Food Bioprocess Technol DOI 10.1007/s11947-011-0533-7
COMMUNICATION
Chemical and Phytochemical Properties and Antioxidant Activities of Three Pomegranate Cultivars Grown in South Africa Olaniyi A. Fawole & Umezuruike L. Opara & Karen I. Theron
Received: 9 December 2010 / Accepted: 1 February 2011 # Springer Science+Business Media, LLC 2011
Abstract A comparative study of chemical contents and antioxidant activities of three pomegranate cultivars (‘Arakta’, ‘Bhagwa’ and ‘Ruby’) grown in South Africa was conducted. Fresh pomegranate juice (PJ) of each cultivar were assessed for soluble solid contents (SSCs), pH and titratable acidity (TA), while extracted juice samples were evaluated for total phenolic (TP), including total tannins (TT), proanthocyanidins (Pcy), total flavonoids, anthocyanins and gallic acids (GA) using spectrophotometric methods. The antioxidant properties of the juice samples were evaluated against stable 2, 2– diphenyl–1–picryl hydrazyl, as well as in ferric reducing ability of plasma (FRAP) and QuantiChrom™ (TAC) antioxidant assays. There were significant differences in the chemical properties of the cultivars. SSC, TA and pH varied between the range of 14.07–15.10 °Brix, 0.22– 0.28 g/100 ml and 3.32–3.64, respectively. ‘Bhagwa’ had the highest TP (449.9 mg/100 ml), 1.3-fold and 1.6-fold higher than ‘Arakta’ and ‘Ruby’, respectively. The strongest total antioxidant activity was exhibited by ‘Bhagwa’ with an antioxidant index of 95.7%, followed by ‘Arakta’ (93.2%) and ‘Ruby’ (79.9%). PJ phytochemical properties (TP, TT, Pcy, GA) and antioxidant activity (FRAP and TAC) were significantly correlated (r2 = 0.509–0.885) with each other. Keywords Pomegranate . Antioxidants . Polyphenols . FRAP . TAC . Gallic acid . South Africa O. A. Fawole : U. L. Opara (*) : K. I. Theron Postharvest Technology Research Laboratory, Department of Horticultural Science, Faculty of AgricSciences, University of Stellenbosch, Private Bag X1, Stellenbosch 7602, South Africa e-mail:
[email protected]
Introduction Several epidemiological and intervention studies have reported positive correlations between fruit and vegetable intake and prevention of non-communicable diseases such as cardiovascular disease, many forms of cancer, type 2 diabetes mellitus and slowing of the ageing process (Lampe 1999; World Cancer Research Fund/ American Institute for Cancer Research 2007). Scientific evidence linking increasing consumption of fresh fruit and vegetables to improved human health have been attributed mainly to their contents of beneficial phytochemicals and other micro-nutrients (Opara and Al-Ani 2010) which act as antioxidants by scavenging the oxidation caused by free radicals that lead to cells and DNA damage and many degenerative disorders (Lampe 1999; Boyer and Liu 2004). The phytochemistry and pharmacological actions of pomegranate (Punica granatum L.) components suggest a wide range of clinical applications for the treatment and prevention of cancer, as well as other diseases where chronic inflammation is believed to play an essential etiologic role (Lansky and Newman 2007). These bioactivities are attributed to high level of antioxidant polyphenol content in pomegranate. Increasing largescale commercialisation and awareness of pomegranate fruit as a medicinal food and dietary supplement has tremendously facilitated its availability and interest among consumers. Commercial orchards of pomegranate are grown in countries such as Iran, India, Egypt, China, Israel, Tunisia, Syria, Lebanon, Turkey, Greece, Cyprus, Italy, France, Spain, Chile, Portugal, USA, Oman and most recently in South Africa (Al-Said et al. 2009; Holland et al. 2009; Opara et al. 2009; Bchir et al. 2010a). The commercialisation of pomegranate has consequently resulted in the introduction and spread of
Food Bioprocess Technol
different cultivars and selections throughout different regions and continents. Due to inherent genetic variability among pomegranate cultivars and the influence of climatic conditions on the chemical and antioxidant values of the fruits (Al-Said et al. 2009; Holland et al. 2009; Opara et al. 2009), scientific assessment of commercially important cultivars grown in different countries is essential for the proper selection, postharvest handling (Caleb et al. 2011) and marketing of fruit with desirable quality attributes (Opara 2009; Schoorl and Holt 1985) for fresh consumption as fresh or processed products. The goal of the present study was to investigate the chemical and antioxidant properties of three commercially grown pomegranate cultivars in South Africa. The specific objectives were to evaluate the chemical (soluble solid contents, pH and acidity) and phenolic contents and the antioxidant capacity of fruit juice.
10,000 rpm for 10 min at 4 °C. The supernatant was gently collected into a clean tube and stored at 4 °C. Phytochemical Analysis Determination of Total Phenolic Compounds Total phenolic (TP) content in juice sample was determined using the Folin-Ciocalteu (Folin-C) colourimetric method as described by Makkar et al. (2007). TP concentrations were determined spectrophotometrically at 750 nm by adding Folin-Ciocalteu reagent to the juice sample, and expressed as the mean ± S.E (milligrammes) of gallic acid equivalents (GAEs) per 100 ml crude juice for three replicates. Quantification of Total Tannins
Materials and Methods
Total tannin (TT) content was quantified according to Makkar et al. (2007). TT content was expressed as the mean ± S.E (milligrammes) GAEs per 100 ml crude juice for three replicates.
Plant Materials and Fruit Processing
Butanol–HCl Assay for Proanthocyanidins
Commercially ripened pomegranate fruits (cv. ‘Arakta’, ‘Bhagwa’ and ‘Ruby’) were collected from Houdconstant Pack-house Porterville, Western Cape (33°01′00″S, 18° 58′59″E) and transported in an air-conditioned vehicle to the Postharvest Technology Laboratory, Stellenbosch University. Three replicate samples, each containing three fruit, were used for all analyses. Each fruit was handpeeled and the arils juiced using a LiquaFresh juice extractor (Mellerware, South Africa).
Proanthocyanidin (Pcy) content was determined as described by Vermerris and Nicholson (2006). Briefly, PJ sample was mixed with Butanol–HCl–Iron reagent and heated in a water bath at 95 °C for 60 min. The absorbance was read at 530 nm using a UV–vis spectrophotometer (Thermo Scientific technologies, Madison, USA). Pcy contents were expressed as mean ± S.E cyanidin equivalents (milligrammes of CyE per 100 ml crude juice) from the standard curve. Determination of Total Flavonoids
Titratable Acidity, Soluble Solid Content and pH Titratable acidity (TA) was measured by titration to an endpoint of pH 8.2 using a Metrohm 862 compact titrosampler (Herisau, Switzerland). Soluble solid content (SSC, degrees Brix) was measured using a digital refractometer (Atago, Tokyo, Japan). The pH values were measured at room temperature using a pH metre (Crison, Barcelona, Spain). All values were presented as mean ± S.E.
PJ sample (250 μl) was mixed with 5% sodium nitrite solution (75 μl) and subsequently with aluminium chloride (10%, 150 μl), sodium hydroxide (1 M, 500 μl) and distilled water (775 μl). The absorbance of the mixture was measured spectrophotometrically at 510 nm (Yang et al. 2009). Total flavonoid (TF) content was expressed as the mean ± S.E (milligrammes) of GAEs per 100 ml crude juice in triplicates. Rhodanine Assay for Gallic Acid Content
Preparation of Fruit Sample Juice samples for phenolic compositions and antioxidant activity were prepared by mixing 2 ml of pomegranate juice (PJ) with 10 ml of cold 50% aqueous methanol in a centrifuge tube. The mixture was vortexed and sonicated in cold water for 5 min before being centrifuged at
The amount of gallic acid (GA) content in PJ samples was carried out as described by (Makkar 2000). Absorbance was read at 520 nm using a UV–vis spectrophotometer. GA content was calculated from the standard curve of analytical gallic acid and expressed as GAEs per 100 ml crude juice.
Food Bioprocess Technol
Quantification of Total Anthocyanins
QuantiChrom™ Antioxidant Assay for Total Antioxidant Capacity
Total anthocyanin content (Acy) was quantified according to Wrolstad (1993) using the pH differential method. In triplicates, diluted PJ sample (1 ml) was mixed with 9 ml of pH 1.0 and pH 4.5 buffers, separately. The absorbance of the mixtures was measured at 520 and 700 nm using a UV– vis spectrophotometer. Acy was expressed as cyanidin 3glucoside (molar extinction coefficient of 26,900 and a molecular weight of 449.2) equivalent per 100 ml crude juice (mg of C3gE/100 ml). Determination of Antioxidant Activities Free Radical-Scavenging Activity PJ sample was tested against stable 2,2-diphenyl-1picrylhydrazyl (DPPH) in the DPPH assay according to Karioti et al. (2004). Briefly, under dim light, PJ sample (15 μl) was diluted with methanol (735 μl) followed by the addition of methanolic DPPH solution (0.1 mM, 750 μl). The mixtures were incubated in the dark at room temperature for 30 min, before the absorbance was measured at 517 nm. The free radical-scavenging activity of PJ was expressed as ascorbic acid (millimoles) equivalent per millilitre of crude PJ (mM AAE/ml PJ; n=3).
For the measurement of the total antioxidant capacity (TAC) of PJ sample, the QuantiChrom™ antioxidant assay kit (DTAC-100) from BioAssay Systems (BioAssay Systems, Hayward, CA) was used. The assay kit is designed for the quantitative colorimetric determination of total antioxidant capacity in food and drug industry. In the assay, the total antioxidant capacity is a measure of the amount of copper (II) (Cu2+) reduced by antioxidants to copper (I) (Cu+) such that the resulting Cu+ specifically forms a deep blue complex with a dye reagent. The colour intensity at 570 nm is proportional to the TAC of the sample being assayed. The absorbance was measured using a Multiskan FC plate reader (Thermo Scientific technologies, China). Results were expressed as Trolox (millimoles) equivalents per millilitre of crude PJ (mM TE/ml PJ) for three replications. Statistical Analysis One-way analysis of variance (ANOVA) and correlation coefficients (r) were carried out using SPSS version 10 software (SPSS Inc., Chicago, USA). Bar chats were plotted using GraphPad Prism software version 4.03 (GraphPad Software, Inc., San Diego, USA).
Ferric Ion-Reducing Antioxidant Power Results and Discussion The method of Benzie and Straino (1996) was employed for measuring the ferric ion-reducing antioxidant power (FRAP) of PJ sample with few modifications. In triplicates, diluted PJ sample (150 μl) was added to 2,850 μl of the FRAP solution (300 mM acetate buffer (pH 3.6), 10 mM 2,4,6tripyridyl-s-triazine (TPTZ) and 20 mM ferric chloride) before being incubated in a dark condition for 30 min. The reduction of the Fe3+–TPTZ complex to a coloured Fe2+– TPTZ complex at low pH by PJ sample was monitored by measuring the absorbance at 593 nm using a UV–vis spectrophotometer. Results were expressed as Trolox (millimoles) equivalents per millilitre of crude PJ (mM TE/ml PJ).
Titratable Acidity, Soluble Solid Content and pH In general, juice characteristics of the pomegranate cultivars investigated were significantly (P
