Innovative Food Science and Emerging Technologies 10 (2009) 413–419

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Innovative Food Science and Emerging Technologies

Antioxidant and anticancer activities of high pressure-assisted extract of longan

a r t i c l e i n f o a b s t r a c t

Article history: Longan fruit pericarp was extracted with 50% ethanol employing high pressure (500 MPa) and conventional
Received 7 January 2009 extraction methods. Total phenolic contents of high pressure-assisted extract of longan (HPEL) and
Accepted 5 April 2009 conventional extract of longan (CEL) were 20.8 and 14.6 mg gallic acid equivalents/g dry weight,
Editor Proof Receive Date 23 April 2009 respectively. Subsequently, the antioxidant activities of these extracts were analyzed employing various
antioxidant model systems including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity,
Keywords: superoxide anion radical scavenging activity, total antioxidant capacity, and lipid peroxidation inhibitory
Anticancer activity. In addition, anticancer activity was also tested using HepG2, A549, and SGC 7901 cancer cell lines.
Antioxidant activity HPEL showed excellent antioxidant and anticancer activities and were higher than CEL. Three phenolic
High-pressure extraction compounds, namely gallic acid, corilagin, and ellagic acid, were identified and quantified by external
Longan fruit standard methods. Compared with CEL, HPEL exhibited higher extraction effectiveness in terms of higher
extraction yield, higher phenolic content, and higher antioxidant and anticancer activity with shorter
extraction time.
Industrial relevance: This study was focused to evaluate the antioxidant and anticancer activity of longan fruit
pericarp by high-pressure treatment. The high-pressure treatment provided a better way of utilizing longan
fruit pericarp as a readily accessible source of the natural anticancer and antioxidant in food and
pharmaceutical industry.
© 2009 Elsevier Ltd. All rights reserved.

1. Introduction acid derivatives (Halliwell, 2007; Li & Jiang, 2007). In recent years,
much attention has also been paid to potential anticancer components
Antioxidants are the compounds which, when added to lipids and from fruits and vegetables. Epidemiological studies have indicated the
lipid-containing foods, can increase their shelf-life by retarding the relationship between polyphenol intake and reduced risk of certain
process of lipid peroxidation, which is one of the major reasons for the cancers. Some flavones and polyphenols have been demonstrated to
deterioration of food products during processing and storage. decrease various types of experimental carcinogenesis (Zhao, Yang,
Synthetic antioxidants such as butylated hydroxyanisole (BHA) and Wang, Liu, Yu, & Jiang, 2007).
butylated hydroxytoluene (BHT) have been restricted for use in foods, Different traditional extraction techniques such as Soxhlet, heat
as these are suspected to be highly carcinogenic (Namiki, 1990). As a reflux, boiling, distillation, and hot water extraction have been widely
result, the importance of exploiting natural antioxidants, especially of used to isolate antioxidants and anticancer compounds from plants,
plant origin, has deeply increased in recent years. Numerous isolated but none of them can be considered as an optimal method for this
plant constituents as well as crude extracts of fruits and vegetables purpose (Zhang, Junjie & Changzhen, 2004). Of late, a high hydrostatic
have been recognized as natural antioxidants that possess beneficial pressure method that works with pressures ranging from 100 to
effects against free radicals in biological systems (Prasad, Divakar, 800 MPa, has been widely used in ceramics, pharmaceutics,
Shivamurthy, & Aradhya, 2005). This beneficial effect of fruits and metallurgy, plastic making, civil engineering, and in the food industry.
vegetables can be attributed to various phenolic compounds present The application of high hydrostatic pressure was first used as an
in them, possessing antioxidant activity. These phenolic compounds extraction technique in plant systems by Zhang et al. (2004). The use
include phenolic acids, anthocyanins, flavonoids, and hydrocinnamic of high pressure enhances mass transfer rates, which increases cell
permeability as well as secondary metabolite diffusion (Dornenburg &
Knoor, 1993). High-pressure extraction (HPE) has been successfully
⁎ Corresponding author. Tel.: +86 20 37252525; fax: +86 20 37253821. used for extraction of icariin from Epimedium, polyphenols from green
E-mail address: ymjiang@scib.ac.cn (Y. Jiang). tea (Zhang et al., 2004), flavones and salidroside from Rhodiola

1466-8564/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ifset.2009.04.003
414 K.N. Prasad et al. / Innovative Food Science and Emerging Technologies 10 (2009) 413–419

sachalinensis (Zhang, Bi, & Liu, 2007), anthocyanins from grapes extraction the solution was filtered and stored. The extraction yield for
(Corrales, Toepfl, Butz, Knorr, & Tauscher, 2008), flavonoids from conventionally extracted longan (CEL) was 14.8 ± 0.44%.
lychee (Prasad, Yang, Zhou, Ruenroengklin, & Jiang, 2009a). High-
pressure techniques could shorten processing times and obtain higher 2.5. Determination of total phenolic contents
extraction yields (Ahmed & Ramaswamy, 2006; Zhang et al., 2004).
Longan (Dimocarpus longan Lour.) belongs to the Sapindaceae The filtrates obtained from each extraction were freeze dried
family and is a highly attractive fruit that is extensively distributed in (Savant, Vapornet VN 100, Labequip Ltd., Markham, Ontario, Canada)
the Southern part of China. Longan fruit pericarp (LFP) contains high and stored at −20 °C until further analysis. Total phenolic contents of
amounts of bioactive compounds, such as phenolic acids, flavonoids, the extracts were analyzed by the method of Singleton and Rossi
hydrolysable tannins, and polysaccharides (Rangkadilok et al., 2007; (1965) and then expressed as gallic acid equivalents (GAE)/g. The
Yang, Zhao, Shi, Yang, & Jiang, 2008). These exhibit antibacterial, total phenolic content was determined using a standard gallic acid
antiviral, antioxidant, anti-inflammatory and anti-carcinogenic prop- calibration curve.
erties (Bravo, 1998; Pan et al., 2008; Rangkadilok et al., 2007; Yang
et al., 2008). There are a few reports on LFP extraction methods like 2.6. Evaluation of antioxidant capacity by phosphomolybdenum method
Soxhlet, microwave extraction (Pan et al., 2008), ultrasonic extraction
(Yang et al., 2008), ultra-high pressure extraction (Yang, Jiang, Rei, The antioxidant capacity of HPEL, CEL and BHT was determined
Zhao, & Jian, 2009), yet there is no study concerning the use of the HPE according to the method of Prieto, Pineda, and Aguilar (1999) with
to extract polyphenolic compounds from longan fruit pericarp. The some modifications. An aliquot of 0.1 mL of sample solution was
objective of this study is to test the possibility of using high pressure- combined with 1 mL reagent solution (0.6 M sulphuric acid, 28 mM
assisted extraction as a rapid and effective method for extracting sodium phosphate and 4 mM ammonium molybdate). The tubes were
polyphenolic compounds showing antioxidant and anticancer activity. capped and incubated in boiling water for 90 min. After the mixture
The total phenolics, extraction yield, antioxidant and anticancer had cooled, the absorbance of the mixture was measured at 695 nm
activities of HPEL were compared to CEL to confirm the advantages using a spectrophotometer (UV-2802, Unico Co. Ltd., Shanghai, China)
of the high pressure-assisted method. against a blank. A typical blank solution contained 1 mL of reagent
solution and the appropriate volume of the same solvent used for
2. Materials and methods dissolving the sample, and it was incubated under the same
conditions as the rest of the sample. The readings were taken at 15,
2.1. Chemicals and reagents 30, 60, and 90 min time intervals. The antioxidant activity was
expressed as the absorbance of the sample. The higher the absorbance
1,1-diphenyl-2-picrylhydrazyl (DPPH), ascorbic acid, butylated value, the higher is the antioxidant activity.
hydroxy toluene (BHT), nitro blue tetrazolium (NBT), methionine,
riboflavin, gallic acid, thiobarbituric acid (TBA), MTT [3-(4,5-dimethyl 2.7. Lipid peroxidation inhibitory activity
thiazole-2yl)-2,5-diphenyl tetrazolium bromide] were purchased
from Sigma Chemical Co. (St. Louis, MO, USA). Trypsin RPMI-1640 The lipid peroxidation inhibitory activity of HPEL was determined
medium (pH 7.4) was obtained from Gibco, NY, USA. Fetal bovine according to the method of Tsuda et al. (1994) with slight
serum was from Ebioway (Guangzhou, China). All other chemicals and modifications. Liposome sample (egg lecithin 6 mg/mL phosphate
solvents used in this study were of analytical grade and obtained from buffer, pH 7.4) was sonicated using an Ultrasonicator (SB-5200DTD,
Tianjin Reagent Company (Tianjin, China). Xinzhi Biotech Co., Ningbo, China, 40 kHz) for 1 h. Then 0.1 mL of HPEL
samples was dissolved in 50% ethanol at different concentrations and
2.2. Material was added to 0.5 mL of the liposome mixture. The control was made
without the test samples. Lipid peroxidation was induced by adding
Fresh fruits of longan (Dimocarpus longan Lour.) cv. Shixia at the 10 μL of FeCl3 (200 mM) and 10 μL of L-ascorbic acid (200 mM). After
commercially mature stage were chosen from a commercial orchard incubating for 1 h at 37 °C, the reaction was stopped by adding 2 mL of
and then selected for regularity of shape and color. The fruits were 0.25 N HCl containing 15% trichloroacetic acid (TCA) and 0.375% TBA
washed by tap water and the pericarp was manually peeled, dried in a (Thiobarbituric acid). The reaction mixture was boiled for 15 min,
hot air oven at 60 °C for 24 h, and grounded into powder. cooled and centrifuged, and the absorbance of the supernatant was
measured at 532 nm. The blank consisted of all the reagents but
2.3. Extraction assisted by high pressure without the lipid. The lipid peroxidation inhibitory activity was
calculated as inhibitory activity (%) = (1 − absorbance of sample /
The high-pressure equipment was from Kefa Food Equipment Ltd, absorbance of control) × 100. The lipid peroxidation inhibitory activity
Baotou, China. Pressure was generated by a hydraulic pump in of BHT and CEL was also assayed for comparison. The EC50 (50% of the
combination with a pressure intensifier. The pressure transmitting radicals scavenged by the test sample) values were also determined.
medium was dioctyl sebacate. The pressure chamber was controlled at The lower the EC50 value, the higher was the antioxidant activity.
constant temperature (30 °C) by a water bath. All processing
conditions, including pressure, time, and temperature, were con- 2.8. Radical scavenging activity using DPPH method
trolled and programmed using a computer. Longan samples (10 g)
were mixed with 500 mL of 50% ethanol and were pressurized at The radical scavenging activities of the extracts were determined
500 MPa for 2.5 min. After de-pressurization, the extracted solution as described by Moon and Terao (1998) with little modification.
was collected, filtered and stored. The yield for high-pressure-assisted Different concentrations (10, 20, 40, 80, and 100 μg/mL) of HPEL, CEL
extraction of longan (HPEL) was found to be 17.92 ± 0.21%. and BHT were placed in different test tubes and were mixed with
0.8 mL of Tris–HCl buffer (pH 7.4), and then 1 mL of DPPH (500 μM in
2.4. Conventional extraction ethanol) was added. The reaction mixture was shaken vigorously and
incubated at 28 °C in a dark room for 40 min. The control was prepared
The conventional extraction was carried out by weighing 10 g of as above without any extract, and ethanol was used for the baseline
longan powder into Erlenmeyer flasks containing 50% ethanol (500 mL). correction. The changes in the absorbance of the samples were
Extractions were carried out for 12 h using a magnetic stirrer. After measured at 517 nm using a spectrophotometer. The inhibition of
 K.N. Prasad et al. / Innovative Food Science and Emerging Technologies 10 (2009) 413–419 415

DPPH radicals by the test samples was calculated as scavenging Corporation, Japan). The separation was performed using a Vydac C18
activity (%) = (Control OD − sample OD / control OD) × 100. The EC50 column (218 TP, 250 mm × 4.6 mm, 5 μm size particle, Sigma-Aldrich,
(50% of DPPH radicals scavenged by the test sample) values were also St-Louis, MO, USA), operated at 25 °C. Mobile phases contained 0.4%
calculated. formic acid (A) and methanol (B) with flow rates of 1 mL/min. The
injection volume was 10 μL. The gradient system started from 0 min
2.9. Superoxide anion radical scavenging activity (100% A) to 2 min (95% A), 5 min (70% A), 8 min (66% A), 11 min
(45% A), 14 min (45% A), and 17 min (100% A) and then maintained
Superoxide anion radical scavenging activity was analyzed by the until 20 min had elapsed. The peaks of the phenolic compounds were
method of Duan, Wu, and Jiang (2007). All solutions were prepared in monitored at 270 nm. Phenolic acid standards such as gallic acid,
0.2 M phosphate buffer (pH = 7.4). The photo-induced reactions were corilagin and ellagic acid were used for identification of phenolic
performed in an aluminum foil-lined box with two 30 W fluorescent acids. The identified phenolic acids were quantified on the basis of
lamps. The HPEL and CEL extracts were mixed with 3 mL of reaction their peak area and compared with a calibration curve obtained with
buffer solution (1.3 μM riboflavin, 0.02 M methionine, and 5.1 μM nitro the corresponding standards and then expressed as mg/g DW.
blue tetrazolium, pH = 7.4). The reaction solution was illuminated for
20 min at 25 °C and the absorbance was then measured at 560 nm. 2.13. Statistical analyses
BHT was used as the positive control and the reaction mixture without
any sample was used as the control. The superoxide anion radical Data were expressed as means ± standard deviations (SD) of three
scavenging activity was calculated as scavenging activity (%) = (1 − replicated determinations and then analyzed by SPSS V.13 (SPSS Inc.,
absorbance of sample / absorbance of control) × 100. The EC50 (50% Chicago, USA). One way analysis of variance (ANOVA) and Duncan's
of the radicals scavenged by the test sample) values were also new multiple-range test were used to determine the differences of
obtained. means. Differences between the means at the 5% level were
considered to be significant.
2.10. Cell line and culture
3. Results
HepG2 (human hepatocellular liver carcinoma), A-549 (human
lung adenocarcinoma) and SGC-7901 (human gastric carcinoma) cell 3.1. Total phenolic content
cultures were obtained from Biomedicine Research and Development
Centre of Jinan University (Guangzhou, China). The cell lines were In our previous studies an ethanol concentration of 50% and
cultured in growth medium (RPMI-1640 medium, pH 7.4), supple- 500 MPa for 2.5 min for HPE was found to be the optimal extraction
mented with 10% fetal bovine serum (FBS) and antibiotics [(penicillin conditions (Prasad, Yang, Yi, Zhao, & Jiang, 2009b). Phenolics present
(100 units/mL) and streptomycin sulfate (100 μg/mL)]. The cell lines in fruits and vegetables have received considerable attention because
were grown in 50 cm2 tissue culture flasks (Corning, NY, USA) and of their potential antioxidant activity (Pan et al., 2008). Phenolic
used for cytotoxicity assay. compounds undergo a complex redox reaction with the phospho-
tungstic and phosphomolybdic acids present in the Folin–Ciocalteu
2.11. Cytotoxicity assay reagent (Amin & Yazdanparast, 2007). In the present study, the
antioxidant potential of HPEL was revealed by expressing antioxidant
The cytotoxicity assay of the samples was determined according to activity in terms of phenolic content, compared to CEL employing
the method of Su et al. (2008) by MTT assay. In brief, cells were plated the Folin–Ciocalteu method. The total phenolic content of HPEL was
at 2 × 104 cells per well in 96 well microtiter plates (Costar 3599, 20.8 ± 1.6 mg/g while CEL was 14.6 ± 0.2 mg/g dry weight, expressed
Corning, NY, USA) with 100 μL RPMI-1640 growth medium and as gallic acid equivalents. HPE increased the phenolic compounds'
incubated for 24 h at 37 °C, with 5% CO2 in a humidified atmosphere recovery approximately one and a half times more than CE.
(Incu-Safe, Sanyo, Japan), during which period a partial monolayer
was formed. Later, the medium was removed and fresh growth 3.2. Total antioxidant capacity by the phosphomolybdenum method
medium containing different concentrations (50, 25, 12.5, 6.5, and
3.125 μg/mL) of the test samples (HPEL, CEL and cisplatin) was added Total antioxidant capacity of HPEL and CEL was determined by the
separately. After 2 days of incubation at 37 °C, 5% CO2, medium was formation of phosphomolybdenum complexes. This assay is based on
removed and MTT reagent (0.1 mg/mL) was added. After incubating the reduction of Mo (VI) to Mo (V) by antioxidant compounds and
at 37 °C for 4 h, the MTT reagent was removed and DMSO (100 μL) was subsequent formation of a green phosphate–Mo (V) complex at acidic
added to each well and shaken for another 15 min. The absorbance pH which has its maximum absorption at 695 nm. A high absorbance
was then determined by an ELISA reader (Bio-Rad, USA) at a value indicates that the sample possessed good antioxidant activity
wavelength of 492 nm. Control wells received only the media without (Pan et al., 2008). In this assay, the total antioxidant activities of HPEL
the test samples. The conventional anticancer drug, cisplatin, was and CEL were measured and compared with BHT (Fig. 1). According to
used as a positive control in this study. The inhibition of cell growth by the results, HPEL had significant total antioxidant activities and the
the samples tested was calculated as the percentage anticancer effects increased with increasing reaction time and increasing
activity and was calculated using the following formula: percentage concentration. The total antioxidant of HPEL at the concentration of
anticancer activity (Ac − As / Ac) × 100%. Ac and As refer to the 50 μg/mL at 90 min was 0.69 ± 0.1, while CEL and BHT was 0.50 ± 0.1
absorbance of control and the sample, respectively. and 0.94 ± 0.2, respectively. The total antioxidant of HPEL was
superior to CEL at all the concentrations tested. This showed that
2.12. Phenolic compound analysis by high performance liquid HPEL possessed better antioxidant activity than CEL. Generally,
chromatography (HPLC) phenolics, carotenoids, flavonoids, and ascorbic acid possess good
total antioxidant activities (Jayaprakasha, Girennavar, & Patil, 2008).
Phenolic compounds were identified using the HPLC method
(Rangkadilok, Worasuttayangkurn, Bennett, & Satayavivad, 2005) and 3.3. Lipid peroxidation inhibitory activity
quantified by external standard method. HPEL and CEL extracts were
re-dissolved in 100 μL of 50% ethanol and membrane-filtered To evaluate the antioxidant activity of HPEL and CEL (to inhibit
(0.45 μm). The samples were analyzed using an LC-20 AT (Shimadzu lipid peroxidation in biological systems), a liposome model system
416 K.N. Prasad et al. / Innovative Food Science and Emerging Technologies 10 (2009) 413–419

Fig. 1. Total antioxidant activity by phosphomolybdenum method of HPEL compared to CEL and BHT. Results are mean ± SD of three parallel measurements. Higher absorbance value
indicates higher antioxidant activity. HPEL, high pressure-assisted extract of longan; CEL, convention-assisted extract of longan; BHT, butylated hydroxy toluene.

was used. Thiobarbituric acid reacts with malondialdehyde (MDA) to HPEL, CEL, and BHT quenched DPPH radicals in a dose-dependent
form a diadduct, a pink chromogen, which can be detected spectro- manner: [r2 = 0.9702] (p b 0.05) for HPEL; [r2 = 0.9723] for CEL and
photometrically at 532 nm. Malondialdehyde is the major product of [r2 = 0.9966] (p b 0.05) for BHT. The EC50 values (concentration of
lipid peroxidation and has been studied widely as an index of lipid sample required to scavenge 50% of DPPH radicals) were found to be
peroxidation and as a marker for oxidative stress (Amin & Yazdanpar- the least in BHT (8.31 ± 1.5), followed by HPEL, (39.05 ± 2.1) and CEL
ast, 2007). The lipid peroxidation inhibitory activity of HPEL and CEL (57.46 ± 1.3 μg/mL) respectively (Table 1). These results indicated
as well as BHT at different concentration is given in Table 1. The that the scavenging effect of HPEL on DPPH radical was superior to
inhibitory activity of HPEL was higher at each concentration compared CEL, which might be attributed to their electron-donating ability.
with CEL, and its inhibitory activity was dose-dependent. EC50 values
for HPEL, CEL, and BHT were 40.23 ± 2.7, 58.03 ± 2.5, and 17.45 ± 3.5. Superoxide anion scavenging activity
4.8 μg/mL, respectively. The lower the EC50 value, the higher is the
antioxidant activity. Generally, phenolics inhibit the lipid peroxidation Superoxide anion plays an important role in plant tissues and is
by chain termination through scavenging the peroxyl radicals and also involved in the formation of other cell-damaging free radicals (Duan
by electron donation (Prasad et al., 2005). Since, HPEL and CEL contain et al., 2007). The relative scavenging effects of HPEL, CEL, and BHT
abundant phenolics, the mechanism of lipid peroxidation in this study towards superoxide anion radicals are shown in Table 1. HPEL and CEL
might be through electron donation or by scavenging the peroxyl exhibited excellent superoxide anion scavenging activity as compared
radicals. with BHT. The EC50 values were found to be the least in HPEL (53.66 ±
2.7, p b 0.05), followed by CEL (84.76 ± 2.4, p b 0.05) and BHT (470.68 ±
3.4. Scavenging activity on DPPH radicals 1.0 μg/mL), respectively. Therefore the superoxide anion scavenging
activity, in increasing order was, HPEL N CEL N BHT. It is known that the
Antioxidants either transfer an electron or a hydrogen atom to hydroxyl group of the phenolics contributes to superoxide anion
DPPH, thus neutralizing its free radical character (Pan et al., 2008). scavenging activity by their electron donation (Bravo, 1998). Similarly,

Table 1
Antioxidant activity of longan samples obtained with different extraction methods.

Sample Concentration Lipid peroxidation DPPH radical scavenging activity Superoxide scavenging activity
(µg/mL) Inhibition (%) EC50 (µg/mL) Inhibition (%) EC50 (µg/mL) Inhibition (%) EC50 (µg/mL)
HPEL 10 27 ± 3 40.23 ± 2.7 28 ± 1.3 39.05 ± 2.1 37 ± 3.5 53.66 ± 2.7
20 38 ± 1.2 41.7 ± 3.3 42.7 ± 3.3
40 51 ± 3.1 53.4 ± 2.3 47.7 ± 4.3
80 78 ± 0.6 76.6 ± 1.8 58 ± 1.4
100 88 ± 6 82 ± 2 60.2 ± 1.1
CEL 10 19 ± 4 58.03 ± 2.5 12 ± 1.3 57.46 ± 1.3 18 ± 2.1 84.76 ± 2.4
20 28.1 ± 0.3 27.7 ± 1 28.5 ± 2.1
40 40.2 ± 1.8 41.3 ± 1.8 35.9 ± 2.5
80 64.4 ± 2.1 68.5 ± 2 50.7 ± 2.7
100 74 ± 4.5 75 ± 0.7 52.6 ± 2.3
BHT 10 42 ± 4 17.45 ± 4.8 55 ± 0.4 8.31 ± 1.5 13 ± 1.8 470.68 ± 1.0
20 53.6 ± 3.2 59.9 ± 1.2 14.8 ± 0.7
40 64.4 ± 7.2 66.4 ± 2.4 16.3 ± 0.3
80 86.1 ± 5.3 79.4 ± 2.1 19.5 ± 1.1
100 92 ± 4.7 85 ± 1.5 20.3 ± 1.5

HPEL, high pressure-assisted extract of longan; CEL, convention-assisted extract of longan and BHT, butylated hydroxy toluene. EC50 represents 50% of the radicals scavenged by the
test sample.
 K.N. Prasad et al. / Innovative Food Science and Emerging Technologies 10 (2009) 413–419 417

Table 2 4. Discussion
Anticancer activity of longan samples obtained with different extraction methods.

Sample Anticancer activity (%) The extraction of bioactive compounds can be described as a mass
SGC 7901* HepG2** A549** transport phenomenon where solids contained in plant structures
CEL 30.58 ± 1.1c − 0.54 NA migrate into the solvent, up to their equilibrium concentration. Mass
HPEL 37.6 ± 2.6a − 0.22 11.96 ± 0.80b transport phenomenon can be increased by heating, changes in
Cisplatin 34.26 ± 3.2b 55.34 ± 2.2 49.57 ± 2.1a concentration gradients, and under the influence of new technologies
NA, no activity; SGC 7901 (human gastric carcinoma); HepG2 (human hepatocellular such as ultrasonics, high pressure, and pulsed electric field (Corrales
liver carcinoma), A 549 (human lung adenocarcinoma); * Concentration of the sample et al., 2008). Increased extraction yields caused by high pressure were
(50 μg/mL) and ** Concentration of the sample (100 μg/mL). CEL, convention-assisted probably due to its aptitude to deprotonate charged groups and to
extract of longan; HPEL, high pressure-assisted extract of longan and cisplatin, positive
disrupt salt bridges and hydrophobic bonds in cellular membranes,
control. For each treatment, the means within a row followed by different letters were
significantly different at the 5% level. which may lead to a higher permeability (Dornenburg & Knoor, 1993).
Based on phase behavior theory, the pressurized cells exhibit
HPEL and CEL obtained in this study might scavenge the superoxide increased permeability as pressure increases, which might account
anion in a similar manner since they are rich in phenolics. for the increased extraction yield by HPE (Zhang et al., 2007).
The antioxidant activity of HPEL and CEL is in accord with the
3.6. Anticancer activity amount of phenolics present in the extracts. Also, HPEL samples
contain substantial amounts of phenolics as compared to those from
The anticancer activities of the HPEL, CEL, and cisplatin were CEL, and it's the extent of phenolics present in this extract that is
investigated using an MTT assay on three human cancer cell lines, responsible for its marked antioxidant activity as proposed through
SGC-7901, HepG2, and A-549. A mitochondrial enzyme in living cells, various in vitro models. Several reports have conclusively shown a
succinate-dehydrogenase, cleaves the tetrazolium ring, converting the close relationship between total phenolic content and antioxidant
MTT to an insoluble purple formazan. Therefore, the amount of activity (Duan et al., 2007; Li & Jiang, 2007; Prasad et al., 2005). The
formazan produced is directly proportional to the number of viable phenolic compounds exhibit extensive free radical scavenging
cells (Lee, Hwang, & Lim, 2004). Table 2 shows that HPEL significantly activities, through their reactivity as hydrogen or electron-donating
inhibited (p b 0.05) cancer cell growth of SGC 7901 and A549 cell lines, agents, and metal ion chelating properties (Hollman & Katan, 1999;
compared to CEL. At a concentration of 50 μg/mL, the percentage Rice-Evans, Miller, & Paganga, 1996). Gallic acid and ellagic acid, which
anticancer activity of HPEL (37.6 ± 2.6) on the SGC 7901 cell lines are reported from LFP, have shown selective cytotoxicity, antiproli-
tested, were significantly higher than cisplatin (34.26 ± 3.2). The ferative activity, and induced apoptosis in cancer cell lines (Kawada et
negative activities of HPEL and CEL to HepG2 cell lines mean that they al., 2001; Losso, Bansode, Trappy, Bawadi, & Truax, 2004). Phenolic
did not inhibit cancer cell growth, but apparently increased the compounds might also be involved in xenobiotic metabolizing
growth rate of cancer cells. However, more confirmative studies will enzymes that alter metabolic activation of potential carcinogens.
be essential in the future to confirm this. Similar results have been Some phenolics may also alter hormone production and inhibit
reported earlier by Lee et al. (2004) where the samples did not inhibit aromatase to prevent the development of cancer (Zhao et al., 2007).
cancer cells but rather increased their growth rate. Our results are also So, there should be a close correlation between the content of
in agreement with those by Liu et al. (2008) where SGC 7901 is phenolic compounds and antioxidant and anticancer activities (Bravo,
sensitive to cisplatin treatments, which was confirmed by our results, 1998).
while the A 549 cell line was resistant to anticancer compounds. LFP contains large amounts of polar compounds like phenolic
acids, flavonoids, and polysaccharides, which are good antioxidant
3.7. Identification and quantification of polyphenolic compounds and anticancer compounds. Using high pressure, these compounds are
probably extracted more due to the increases in solvent strength,
Calibration curves of the standards were linear with r2 value of solvent density, and solubilities of polar compounds. Also, a decrease
0.9175, 0.9024 and 0.9994 for gallic acid, corilagin and ellagic acid, in the dielectric constant of water caused by high pressure could lead
respectively. The HPLC profiles of phenolic compounds extracted from to a decrease in the polarity of the media which may contribute to the
longan fruit pericarp tissues are presented in Fig. 2. Three phenolic higher phenolic content and also higher antioxidant and anticancer
compounds, namely gallic acid, corilagin, and ellagic acid, were activities (Corrales et al., 2008). Moreover, high pressure provides the
identified and quantified by external standard method. Corilagin had possibility of inactivating degrading enzymes, which may explain the
the highest content among the three phenolic compounds. The total higher yields and also higher antioxidant and anticancer activity
phenolic content of the HPEL sample was higher as compared to compared with other methods. High pressure also has the ability to
the CEL sample (Fig. 3). This present study is in agreement with reduce the pH of the solvent during extraction, not only because of the
Rangkadilok et al. (2005) and Sun, Shi, Jiang, Xue, and Wei (2008), higher release of phenolics into the solvent but also because of the
where gallic acid, corilagin, and ellagic acid were identified from deprotonation of molecules present in the extracts. This reduction in
longan pericarp. pH might also enhance the extraction of bioactive compounds, since

Fig. 2. HPLC profile of phenolic compounds from pericarp tissues of longan fruit.
418 K.N. Prasad et al. / Innovative Food Science and Emerging Technologies 10 (2009) 413–419

Fig. 3. Phenolic contents of longan fruit obtained by different extraction methods. HPEL, high pressure-assisted extract of longan; CEL, convention-assisted extract of longan. Values
are means ± standard deviations of three replicate analyses.

most of the compounds are more stable at pH less than four (Corrales References
et al., 2008; del Valle, Rogalinski, Zetzl, & Brunner, 2005).
Our results are in agreement with other authors who have Ahmed, J., & Ramaswamy, H. S. (2006). High pressure processing of fruits and
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with higher antioxidant activity of Peumus boldus leaves by using high Amin, A., & Yazdanparast, R. (2007). Antioxidant and free radical scavenging potential
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This work was supported by the National Natural Science Chemistry, 106, 1264–1270.
Foundation of China (Grant Nos. 30425040 and 30700557) and Prasad, N. K., Divakar, S., Shivamurthy, G. R., & Aradhya, S. M. (2005). Isolation of a free
radical scavenging antioxidant from water spinach (Ipomoea aquatica Forsk).
International Foundation of Science (No. F/4451-1), CAS/SAFEA
Journal of the Science of Food and Agriculture, 85, 1461–1468.
international partnership program for creative research teams and Prasad, N. K., Yang, B., Ruenroengklin, N., Zhao, M., & Jiang, Y. (2009a). Application of
Science Foundation of South China Botanical Garden (No. 200807). ultrasonication or high pressure extraction of flavonoids from litchi fruit pericarp.
The author is also thankful to Prof. Xiaoyi Wei, South China Botanical Journal of Food Process Engineering. doi:10.1111/j.1745-4530.2008.00247.x.
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Garden, Guangzhou, China for donating ellagic acid and corilagin extraction yield, total phenolic content and antioxidant activity of longan fruit
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