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Journal of Food Biochemistry ISSN 1745-4514

PHENOLIC ACIDS, FLAVONOID PROFILE AND ANTIOXIDANT ACTIVITY IN MANGOSTEEN (GARCINIA MANGOSTANA L.) PERICARP jfbc_575

627..633

A.S. ZARENA and K. UDAYA SANKAR1 Food Engineering Department, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore 570020, India

1

Corresponding author. TEL: 91-821-2514874; FAX: 91-821-2517233; EMAIL: [email protected] Accepted for Publication May 2, 2011 doi:10.1111/j.1745-4514.2011.00575.x

ABSTRACT Plants contain natural antioxidant constituents such as phenolic compounds, which have attracted a great deal of public and scientific interest because of their healthpromoting effects as antioxidants. A high-performance liquid chromatography procedure for separating 15 polyphenols was used for the determination of phenolic acids and flavonoids in the pericarp of the mangosteen fruit. Sequential hydrolysis of the pericarp showed that phenolic acids can be released by hydrolyzing the mangosteen pericarp under basic or acidic conditions; however, the former was more efficient in the release of phenolic acids than the latter. The base hydrolyzed fraction also showed to be the most potent antioxidant and free radical scavenger compared with acid hydrolyzed fraction or unhydrolyzed fraction, which may be accounted for the high polyphenolic content.

PRACTICAL APPLICATIONS The outcome of this study shows that mangosteen fruit pericarp extracts contain phenolic acids, which are believed to be effective nutrients in the prevention of oxidative stress. This experiment also proved that simple high-performance liquid chromatography offers a very good alternative for identification of phenolic compounds instead of silyl derivatization. The mass spectrometry of the phenolic compounds is used together with the aforementioned technique as a more sensitive, easy method to detect and study the compositional profile of the extracts as well as the structure and functional relationship of the components.

INTRODUCTION Phenolic acids, in general, are phenols with one carboxylic acid group virtually derived from benzoic and cinnamic acids. Hydroxybenzoic acids have the carboxylic acid group directly attached to the ring while hydroxycinnamic acids have a three carbon side chain. The different phenolic acids differ in the number and position of the hydroxyl and methoxyl groups attached to the aromatic ring (Robbins 2003). The most common hydroxycinnamic acids are caffeic, p-coumaric and ferulic acids, which frequently occur in foods as simple esters with quinic acid or glucose (Mattila and Hellström 2007). Probably the most well-known bound hydroxycinnamic acid is cholorogenic acid, which is combined from caffeic and quinic acids. Unlike hydroxycinnamates, hydroxyJournal of Food Biochemistry 36 (2012) 627–633 © 2011 Wiley Periodicals, Inc.

benzoic acid derivatives are mainly present in foods in the form of glucosides; the most common forms are p-hydroxybenzoic acid, vanillic and protocatechuic acids (Manach et al. 2004). Most of the prophylactic activities of phenolic acids have been ascribed to their antioxidant, antimutagenic, antiproliferative and antimicrobial properties (Kampa et al. 2004); because of the wide spectrum of biological activity of phenolic acids, they are routinely consumed in significant amount in human diet. Phenolic acids are responsible for the color, flavor and oxidative stability of fresh and processed products; therefore, they are continually investigated by food technologists (Luzia et al. 1997). The presence of the CH = CH–COOH group in the hydroxycinnamic acids is considered to be key for the significantly higher antioxidative efficiency than 627

ANALYSIS OF PHENOLIC ACID BY HPLC–ESI–MS

2the COOH in the hydroxybenzoic acids (White and Xing 1997). Mangosteen fruits pericarp is rich in xanthones, anthocyanins and oligomeric proanthocyanidin (Fu et al. 2007; Zarena et al. 2010). To the best of our knowledge, there are very few reports on phenolic acid profiles of mangosteen pericarp. The phenolic acids are mainly located in the pericarp of the mangosteen fruit. The pericarps of the fruit are often treated as waste. Because of the large number of structurally similar polyphenolics in plants, the analysis of individual polyphenolics is difficult and complicated. The objective of the present study is to determine the contents of individual phenolic acids in different hydrolyzed mangosteen pericarp (dried) fraction by liquid chromatography–mass spectrometry and to measure antioxidative activities.

MATERIALS AND METHODS

A.S. ZARENA and K.U. SANKAR

and was collected in a round-bottom flask and evaporated at 20C in a rotavapor and reconstituted with methanol water (85:15) and analyzed for free phenolic acid (FPA) content by high-performance liquid chromatography (HPLC). An aliquot of the said methanolic extract was hydrolyzed by adding 5 mL of 10 M NaOH to the extract and the mixture was flushed with nitrogen and kept in a screw capped tube overnight at ambient temperature. The pH of the extract was adjusted to 2 by the addition of 10 mL of 6 N HCl, and the liberated phenolic acids were extracted with ethyl acetate and were evaporated to dryness. The resulting fraction contained base-hydrolyzed phenolic acids (BHPAs). The remaining water phase was treated with 5 mL of concentrated HCl and the mixture was stirred for 30 min in a hot water bath at 85C for 20 min. The solution was again partitioned with ethyl acetate, which contains acid-hydrolyzed phenolic acids (AHPAs). All fractions were reconstituted in methanol and were analyzed by HPLC.

Materials Gallic acid, gentisic acid, 4-hydroxybenzoic acid, caffeic acid, vanillic acid, syringic acid, coumaric acid, ferulic acid, transcinnamic acid and linolenic acid were purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO). Ascorbic acid, butylated hydroxyanisole (BHA), 1,1-diphenyl-2picrylhydrazyl (DPPH), acetic acid, anhydrous sodium carbonate, methanol and water used for preparing mobile phases were purchased from Merck (Mumbai, India). Folin– Ciocalteu reagent was from Loba Chemie (Mumbai, India). All other reagents were of analytical grade.

Preparation of Extracts The mangosteen pericarp is rich in xanthones and anthocyanins; these may act as interfering materials during polyphenolic extraction procedure. Xanthones and other lipophilic compounds were removed from dried pericarp of mangosteen powder by precipitating with cold dichloromethane for 24 h. Phenolic acid extraction in the aforementioned crude extract was carried out as proposed by Luthria and PastorCorrales (2006) with slight modification. Precisely weighed amount of dry ground mangosteen pericarp sample (0.5 g) was treated with methanol water (85:15) with 1% acetic acid. The mixture was crushed using mortar and pestle and sonicated for 30 min, and was filtered through a 0.45-mm polytetrafluoroethylene syringe filter. A known volume of filtered extract was loaded onto C-18 sep-pak cartridge (preconditioned by sequentially passing 10 mL ethylacetate, 10 mL absolute methanol and 10 mL of 0.01 N aqueous HCl through the cartridge). The adsorbed anthocyanin in the cartridge was eluted with 6 mL of acidic methanol and collected separately. The cartridge was rinsed with 40 mL ethyl acetate to elute polyphenolic compounds other than anthocyanins 628

HPLC Analysis Phenolic acids were analyzed in Shimadzu LC 8A (Shimadzu Corporation, Kyoto, Japan) system. Separation of individual phenolic acid was carried out using a Bondapak C18 column (300 mm ¥ 3.9 mm i.d.; particle size, 5 mm) at ambient temperature. The mobile phase consisted of water : methanol : acetic acid (85:14:1). The flow rate was 1.0 mL/min and the injection volume was 20 mL. The detector was set at 280 and 320 nm. About 10 mg of a standard of each kind of phenolic acid weighed accurately was dissolved into a 10-mL volumetric flask in 1:1 ethanol to obtain stock solutions. For calibration curves, the stock solution was diluted with 1:4 ethanol to obtain the concentration sequence. Seven concentration levels were used in calibration curves (20, 15, 10, 5, 2.5, 1.0 and 0.5 mg/L). The mean areas generated from the standard solutions were plotted against concentration to establish calibration equations and the peak areas in the sample chromatograms were correlated with the concentrations according to the calibration curve. Unknown phenolic acids were tentatively identified by mass spectroscopy.

Direct Infusion Electrospray–Mass Spectrometry (ESI–MS) The fractions (1–2 mL) were directly infused on mass spectrometer for mass ion analysis. The ESI–MS of the mangosteen pericarp was obtained with an alliance waters 2,695 mass spectrometer (Waters Corporation, Micromass Ltd., Manchester, U.K.) operating at ESI negative mode. The following ion optics was used: capillary voltage: 3.00 kV; cone: 100; source temperature: 120C, desolvation temperature: 300C; cone gas flow, 50 L/h; and desolvation gas: 500 L/h. The mass range was from 200 to 900 m/z, scan speed 1,000 amu/s. Journal of Food Biochemistry 36 (2012) 627–633 © 2011 Wiley Periodicals, Inc.


Total Polyphenolic Content The total polyphenolic content of mangosteen extracts was determined using Folin–Ciocalteau reagent as described by Kujala et al. (2000). The total concentration of polyphenolic compounds in mangosteen extracts was measured as gallic acid equivalent (GAE) and expressed as mg/g of dry extract.

DPPH• Free Radical Scavenging Assay The antioxidant activity of the extracts was measured on the basis of the scavenging activity of the stable radical DPPH• according to the method of Wang et al. (2003). The extracts in methanol at different concentration range (20–160 mg/mL) were mixed in the freshly prepared 0.5 mM DPPH• in ethanol and 0.1 M acetate buffer (pH 5). Absorbance at 517 nm was determined after 30 min. The scavenging activity was calculated using the following equation:

% DPPH scavenging activity ⎡ (A of control − A 517 of sample) ⎤ = ⎢ 517 ⎥ × 100 A 517 of control ⎣ ⎦

Lipid Peroxidation by Thiobarbituric Acid (TBA) Assay Lipid peroxidation induced by ferrous sulfate–ascorbic acid system in linolenic acid micelles was estimated by the method of Shimazaki et al. (1984). An aliquot of extracts (20–120 mg/ mL) was incubated with ferrous sulfate and ascorbic acid (10:100 mmol) in a final volume of 0.5 mL of Tris buffered saline (10 mM Tris, pH 7.4, 150 mM NaCl); the reaction mixture was incubated at 37C for 1 h. The reaction mixtures were treated with 1% TBA and incubated in a hot water bath for 15 min. BHA was used as positive controls while the negative control was without any antioxidant or extract. The color developed was measured spectrophotometrically at 535 nm.

% Lipid peroxidation inhibition ⎡ (A of control − A 535 of sample) ⎤ = ⎢ 535 ⎥ × 100 A 535 of control ⎣ ⎦

RESULTS AND DISCUSSION The total phenolic content in BHPA fraction showed 294.4 ⫾ 2.3 mg/g GAE. The phenolic content of the BHPA fraction was higher than that of the FPA fraction (209.1 ⫾ 2.04 mg/g GAE) and AHPA fraction (18.6 ⫾ 0.04 mg/g GAE), indicating that bound phenolic acids can be hydrolyzed using an acid or an alkali to release hydrolyzable phenolic acids. Journal of Food Biochemistry 36 (2012) 627–633 © 2011 Wiley Periodicals, Inc.


The Folin–Ciocalteu method gives an overall comparative results with other plant extracts but the determination of phenolic compounds is not specific as its reactivity is different for different polyphenolics, whereas HPLC analysis provides more precise information about individual compounds. The phenolic compounds identified in the different fraction in mangosteen pericarp are listed in Table 1 and a typical HPLC chromatogram of the base hydrolyzed fraction is presented in Fig. 1A. Individual phenolic acid in each fraction was identified and quantified in comparison with authentic standards and the quantification is represented as mg/g dry weight of the extract. A comparative HPLC chromatogram recorded at 280 nm for the FPA, BHPA and AHPA fractions is presented in Fig. 1B. Caffeic acid (1.51 mg/g), t-cinnamic acid (0.73 mg/g), vanillic acid (0.71 mg/g), sinapic acid (0.71 mg/g) and syringic acid (0.63 mg/g) were the predominant phenolic acids in the BHPA fraction. Gallic acid (2.77 mg/g) was the only prominent peak observed in the AHPA fraction with vanillic acid and caffeic acid present in trace amount. Robbins (2003) has reported that cinnamic acid derivatives, p-coumaric, caffeic and ferulic acids, were found to degrade under hot acidic conditions. This may partially explain why acid hydrolysis gave a low yield of hydrolyzable phenolic acids. In FPA fraction, the percent of gallic acid content (0.21 mg/g) was predominately higher than BHPA extract. There were three unknown peaks at relative retention time of 0.60 (0.08 mg/ g), 0.73 (0.67 mg/g) and 1.42 min (0.13 mg/g), which were absent in the BHPA and AHPA extracts. Gentisic acid, 4-hydroxybenzoic acid, vanillic acid and sinapic acid, which were present in BHPA extract, were not detected in the FPA extract. These differences in the phenolics may be attributed to the presence of different extractable components Zadernowski et al. (2009) have reported the presence of protocatechuic acid as the major phenolic acid in peel and rind of the mangosteen, as detected by gas chromatography method. Their study also reported the absence of p-coumaric acid and sinapic acid in the peel or rind of the FPA fraction of lyophilized mangosteen; however, p-coumaric acid was detected in the rind of the bound phenolic acid fraction. In our present study, we found both the phenolic acids to be present in BHPA fraction (0.31 and 0.71 mg/g) and only p-coumaric acid was detected in FPA fraction (0.05 mg/g). The p-coumaric acid and sinapic acid are known to be involved in the lignin synthesis in the mangosteen pericarp, which results in hardening of the pericarp (Bunsiri et al. 2003). Apart from phenolic acid, we were able to identify two flavonoids in FPA and BHPA fractions. While catechin (2.04 mg/g) was the main flavonol identified in the FPA, extract quercetin (2.27 mg/g) was the major flavonol identified in BHPA fraction, and no flavonoids were detected in AHPA fraction. 629



Phenolic compound

RRT (min)

[M-H]- (m/z)

FPA (mg/g)

BHPA (mg/g)

UK Gallic acid* Protocatechuic acid Gentisic acid* 4-hydroxybenzoic acid* Veratric acid UK Vanillic acid* UK Caffeic acid* Syringic acid* p-coumaric acid Sinapic acid UK Mandelic acid Ferulic acid* t-cinnamic acid* Epicatechin* Quercetin

0.29 0.34 0.40 0.46 0.50

169 153 153 137

ND 0.21 ⫾ 0.04 0.13 ⫾ 0.02 ND ND

0.16 ⫾ 0.07 0.09 ⫾ 0.01 0.59 ⫾ 0.32 0.13 ⫾ 0.08 0.36 ⫾ 0.11

0.02 ⫾ 0.01 0.08 ⫾ 0.01 ND 0.67 ⫾ 0.24 0.47 ⫾ 0.20 0.22 ⫾ 0.05 0.05 ⫾ 0.01 ND 0.13 ⫾ 0.02 0.04 ⫾ 0.01 0.13 ⫾ 0.08 0.02 ⫾ 0.01 2.04 ⫾ 0.83 0.20 ⫾ 0.03

0.27 ⫾ 0.01 ND 0.71 ⫾ 0.36 ND 1.51 ⫾ 0.62 0.63 ⫾ 0.28 0.31 ⫾ 0.19 0.71 ⫾ 0.31 ND 0.43 ⫾ 0.12 0.13 ⫾ 0.09 0.73 ⫾ 0.37 0.77 ⫾ 0.31 2.27 ⫾ 0.82

0.57 0.60 0.68 0.73 0.84 1.00 1.15 1.26 1.42 1.61 1.78 2.00 2.64 3.07

181 167 179 197 163 223 151 193 147 289 301

AHPA (mg/g)

TABLE 1. PHENOLIC ACIDS CONTENT IN MANGOSTEEN PERICARP FRACTIONS (MG/G, DRY WEIGHT BASIS)

2.77 ⫾ 0.74

0.56 ⫾ 0.21 0.24 ⫾ 0.16

* Identification based on authentic standard and mass spectrometry spectra. Values are mean ⫾ standard deviation of two experiments. RRT, relative retention time; FPA, free phenolic acid; BHPA, base-hydrolyzed phenolic acid; AHPA, acid-hydrolyzed phenolic acid; UK, unknown; ND, not detected.

As identification of all the peaks was not accomplished by reversed phase-HPLC, direct ESI ionization (negative mode) was used to identify the phenolic compounds of the extracts. The full scan negative mass spectra (Fig. 2) allowed the identification of molecular masses of the mangosteen pericarp fractions. Each compound gave reasonably intense [M-H]ions. The ESI–MS fingerprint of FPA and BHPA fractions showed the presence of trace amounts of piperonylic acid (m/z 165) and tentatively identified flavonoid such as quercetin-3-glucoside (m/z 463), quercetin pentoside (m/z 433), epigallocatechin (m/z 457) and methyl epicatechin gallate (m/z 455). The results indicate hydrolysis conditions such as acid or alkaline, or, in different sequence, can significantly affect the total yield and profile of phenolic acids. The separation of each polyphenolic is based on the polarity differences among polyphenolics with structural similarities and use of various combinations of mobile and stationary phases and the specific column used. Hydrolysis process is also adopted widely in order to obtain maximum yield during phenolic extraction from the pericarp of litchi (Ruenroengklin et al. 2009), longan (Rangkadilok et al. 2005) and pineapple peel (Tilay et al. 2008) in order to determine bound phenols.

Antioxidant Activity of the Extract Fractions The antioxidant activity of the plant products is associated with their bioactive compounds, mainly phenolics, because of 630

their ability to scavenge free radicals. There is hardly any study on the antioxidant activity of phenolic acids present in mangosteen fruit as most of the research have concentrated on the major secondary metabolite xanthones. The DPPH• assay provides basic information on the antiradical activity of the extracts. The DPPH radical has been widely used to test the ability of the extracts as free-radical scavengers or hydrogen donors (Sanchez-Moreno 2002). Therefore, the extracts and their hydrolysis products were evaluated for their antioxidant activities. In the present work, as shown in Fig. 3A, the BHPA fractions had greater antioxidant activities than FPA extract, and the lowest activity was determined in AHPA fraction. Percent DPPH radical scavenging activities of all the extracts were dose dependent. Lipid peroxidation is an oxidative alteration of polyunsaturated fatty acids in the cell membranes, which generates a number of degradation products. Malonaldehyde is one of the products of lipid peroxidation and has been studied widely as an index of lipid peroxidation and a marker of oxidative stress. Figure 3B shows the lipid peroxidation inhibition activity of the three fractions. As in DPPH• assay, the lipid peroxidation assay showed BHPA and FPA fractions to have higher inhibition, while the acid hydrolyzed fractions showed the lowest activity (20.2%) only when the concentration was above 200 mg. The antioxidant activity of the FPA and BHPA fractions inhibited the lipid peroxidation or scavenged the DPPH• radical almost the same as the standard antioxidant BHA. Journal of Food Biochemistry 36 (2012) 627–633 © 2011 Wiley Periodicals, Inc.



FIG. 1. (A) HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) CHROMATOGRAM OF BASE-HYDROLYZED PHENOLIC ACID (BHPA) FRACTION IN MANGOSTEEN PERICARP DETECTED AT 280 NM: UNKNOWN (1), GALLIC ACID (2), PROTOCATECHUIC ACID (3), GENTISIC ACID (4), 4-HYDROXYBENZOIC ACID (5), VERATRIC ACID (6), VANILLIC ACID (7), CAFFEIC ACID (8), SYRINGIC ACID (9), P-COUMARIC ACID (10), SINAPIC ACID (11), MANDELIC ACID (12), FERULIC ACID (13), T-CINNAMIC ACID (14), EPICATECHIN (15), QUERCETIN (16). (B) HPLC PROFILE OF PHENOLIC ACID EXTRACTED FROM SEQUENTIAL HYDROLYSIS OF MANGOSTEEN PERICARP FREE PHENOLIC ACID (SOLID LINE), BHPA (DOTTED LINE) AND ACID-HYDROLYZED PHENOLIC ACID (DASHED LINE)

Correlation existed between the DPPH• value and the total phenolic content of the mangosteen fraction, with the correlation coefficient (r) = 0.89 (y = 1.28x), P ⱕ 0.01; r = 0.85 (y = 1.19x), P ⱕ 0.01; and r = 0.961 (y = 4.53x), P ⱕ 0.001 for FPA, BHPA and AHPA fractions, respectively. Similarly, a

strong positive correlation was obtained in lipid peroxidation assay with r = 0.99 (y = 1.34x), P ⱕ 0.001 and r = 0.98 (y = 1.25x), P ⱕ 0.001 for FPA and BHPA fractions. Indeed, it is worth noting that the extracts with the highest concentration of total phenolics as determined by the Folin-Ciocalteu

FIG. 2. INFUSION ELECTROSPRAY–MASS SPECTROMETRY FINGERPRINTS OF BASE-HYDROLYZED PHENOLIC ACID FRACTION OF MANGOSTEEN PERICARP (FULL SPECTRA)

Journal of Food Biochemistry 36 (2012) 627–633 © 2011 Wiley Periodicals, Inc.

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100 % Lipid peroxidation inhibition

% DPPH scavenging activity

100 80 FPA BHPA AHPA BHA

60 40 20 0 0

A

40

80

120

Concentration (μg/mL)

80 60 40

FPA BHPA BHA

20 0 0

160

B

20

40

method were also the ones with the highest phenolic acid content as well as the source that showed good antioxidant activity. In the present study, the antioxidant activity of mangosteen pericarp extract fractions only indicates the total antioxidant capacity of each fraction and their contribution to the total antioxidant activity and not the role of individual phenolic acids. The free radical scavenging activity depends not only on the phenolic content but also on the type of phenolic compounds. The analytical procedures can significantly affect the antioxidant activity of phenolic acids because of the types of phenolic acids and their contents through different sample preparations (Nuutila et al. 2002).

CONCLUSION In the present study,we found that the majority of the phenolic acids existed in a bound form. After alkaline hydrolysis, many phenolic acids were detected in mangosteen pericarp belonging to hydroxybenzoic as well as hydroxycinnamic acid family. BHPA and FPA showed good antioxidant activity in the DPPH• and lipid peroxidation system.AHPA fraction showed reduced antioxidant activity and also low phenolic acid content. Indeed, a good correlation was obtained in our work between the total phenolic content of the different pericarp fractions tested and DPPH• or lipid peroxidation.The present method is simple, easy to use and effective enough for identification and quantification of major phenolic compounds in mangosteen pericarp. The outcome of this study shows that the mangosteen pericarp extracts are rich in polyphenols – a natural antioxidant, which are believed to be effective nutrients in the prevention of oxidative degradation of lipids and as radical scavengers, and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries.

ACKNOWLEDGMENT The first author, Ms. A.S. Zarena, acknowledges the CSIR, India for the Senior Research Fellowship. 632

60

80

100

120

Concentration (μg/mL)

FIG. 3. (A) DPPH• RADICAL SCAVENGING (%) ACTIVITY OF DIFFERENT MANGOSTEEN PERICARP FRACTIONS. (B) INHIBITION (%) OF LIPID PEROXIDATION BY DIFFERENT MANGOSTEEN PERICARP FRACTIONS Values are mean ⫾ standard deviation of three experiments.

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