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: c.scarlett@newcastle.edu.au
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
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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
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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
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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. Journal of Agricultural and Food Chemistry, 52, 7970–7981.
Aravind, G., Debjit, B., Duraivel, S. & Harish, G. (2013). Traditional and medicinal uses of carica papaya. Journal of Medicinal
Plants Studies, 1, 7–15.
Canini, A., Alesiani, D., D’Arcangelo, G. & Tagliatesta, P. (2007).
Gas chromatography–mass spectrometry analysis of phenolic compounds from Carica papaya L. leaf. Journal of food composition
and analysis, 20, 584–590.
Gurung, S. & Skalko-Basnet,
N. (2009). Wound healing properties
of Carica papaya latex: in vivo evaluation in mice burn model.
Journal of Ethnopharmacology, 121, 338–341.
Hu, T., Guo, Y.-Y., Zhou, Q.-F. et al. (2012). Optimization of ultrasonic-assisted extraction of total saponins from Eclipta prostrasta
L. using response surface methodology. Journal of Food Science,
77, C975–C982.
Javanmardi, J., Stushnoff, C., Locke, E. & Vivanco, J.M. (2003).
Antioxidant activity and total phenolic content of Iranian Ocimum
accessions. Food Chemistry, 83, 547–550.
Ladas, E.J., Jacobson, J.S., Kennedy, D.D., Teel, K., Fleischauer,
A. & Kelly, K.M. (2004). Antioxidants and cancer therapy: a systematic review. Journal of clinical oncology, 22, 517–528.
Lee, J.H., Jeon, J.K., Kim, S.G., Kim, S.H., Chun, T. & Imm,
J.-Y. (2011). Comparative analyses of total phenols, flavonoids,
saponins and antioxidant activity in yellow soy beans and mung
beans. International Journal of Food Science & Technology, 46,
2513–2519.
Li, X., Lin, J., Han, W. et al. (2012). Antioxidant ability and mechanism of rhizoma Atractylodes Macrocephala. Molecules, 17, 13457–
13472.
Mahmood, A.A., Sidik, K. & Salmah, I. (2005). Wound healing
activity of Carica papaya L. aqueous leaf extract in rats. International Journal of Molecular Medicine and Advance Sciences, 1,
398–401.
Molan, A.-L., Faraj, A.M. & Mahdy, A.S. (2012). Antioxidant activity and phenolic content of some medicinal plants traditionally
used in Northern Iraq. Phytopharmacology, 2, 224–233.
Naczk, M. & Shahidi, F. (2006). Phenolics in cereals, fruits and vegetables: occurrence, extraction and analysis. Journal of Pharmaceutical and Biomedical Analysis, 41, 1523–1542.
Otsuki, N., Dang, N.H., Kumagai, E., Kondo, A., Iwata, S. & Morimoto, C. (2010). Aqueous extract of Carica papaya leaves exhibits
anti-tumor activity and immunomodulatory effects. Journal of
Ethnopharmacology, 127, 760–767.
© 2014 Institute of Food Science and Technology
Owoyele, B., Adebukola, O., Funmilayo, A. & Soladoye, A. (2008).
Anti-inflammatory activities of ethanolic extract of Carica papaya
leaves. Inflammopharmacol, 16, 168–173.
Pisoschi, A.M. & Negulescu, G.P. (2011). Methods for total antioxidant activity determination: a review. Biochemistry and Analytical
Biochemistry, 1, 1–10.
Prieto, P., Pineda, M. & Aguilar, M. (1999). Spectrophotometric
quantitation of antioxidant capacity through the formation of
a phosphomolybdenum complex: specific application to the
determination of vitamin E. Analytical Biochemistry, 269,
337–341.
Ragupathi Raja Kannan, R., Arumugam, R., Thangaradjou, T. &
Anantharaman, P. (2013). Phytochemical constituents, antioxidant
properties and p-coumaric acid analysis in some seagrasses. Food
Research International, 54, 1229–1236.
Runnie, I., Salleh, M.N., Mohamed, S., Head, R.J. & Abeywardena,
M.Y. (2004). Vasorelaxation induced by common edible tropical
plant extracts in isolated rat aorta and mesenteric vascular bed.
Journal of Ethnopharmacology, 92, 311–316.
Satrija, F., Nansen, P., Murtini, S. & He, S. (1995). Anthelmintic
activity of papaya latex against patent Heligmosomoides polygyrus infections in mice. Journal of Ethnopharmacology, 48,
161–164.
Scarlett, C.J., Salisbury, E.L., Biankin, A.V. & Kench, J. (2011). Precursor lesions in pancreatic cancer: morphological and molecular
pathology. Pathology, 43, 183–200.
Shi, J., Arunasalam, K., Yeung, D., Kakuda, Y., Mittal, G. & Jiang,
Y. (2004). Saponins from edible legumes: chemistry, processing,
and health benefits. Journal of Medicinal Food, 7, 67–78.
Starley, I.F., Mohammed, P., Schneider, G. & Bickler, S.W. (1999).
The treatment of paediatric burns using topical papaya. Burns, 25,
636–639.
Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L. &
Hawkins Byrne, D. (2006). Comparison of ABTS, DPPH, FRAP,
and ORAC assays for estimating antioxidant activity from
guava fruit extracts. Journal of Food Composition and Analysis, 19,
669–675.
Vuong, Q.V., Hirun, S., Roach, P.D., Bowyer, M.C., Phillips, P.A.
& Scarlett, C.J. (2013). Effect of extraction conditions on total phenolic compounds and antioxidant activities of Carica papaya leaf
aqueous extracts. Journal of Herbal Medicine, 3, 104–111.
Zunjar, V., Mammen, D., Trivedi, B.M. & Daniel, M. (2011). Pharmacognostic, physicochemical and phytochemical studies on Carica
papaya Linn. leaves. Pharmacognosy Journal, 3, 5–8.
International Journal of Food Science and Technology 2015
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