enriched Carica papaya leaf extracts
Transcription
enriched Carica papaya leaf extracts
International Journal of Food Science and Technology 2015, 50, 169–177 169 Original article Antioxidant and anticancer capacity of saponin-enriched Carica papaya leaf extracts Quan V. Vuong,1,2 Sathira Hirun,1,2 Tiffany L.K. Chuen,1,2 Chloe D. Goldsmith,1,2 Shane Murchie,2 Michael C. Bowyer,1,2 Phoebe A. Phillips3 & Christopher J. Scarlett1,2,4* 1 Pancreatic Cancer Research, Nutrition Food & Health Research Group, University of Newcastle, 10 Chittaway Road, Ourimbah, NSW, Australia 2 School of Environmental and Life Sciences, University of Newcastle, 10 Chittaway Road, Ourimbah, NSW, Australia 3 Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, Prince of Wales Clinical School, Faculty of Medicine, The University of New South Wales, High Street, Kensington, NSW, Australia 4 Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW, Australia (Received 9 April 2014; Accepted in revised form 15 June 2014) Summary The papaya (Carica papaya) leaf (PL) contains high levels of saponins and polyphenolic compounds, and historically, it has been used as a folk medicine for numerous ailments, including cancer. PL is traditionally prepared by hot water extraction; however, optimised extraction conditions have not been assessed. This study optimised conditions for the extraction of saponins from PL and assessed their antioxidant capacity and antipancreatic cancer activity. Optimisation was achieved using response surface methodology. Saponins and total phenolic compounds were assessed for their antioxidant, free radical scavenging, ionreducing capacity, and antipancreatic cancer activity. Optimal aqueous extraction conditions were 85 °C, 25 min. and a water-to-leaf ratio of 20:1 mL g1. Ethanol extracts demonstrated higher antioxidant, free radical scavenging and ion-reducing capacity, as well as antipancreatic cancer activity. This study revealed that the PL contains numerous bioactive compounds, with significant anticancer activity warranting further studies on the isolation and characterisation of individual bioactive compounds from the PL. Keywords Antioxidant, Carica papaya leaf, pancreatic cancer, saponins. Introduction In many parts of the world, especially in remote areas of Asian countries, Carica papaya L. (papaya or paw paw) leaf has been used as a folk medicine for a variety of ailments such as healing of burns, relief of asthma symptoms, treatment of intestinal worms, treatment of digestion problems, fever control and treatment of amoebic dysentery (Starley et al., 1999; Canini et al., 2007; Zunjar et al., 2011). Papaya leaf has also been used to increase appetite, ease menstrual pain and relieve nausea (Aravind et al., 2013). Furthermore, papaya leaf juice has been consumed by people living on the Gold Coast of Australia, with some anecdotes of successful cases being reported for its purported anticancer activity (Otsuki et al., 2010). Additionally, the tender leaf has been consumed as an *Correspondent: Fax: +61 2 4348 4145; e-mail: [email protected] doi:10.1111/ijfs.12618 © 2014 Institute of Food Science and Technology alternative to traditional leafy vegetables and as an additive to tenderise meat (Aravind et al., 2013). Recent scientific reports suggest that papaya leaf extract and its latex can be utilised to treat skin lesions (Mahmood et al., 2005; Gurung & Skalko-Basnet, 2009), lower the risk of cardiovascular disease (Runnie et al., 2004), act as an anti-inflammatory (Owoyele et al., 2008) and an anthelmintic against intestinal nematode (Satrija et al., 1995). A recent study (Otsuki et al., 2010) found that papaya leaf extract could prevent growth of cancer cells, including pancreatic cancer – one of the most devastating forms of cancer (Scarlett et al., 2011). This result suggests that papaya leaf may contain compounds that limit the proliferation of pancreatic cancer cells. However, because the study investigated only one pancreatic epithelioid carcinoma cell line (Panc-1), further study on other types of pancreatic cancer cells is required to substantiate this claim. We recently revealed that the papaya leaf not only contained phenolic compounds but it also had a 170 Bioactivity of saponin enriched papaya extract Q. V. Vuong et al. substantial content of saponins, which was significantly higher than the level of phenolic compounds (Vuong et al., 2013). Saponins, which have been associated with the prevention of cancer, are structurally amphiphilic, containing hydrophilic (carbohydrate) and hydrophobic (steroid or triterpene) moieties (Shi et al., 2004). Papaya leaf has been traditionally consumed in folk medicine preparations as a tea, by brewing it in hot water. To date, previous study has reported the optimised conditions for extracting its saponins. Ethanol has been reported as an effective solvent for the extraction of saponins using concentrations of 70–80% (v/v) (Hu et al., 2012), and again, no study has extracted saponins from papaya leaf using 80% ethanol. Therefore, the aims of this study are to optimise conditions for water extraction of saponins, prepare water and ethanol saponin-enriched extracts, and test their antioxidant capacity and antiproliferative effects of these extracts on pancreatic cancer cell lines. Materials and methods The mature papaya leaves with stems were taken from the Central Coast, New South Wales, Australia and stored at 20 °C prior to processing to minimise deterioration of the polyphenolics. The leaves were then cut into small pieces, frozen in liquid nitrogen and freeze-dried (Thomas Australia Pty. Ltd., Seven Hills, NSW, Australia). Using a blender (John Morris Scientific, Chatswood, NSW, Australia), the dried papaya leaves were ground then sieved to ≤1 mm particle size using a 1 mm EFL 2000 stainless steel mesh sieve (Endecotts Ltd., London, England). Samples were then stored at 5 °C until required. Table 1 Experimental design for optimisation of water extraction of saponin from papaya leaf and observed response. The influence of temperature, time and water-to-leaf ratio on papaya extraction efficiency was assessed Runs A Temperature (°C) B Time (min) C Water-to-leaf ratio (mL g1) Y Extraction efficiency (mg ASE g1) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 70 70 80 70 90 90 80 80 80 80 90 80 70 80 90 15 5 15 25 5 15 5 25 5 15 15 25 15 15 25 100:1 55:1 55:1 55:1 55:1 10:1 10:1 100:1 100:1 55:1 100:1 10:1 10:1 55:1 55:1 29.66 25.50 29.87 28.81 31.51 30.23 27.88 32.84 28.86 30.02 32.00 31.35 26.70 29.89 31.62 reduced pressure. The concentrated extract was then frozen in liquid nitrogen and dried using a FD3 freeze dryer (Thomas Australia Pvt. Ltd., Seven Hills, NSW, Australia) to obtain crude saponin-enriched papaya Experimental design Response surface methodology (RSM) was employed to optimise the conditions for saponin-enriched PL aqueous extracts. The combinatorial effects of temperature, time and water-to-leaf ratio on extraction efficiency were then assessed. A Box–Behnken factorial design with three centre points was used for the experimental design (Table 1). Sample preparation Figure 1 shows the process undertaken to prepare saponin-enriched water and ethanol extracts. Briefly, ground leaves were extracted in either water under optimal conditions identified using RSM (85 °C, 25 min, water-to-leaf ratio of 100:5 mL g1) or 80% (v/v) ethanol (room temperature for 72 h). The extracted solution was filtered and then concentrated using a rotary evaporator (Buchi Rotavapor B-480, Buchi Australia, Noble Park, Vic., Australia) under International Journal of Food Science and Technology 2015 Figure 1 Preparation of water and ethanol crude extracts from papaya leaf. © 2014 Institute of Food Science and Technology Bioactivity of saponin enriched papaya extract Q. V. Vuong et al. extracts as a solid. Extracts were then stored at 18 °C until required. Determination of saponin content The method of Vuong et al. (2013) was used to measure the saponin content of papaya leaf extracts. The appropriately diluted samples (0.5 mL) were mixed with 8% (w/v) vanillin (0.5 mL) and 72% (v/v) H2SO4 (5 mL), cooled on ice (5 min) and then incubated at 60 °C for 15 min. The mixture was then cooled on ice to room temperature (RT) and measured at 560 nm using a UV spectrophotometer (Varian Australia Pty. Ltd., Mulgrave, Vic., Australia). Aescin was used as the standard for the calibration curve, with results expressed as mg of aescin equivalents per g of sample (mg ASE g1). Determination of total phenolic compounds Total phenolic compounds (TPC) was determined as previously described with minor modifications (Vuong et al., 2013). Briefly, the appropriately diluted samples (1 mL) were mixed with 10% (v/v) Folin–Ciocalteu reagent (5 mL) and left at RT for 5 min to equilibrate before adding 7.5% (w/v) Na2CO3 (4 mL) and incubating for a further 1 h in the dark (RT). Solution absorbance was then measured (k = 760 nm). Gallic acid was used as the standard for a calibration curve, and the results were expressed as mg of gallic acid equivalents per g of sample (mg GAE g1). Determination of antioxidant, free radical scavenging and ion-reducing capacity To determine the antioxidant, free radical scavenging and ion-reducing capacity, the lyophilised extracts were diluted in methanol to yield final solution concentrations of 25, 50, 100 and 200 lg mL1. Six different antioxidant assays were then performed, with the potent antioxidant a-tocopherol (90% purity) used for comparative purposes. Antioxidant capacity SSA (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) reagent solution and ABTS (2,20 -azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) assays were employed to test the antioxidant capacity of both extracts. The SSA assay was performed as described by Prieto et al. (1999). The diluted extracts (3 mL) were mixed with 3 mL of SSA reagent, then incubated at 95 °C for 90 min. Absorbance was then measured (k = 695 nm), referenced against the SSA reagent (as a blank). Ascorbic acid was used as the standard, with © 2014 Institute of Food Science and Technology results expressed as lg of ascorbic acid equivalents per g of the PL extract (lg ACE g1). The ABTS assay was used to determine the antioxidant capacity of the extracts based on the studies of Thaipong et al. (2006). A stock solution was initially prepared by mixing 1:1 of 7.4 mM ABTS+and 2.6 mM potassium persulphate solution and incubated for 12 h at RT in the dark. A working solution was prepared fresh by mixing 1 mL stock solution with 60 mL methanol to obtain a solution absorbance of 1.1 0.02 (k = 734 nm). The diluted extract (150 mL) was then mixed with 2850 mL of the working solution then incubated for 2 h in the dark (RT). The solution absorbance was then recorded (k = 734 nm). Results were expressed as total antioxidant capacity (TAC) percentage, which is calculated according to eqn 1. TAC½% ¼ ðAbsorbance of control Absorbance of sampleÞ 100%=Absorbance of control ð1Þ Free radical scavenging capacity The DPPH (1,1-diphenyl-2-picrylhydrazyl) and hydrogen peroxide (H2O2) radical scavenging capacity assays were employed to determine the free radical scavenging capacity of the saponin-enriched extracts. The DPPH assay was performed as described by Vuong et al. (2013) with some modifications. A stock solution of 0.024% (w/v) DPPH in methanol was prepared and stored at 20 °C. The working solution was then prepared by diluting the stock solution (10 mL) with methanol (45 mL) to obtain a solution absorbance at of 1.1 0.02 (k = 515 nm). The diluted sample (0.2 mL) was then mixed with the working solution (3.8 mL) and incubated in the dark for 3 h (RT) before measuring the solution absorbance (k = 515 nm). DPPH free radical scavenging inhibition was expressed as a percentage, calculated as per eqn 1. H2O2 radical scavenging assay was conducted as described by Ragupathi Raja Kannan et al. (2013) with a minor modification. The diluted extracts (1.0 mL) were added to a H2O2 solution prepared in 0.1 M phosphate buffer saline (pH 7.4, 40 mM and 0.6 mL) and incubated at RT for 10 min. Absorbance was then recorded (k = 230 nm), referenced against a blank solution of phosphate buffer without H2O2. Results were expressed as lg of BHT equivalents per g of the extract (lg BHT g1). Ion-reducing capacity Cupric ion-reducing antioxidant capacity (CUPRAC) and ferric-reducing antioxidant power (FRAP) assays were used to determine the ion-reducing capacity of the extracts. International Journal of Food Science and Technology 2015 171 172 Bioactivity of saponin enriched papaya extract Q. V. Vuong et al. The CUPRAC assay of Apak et al. (2004) was used to determine the cupric ion-reducing antioxidant capacity with some modifications. A working solution was prepared by mixing 10 mM CuCl2 (1 mL), 7.5 mM neocuproine (1 mL) and 7.708% (w/v) NH4Ac (1 mL). This solution was then mixed with the diluted samples (1.1 mL) and left at RT for 1.5 h before measuring the absorbance at 450 nm against a blank reagent. The results were expressed as lg of ascorbic acid equivalents per g of sample (lg AAE g1). The FRAP assay described by Thaipong et al. (2006) was employed to determine the ferric-reducing antioxidant power. Reagent A: 300 mM acetate buffer (3.1 g CH3CO2Na3H2O and 16 mL CH3CO2H diluted to 1000 mL), pH 3.6; Reagent B: 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl; and Reagent C:- 20 mM FeCl36H2O solution. The FRAP solution was prepared by mixing reagents A, B and C at a ratio of 10:1:1 prior to use. The diluted extract (150 mL) was then mixed with 2850 mL of the FRAP solution and incubated for 30 min in the dark at RT. Absorbance was then measured (k = 593 nm) against a blank reagent, and the results were expressed as lg of ascorbic acid equivalents per g of sample (lg AAE g1). Determination of pancreatic cell viability Cell culture Human pancreatic cancer cells (Mia-PaCa2 and ASPC-1) were cultured at 37 °C, 5% CO2. Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 2.5% horse serum and L-glutamine (100 lg/mL) was used for MiaPaCa-2 cells, while 10% FBS in RPMI media was used for ASPC-1. Cell Viability Cell viability was determined using the Dojindo Cell Counting Kit-8 (CCK-8: Dojindo Molecular Technologies, Inc., Rockville, MD, USA). Cells were seeded into a 96-well plate at 5 9 103 cells per well and allowed to adhere for 24 h. The cells were then treated with 100 lg mL1 of crude papaya ethanolic extract, crude papaya water extract or gemcitabine (IC50 – 50 nM), or vehicle control. After 72 h, 10 lL of CCK8 solution was added before incubating at 37 °C for 90 min. The absorbance was measured at 450 nm, and cell viability was determined as a percentage of control. All experiments were performed in triplicate. Statistical analysis Response surface methodology (RSM) experimental design and analysis was conducted using JMP software (version 10). The software was also used to establish International Journal of Food Science and Technology 2015 the model equation, to graph the 3-D plot, 2-D contour of the response and to predict the optimum values for the three response variables. A Student’s t-test was used when there were only two treatments to compare. One-way ANOVA and LSD post hoc test were conducted using the SPSS statistical software (version 20). Differences between the mean levels of the components in the different experiments were taken to be statistically significant at P < 0.05. Values given are mean SD for triplicate experiments. Those not sharing the same superscript on top of the columns were significantly different (P < 0.05). Results and discussion Optimisation of water extraction conditions for saponin enrichment Papaya leaf is traditionally brewed in hot water for use in folk medicine; however, no optimised brewing conditions have been described. As previously reported (Vuong et al., 2013), saponins are a major bioactive constituent in the papaya leaf. Results for the optimised conditions for saponin-enriched water extracts are shown in Table 2. We demonstrate that the three major parameters temperature, extraction time and water-to-leaf ratio independently significantly affected saponin extraction efficiency (P < 0.05; Table 2). The analysis of variance of saponin extraction yield, performed using RSM, showed that the regression model had low dispersion (R2 = 0.9333) and there was no significance in the lack of fit (P > 0.05) (Table 2). Therefore, the analysis indicated that the quadratic polynomial model was adequate to describe the effect of the extraction factors on the yield of extracted saponins. The model also showed that temperature had the greatest influence on the saponin yield, followed by extraction time and water-to-leaf ratio (Table 2, Fig. 2). Consequently, the predicted model (Y) for extraction yield of saponins was: Table 2 Statistical analysis of regression equation results Source Degree of freedom F - ratio Probability > F Model A (temperature) B (time) C (water-to-leaf ratio) AB AC BC A2 B2 C2 Lack of fit 9 1 1 1 1 1 1 1 1 1 3 7.7815 35.7445 19.5503 8.5984 3.3862 0.4627 0.0854 1.6345 0.0003 0.4262 193.9726 <0.05* 0.0019* 0.0069* 0.0325* 0.1251 0.5266 0.7819 0.2572 0.9878 0.5427 *Significant (P < 0.05). © 2014 Institute of Food Science and Technology Bioactivity of saponin enriched papaya extract Q. V. Vuong et al. Y ¼ 0:0058A2 þ 0:00007B2 þ 0:0001C2 þ 0:3366A þ 0:7591B þ 0:0525C 0:008AB 0:0006AC þ 0:0003BC 36:6 Response surface methodology predicted a higher saponin content in the extracts when the temperature, extraction time and/solvent sample ratio were increased (Fig. 3). The highest saponin content (32.8 mg ASE g1) was obtained when extracting the leaf at 85 °C for 25 min at a ratio of 100:1 mL g1. The disadvantage of this relatively high dilution ratio is the large amounts of energy required (a product of the high heat capacity of water) to heat the solution during the extraction procedure and the subsequent remove of the solvent during the drying of the extract. Importantly, with five times less water volume, approximately 95% of saponins (31.16 1.77 mg ASE g1), affording significant energy savings. As a consequence, conditions of 85 °C, 25 min and waterto-leaf ratio of 20:1 mL g1 were selected as optimal conditions for aqueous saponins enrichment. To validate the predicted value at these conditions, papaya leaf was extracted in triplicate and analysed. The model extracted 29.24 2.54 mg ASE g1 of saponins, which was not significantly different to the predicted value (P > 0.05). Antioxidant activity of saponin-enriched water and ethanol extracts Saponins and TPC in water and ethanol extracts Figure 2 The 3-D response surface and 2-D contour plots of total saponins affected by extraction temperature, time and water-to-leaf ratio. © 2014 Institute of Food Science and Technology Several solvents (methanol, ethanol, acetone and water) have been to extract bioactive components, with extraction efficiency varying as a function of solvent polarity (Naczk & Shahidi, 2006). The current study used water and 80% ethanol (v/v) as the solvent of choice for the preparation of the saponin-enriched extracts. Figure 4 shows that the ethanol extract contained saponin levels of approximately 368 mg ASE g1; fourfold higher than that of the water extract (87 mg ASE g1). The level of total phenolic compounds was also higher in the ethanol extract (82 mg GAE g1) compared with the water extract (63 mg GAE g1). These findings support results from our previous study (Vuong et al., 2013), whereby the use of ethanol could extract higher levels of saponins from the papaya leaf than water; however, levels of total phenolic compounds were different. This difference could be explained by the concentration of ethanol used for the extraction. Our previous study used 100% ethanol, while the current study used 80% aqueous ethanol, which has been reported to extract increased levels of total phenolic compounds (Naczk & Shahidi, 2006). International Journal of Food Science and Technology 2015 173 174 Bioactivity of saponin enriched papaya extract Q. V. Vuong et al. Figure 3 Prediction profilers of temperature, time and ratio on extraction efficiency of saponins. higher than TPC (Fig. 4), its antioxidant capacity was only slightly higher than that of the water extract. Correlation between saponins and TPC, and antioxidant capacity was assessed, revealing that TPC had a stronger correlation (R2 = 0.99 and 0.99) with antioxidant capacity than saponins (R2 = 0.73 and 0.66; Table 3); thus, TPC contributed more to the antioxidant capacity of the extract than did the saponins. Free radical scavenging capacity Figure 4 Saponins and total phenolic compounds in water and ethanol extracts. Saponin levels were expressed as mg ASE g1 SD, and levels of total phenolic compounds were expressed as mg GAE g1 SD. Antioxidant capacity Antioxidant capacity of water and ethanol extracts are shown in Fig. 5a,b. Total antioxidant and ABTS antioxidant capacity were not significantly different when concentrations of both water and ethanol extracts were increased from 25 to 100 lg mL1; however, antioxidant capacity significantly increased when extract concentrations exceeded 100 lg mL1. At concentrations greater than 100 lg mL1, the papaya leaf ethanol extract demonstrated higher antioxidant capacity than the water extract (Fig. 5a,b). Antioxidant capacity of the water and ethanol extracts possessed approximately one-fifth of those of a-tocopherol, which is known for its high antioxidant properties. These differences can be explained by the relative purity of the assays, with a-tocopherol being of high purity (~90%); whereas, both the water and ethanol extracts were of low purity, being crude extracts. Of note was the fact that although the content of saponins in the ethanol extract was four times International Journal of Food Science and Technology 2015 The DPPH (1,1-diphenyl-2-picrylhydrazyl) and H2O2 radical scavenging capacity assays were used to assess the free radical scavenging capacity of the two extracts. Both assays showed that the water and ethanol extracts had a dose-dependent effect on their free radical scavenging capacity (Fig. 6a,b). As the concentration of the extracts increased from 25 to 200 lg mL1, free radical scavenging capacity significantly increased. At concentrations ≤100 lg mL1, the free radical scavenging capacities of ethanol and water extracts were similar, but at 200 lg mL1, the ethanol extract had a higher free radical scavenging capacity. This data also showed that both water and ethanol extracts had a significantly lower free radical scavenging capacity than that of a-tocopherol. The correlation of saponins and TPC in the two extracts with their free radical scavenging capacity was also analysed. The findings from both DPPH and H2O2 assays revealed that TPC had stronger correlation (R2 = 0.98 and 0.86, respectively) with free radical scavenging capacity than saponin concentration (R2 = 0.58 and 0.32, respectively) (Table 3). Ion-reducing antioxidant capacity The cupric ion-reducing antioxidant capacity (CUPRAC) and ferric-reducing antioxidant power (FRAP) assays were used to determine the ion-reducing capacity of both the water and ethanol extracts. Figure 7a,b showed that both water and ethanol extracts processed similar ion-reducing capacity, which was significantly © 2014 Institute of Food Science and Technology Bioactivity of saponin enriched papaya extract Q. V. Vuong et al. (a) (a) (b) (b) Figure 5 Antioxidant capacity of water and ethanol extracts using SSA assay (a) and ABTS assay (b) in comparison with a-tocopherol. Table 3 Correlation of total phenolic compounds and saponins with antioxidant capacity of the extracts R2 value Antioxidant capacity Saponins TPC Total antioxidant capacity ABTS antioxidant capacity DPPH free radical scavenging capacity H2O2 radical scavenging capacity CUPRAC FRAP 0.7305 0.6593 0.5866 0.3275 0.4830 0.5508 0.9944 0.9901 0.9858 0.8600 0.9490 0.9655 CUPRAC, Cupric ion-reducing antioxidant capacity; FRAP, ferric-reducing antioxidant power. lower than that of a-tocopherol. The findings also showed that cupric ion-reducing capacity of both extracts increased significantly in the concentration range 25–200 lg mL1; whereas, the ferric ion-reducing power of both extracts only increased significantly when their concentration exceeded 100 lg mL1. In addition, TPC in both extracts was found to have a stronger correlation with CUPRAC and FRAP (R2 = 0.94 and 0.96, respectively) than with © 2014 Institute of Food Science and Technology Figure 6 Free radical scavenging capacity of water and ethanol extracts using DPPH assay (a) and H2O2 radical scavenging assay (b) in comparison with a-tocopherol. saponin concentration (R2 = 0.48 and 0.55, respectively). Antioxidants have been linked to the prevention and/or treatment of certain cancers and have been widely used as antioxidant supplements during or after conventional cancer treatment to enhance treatment benefits, alleviate side effects and/or maintain or improve general health and well-being (Ladas et al., 2004). Our study found that the TPC in the papaya leaf extract had a stronger correlation to the antioxidant, free radical scavenging and ion-reducing capacity than saponins content (Table 3). These findings were supported by previous studies (Javanmardi et al., 2003; Lee et al., 2011; Li et al., 2012; Molan et al., 2012) as phenolic compounds were demonstrated to possess high antioxidant capacity due to the presence of redox active groups in their structures (Pisoschi & Negulescu, 2011); whereas, saponins are composed of sapogenin, sugars and organic acids (Li et al., 2012). Although possessing low antioxidant capacity, saponins have been found to demonstrate anticancer properties (Shi et al., 2004; Li et al., 2012). As such, the current study examined the anticancer activity of both water and ethanol extracts against pancreatic cancer cell lines from both primary and metastatic sites. International Journal of Food Science and Technology 2015 175 176 Bioactivity of saponin enriched papaya extract Q. V. Vuong et al. compared to untreated control cells. The ethanol extract was equally as cytotoxic as gemcitabine towards MiaPaCa-2 cells (P = 0.09) and importantly was significantly more cytotoxic than gemcitabine against ASPC-1 cells (P = 0.004), which are inherently resistant to gemcitabine (Table 4). With the response rate of patients with pancreatic cancer to gemcitabine being less than 20%, the development of a novel therapeutic agent against pancreatic cancer is desperately required. These data demonstrate the potential of the bioactive compounds within the crude papaya leaf extract to be further purified and investigated for their antipancreatic cancer properties. (a) (b) Conclusion Figure 7 Ion-reducing capacity of water and ethanol extracts using the CUPRAC (a) and ferric-reducing antioxidant power (FRAP) (b) assays in comparison with a-tocopherol. Table 4 Cell viability (%) of pancreatic cancer cell lines exposed to papaya leaf ethanol and water extracts, compared with gemcitabine MiaPaCa-2 ASPC-1 Water extract Ethanol extract Gemcitabine 95.96 5.15 107.68 4.67c,d 18.96 1.52 45.94 3.51e 23.28 2.97 66.45 4.60 a b P < 0.0001 cf. ethanol extract and gemcitabine. b P = 0.09 cf. gemcitabine. c P < 0.0001 cf. ethanol extract. d P < 0.0004 cf. gemcitabine. e P = 0.0036 cf. gemcitabine. The current study found that the optimal conditions to extract saponins using water and the traditional brewing method were: 85 °C, 25 min and a water-toleaf ratio of 20:1 mL g1. However, 80% (v/v) ethanol proved to be more effective than water in extracting saponins from papaya leaf. Both water and ethanol saponin-enriched extracts possessed similar antioxidant, free radical scavenging and ion-reducing capacity at concentrations ranging from 25 to 100 lg mL1. The ethanol extract was found to have a slightly higher antioxidant capacity than the water extract at a concentration of 200 lg mL1. Ethanol extracts were more effective in inhibiting the proliferation of two pancreatic cancer cell lines and were at least as effective as the chemotherapeutic agent gemcitabine. Therefore, saponins in papaya leaf are bioactive compounds that exhibit potential in limiting the proliferation of certain pancreatic cancer cell lines. Studies aimed at characterising the bioactivity of individual saponins isolated from papaya leaf are currently underway. Acknowledgments a Ethanol extract decreases viability of pancreatic cancer cells The effects of 100 lg mL1 of both water and ethanol extracts on pancreatic cancer cells derived from both primary (MiaPaCa-2) and metastatic (ASPC-1) sites was assessed. The findings were benchmarked against the chemotherapeutic agent gemcitabine, used in the first line of treatment of patients with pancreatic cancer. At 100 lg mL1, the ethanol extract decreased cell viability of MiaPaCa-2 and ASPC-1 pancreatic cancer cells by 81% and 54%, respectively (Table 4), when International Journal of Food Science and Technology 2015 The authors would like to thank Dr Anita C. Chalmers for her critical assessment of the manuscript. We acknowledge the following funding support: Ramaciotti Foundation (ES2012/0104); Cancer Australia and Cure Cancer Australia Foundation (1033781). PAP is supported by a National Health and Medical Research Council Career Development Fellowship. Conflict of interest The authors declare no conflict of interest. References € urek, M. & Karademir, S.E. (2004). Novel Apak, R., G€ ußcl€ u, K., Ozy€ total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the © 2014 Institute of Food Science and Technology Bioactivity of saponin enriched papaya extract Q. V. Vuong et al. presence of neocuproine: CUPRAC Method. 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