with �-tocopherol and ascorbic acid.

Assay Antioxidant properties (mg BHT/g)# Dose dependence of ER extract* R square correlationwith TPC**

ER extract �-Tocopherol Ascorbic acid Linear regression coefficient Intercept

SSA 732.0 ± 14.4a 629.1 ± 21.1b 1416.0 ± 15.0c 5.5 157.6 0.97ABTS 832.8 ± 57.1a 455.0 ± 5.8b 857.8 ± 13.5a 4.7 229.3 0.93DPPH 1403.9 ± 107.1a 950.0 ± 142.2b 1842.8 ± 9.5c 10.9 207.4 0.98H2O2 1447.5 ± 115.6a 948.7 ± 23.2b 215.2 ± 2.6c 10.6 284.8 0.95FRAP 1638.2 ± 97.9a 1204.7 ± 15.4b 1961.2 ± 21.6c 8.9 353.0 0.84CUPRAC 715.7 ± 43.9a 383.2 ± 2.6b 756.6 ± 5.9a 5.8 80.4 0.98

# The values are mean ± standard deviations for triplicate experiments and those not sharing a letter in the same row are significantly different at P < 0.05.* The linear correlation between the does applied (25–200 �g/mL) and the responding antioxidant capacity.

** Correlation of TPC and antioxidant capacity in different antioxidant assays.

Table 5Cell growth inhibition (%) of ER extract across various cancer cell lines. Higher values indicate greater inhibition.

Cell line Cancer cell type Eucalyptus robusta extract

100 �g/mL 200 �g/mL GI50 (�g/mL)

HT29 Colon 84 ± 4 >100 77 ± 2.0U87 Glioblastoma 27 ± 5 64 ± 4 183 ± 8.7SJ-G2 Glioblastoma 70 ± 4 >100 79 ± 5.6SMA Glioblastoma (Murine) 49 ± 2 99 ± 0.5 100 ± 5.0MCF-7 Breast 38 ± 2 89 ± 2 124 ± 4.5MCF10A Breast (Normal) 31 ± 4 92 ± 1 130 ± 5.2A2780 Ovarian 63 ± 2 97 ± 0.5 80 ± 3.0H460 Lung 87 ± 5 >100 77 ± 3.0A431 Skin 57 ± 5 >100 98 ± 7.8Du145 Prostate 45 ± 2 92 ± 3 113 ± 5.5BE2-C Neuroblastoma 80 ± 4 >100 77 ± 4.6MiaPaCa2 Pancreas 40 ± 8 >100 129 ± 5.8

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I50 = concentration that inhibits cell growth by 50%.ote: 100% is indicative of total growth inhibition, while a value greater than 100%

resent, revealing that ER extract contained other bioactive com-ounds however these have yet to be identified. Therefore, thesendings further confirmed that ER powdered extract is an excel-

ent lead material for further purification to obtain key phenolicompounds with potential anti-cancer activity.

.3. Antioxidant capacity of ER powdered extract

The findings from the six different antioxidant assays (Table 4)evealed that the crude ER powdered extract had a comparablentioxidant capacity to that of ascorbic acid (similar in ABTS andUPRAC assays; lower in SSA and FRAP assays, but higher in the2O2 assay). Interestingly, the ER powdered extract was found toave a significantly higher antioxidant capacity than that of �-ocopherol in all six tested antioxidant assays (p < 0.05). Of note,scorbic acid and �-tocopherol used in this study were of highurity (>90%), thus the crude ER extract had potent antioxidantapacity, with the potential of improvement if further purifications undertaken on the crude extract.

Table 4 also indicated that the linear regression coefficientsnd intercepts from six antioxidant assays were positive, revea-ing that the antioxidant capacity of the crude ER extract wasose dependent, and thus the higher the concentrations of theR extract applied, the higher the antioxidant capacity would bebtained. In addition, the data also showed that the R-squared val-es between TPC and antioxidant capacity ranged from 0.8408 to.9859 (Table 4), indicating that TPC had a strong correlation andere a major contributor to the antioxidant capacity of ER extract.

revious studies also reported that TPC were closely linked withntioxidant capacity and were the major antioxidant contributorJavanmardi et al., 2003; Lee et al., 2011; Li et al., 2012; Molan et al.,012).

cative of growth inhibition and cell death.

3.4. Assessment of growth inhibition of crude ER extract oncancer cell lines in vitro

Inhibition of cell growth was demonstrated across the panelof cell lines with varying efficacy (Table 5). The crude ERextract showed the greatest growth inhibition against colon(HT29—GI50 77 ± 2.0 �g/mL), lung (H460–GI50 77 ± 3.0 �g/mL),neuroblastoma (BE2-C–GI50 77 ± 4.6 �g/mL), glioblastoma (SJ-G2—GI50 79 ± 5.6 �g/mL) and ovarian (A2780–GI50 80 ± 3.0 �g/mL)cancer cells. The GI50 for ER extract for the MiaPaCa-2 pancreaticcancer cells was 129 ± 5.8 �g/mL. These data underscore the poten-tial of the ER extract to be further purified and investigated for itsactivity against numerous cancer types, including for pancreaticcancer.

3.5. ER extract decreases viability of pancreatic cancer cells

The ER extract exhibited high antioxidant and free radical scav-enging capacity at the concentration of 100 �g/mL, and preliminaryscreening against numerous cancer cell lines showed cytotoxicityat this concentration (GI50 129 ± 5.8 �g/mL; Table 5). As such, theeffects on cell viability of 100 �g/mL of ER extract on pancreaticcancer cells derived from both primary (MiaPaCa-2) and metastatic(ASPC-1, HPAF-II) sites was assessed. The findings were bench-marked against the chemotherapeutic agent gemcitabine, used inthe first line of treatment of patients with pancreatic cancer. At100 �g/mL, ER extract decreased cell viability of MiaPaCa-2, ASPC-1and HPAF-II pancreatic cancer cells by 86%, 62% and 47%, respec-

tively (Fig. 2), when compared to untreated control cells. The ERextract was significantly more cytotoxic than gemcitabine towardsMiaPaCa-2 cells (p = 0.006), ASPC-1 cells (p = 0.0006) and HPAF-IIcells (p = 0.0002). This is an important observation because ASPC-1

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proanthocyanidins, flavones and flavonols in some plant materials and theirantioxidant activities. Food Chem. 89, 191–198.

ig. 2. Effect of the crude ER extract and gemcitabine on the viability of MiaPaCa-2,SPC-1 and HPAF-II pancreatic cancer cells.

nd HPAF-II cells are inherently resistant to gemcitabine. With theesponse rate of patients with pancreatic cancer to gemcitabineeing less than 20%, the development of novel therapeutic agentsgainst pancreatic cancer are desperately required. These data,emonstrate the potential of the bioactive compounds within therude ER extract to be further purified and investigated for theirnti-pancreatic cancer properties.

. Conclusion

The optimal extraction conditions for maximum yield of TPCrom ER leaves using water were 85 ◦C, 15 min and water-to-leafatio of 20:1 mL/g. At these conditions 124.9 ± 2.54 mg GAE/g of TPCould be extracted from the ER leaves. Spray drying was effectiven producing the powdered extract with a recovery yield of 85%.pproximately 62 g of the powdered extract could be obtained from

kg of fresh leaves. The powdered extract had low moisture con-ent, was water-soluble and had high levels of TPC (407 mg GAE/g).he powdered extract also possessed potent antioxidant capacity,hich were comparable to ascorbic acid and �-tocopherol. Theseata also demonstrated potent anti-cancer activity of the ER extractgainst a variety of cancer cell lines, including pancreatic cancer.mportantly, the ER extract showed significantly increased cytotox-city against pancreatic cancer cell lines derived from both primarynd metastatic sites when compared to the first line chemother-peutic agent gemcitabine, and importantly had less of an effectn normal pancreatic ductal epithelial cells. Thus ER should be vig-rously investigated for both its health promoting and potentialnti-cancer benefits, particularly for pancreatic cancer.

onflict of interest statement

The authors report no declarations of interest.

cknowledgements

We acknowledge the following funding support: Ramaciottioundation (ES2012/0104); Cancer Australia and Cure Cancer

ustralia Foundation (1033781). PAP is supported by a Nationalealth and Medical Research Council Career Development Fellow-

hip.

d Products 64 (2015) 167–174 173

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