Determination of pitaya seeds as a natural antioxidant and source of essential fatty acids

The antioxidant capacity and total phenolic content of seeds of three types of pitaya namely Hylocereus polyrhizus, Hylocereus undatus and Selenicereus megalanthus and the fatty acid content of pitaya seeds oil were examined in this study. The ethanolic extracts of H. polyrhizus seeds showed significantly high (p<0.05) total phenolic (43.9 mg GAE/100g dry weight) and flavonoid (50.8 mg CAE /100g dry weight) contents as compared to H. undatus and S. megalanthus and its aqueous extracts. DPPH assay showed similar trend showing that H. polyrhizus seeds had significantly higher (p<0.05) scavenging capacities (46.6%). FRAP test also indicated that the ethanolic extracts of H. polyrhizus seeds had significantly higher (p<0.05) reducing capacity (59.1mg Trolox/100g dry weight). The main fatty acids of pitaya seeds oil were C16:0, C18:0, C18:1, C18:2 with an exceptional high level of linoleic acid, up to 660 g/kg for S. megalanthus, 540 g/kg for H. undatus and 480 g/ kg for H. polyrhizus. Keyword: antioxidant, pitaya seeds, essential fatty acid, H.polyrhizus, H.undatus, S.megalanthus 1004 Chemah, T. C., Aminah, A., Noriham, A. and Wan Aida, W. M. International Food Research Journal 17: 1003-1010 stage (Nerd and Mizrahi, 1998). Pitaya seeds which are the by-products of juice and wine processing were usually discarded. It is the objective of this study to investigate the content of pitaya seeds and the potential of the seeds as a source of antioxidant. To the best of our knowledge no studies has ever been conducted on the potential of pitaya seeds as an antioxidant and essential fatty acid. Materials and Methods Production of seed powders Seeds from three types of pitaya, Hylocereus undatus, Hylocereus polyrhizus and Selenicereus megalanthus were manually separated from the pitaya pulp in the lab. The seeds were cleaned and washed under runnning water until all the pulp removed. Seeds were later dried in the oven at 40°C overnight and kept in a dessicator until further analysis. Proximate analysis of pitaya seed The proximate analysis was determined according to AOAC (1990) methods. Carbohydrate was determined by difference. Pitaya seeds extraction Pitaya seeds were finely ground before being extracted with aqueous and 50% ethanol. Half gram (0.5g) of seeds sample were mixed with 10 ml of water and 50% ethanol before homogenized at 24000 rpm (Ultra-Turrax, IKA, Germany) for 1 min and centrifuged for 20 minutes at 4000 g (Kubota Model 2420) at room temperature. The extracts were obtained from 2 batches of seeds prepared. Each batch contained 3 replicates. Total phenolic content Total phenolic content were determined using Folin-Ciocalteu reagent, modification method by Wolfe et al. (2003). Gallic acid was used as standard and the concentration of total phenolic compounds in the extracts were calculated by standard curve interpolation. Results were reported as mg gallic acid equivalent/100 g dried sample. Determination of total flavonoid content Flavonoid were measured using a method by Liu and Zhu (2007) with slight modification. Catechin was used as standard and total flavonoid in the extracts were calculated by standard curve interpolation. Results were expressed as mg catechin/100 g dry weight. Antioxidant capacity assay DPPH assay Free radical scavenging activity were measured by the 2,2diphenyl-1-picrylhydrazil (DPPH) according to a modified method by Wu et al. (2006). Sample extracts (100ul) were added into 3.9ml of DPPH reagent (prepared with 24mg of DPPH/L of methanol). The percentage of DPPH scavenging activity is expressed by the following formula, DPPH inhibition=[(Initial absorbance-sample absorbance) x100 Initial absorbance FRAP assay FRAP method were according to Benzie and Strain (1996) which measures the ferric reducing ability of plasma (FRAP). The method is based on the reduction of a ferric 2,4,6-tripyridyl-s-triazine complex (Fe3+-TPTZ) to the ferrous form (Fe2+TPTZ). The stock solutions included 300mM acetate buffer (3.1 g C2H3NaO2.3H2O) and 16mL C2H4O2), pH 3.6, 10mM TPTZ (2, 4, 6-tripyridyl-s-triazine) solution in 40mM HCl, and 20mM FeCl3.6H2O solution. The fresh working solution was prepared by mixing 25mL acetate buffer, 2.5mL TPTZ solution, and 2.5mL FeCl3.6H2O solution and then warmed at 37°C before using. The reagent were added into 100ul sample extracts. The reduced form of blue colour were read at 593nm after 30 minutes. Trolox was used as standard and ferric reducing power of the extracts were calculated by standard curve interpolation. Results were expressed as mg Trolox equivalent/100 g dry weight. Fatty acid methylation of pitaya seeds oil Fatty acid composition (FAC) of the samples was analyzed as fatty acid methyl esters (FAMEs), which were prepared according to IUPAC 2.301 (1987). FAMEs were then analyzed using gas chromatography (Waters) with FID detector. Column used was 60 m x 0.25 mm film 0.25 um (J&W Scientific DB 23). The injection temperature 130°C with flame ionizing detector temperature at 220°C. Nitrogen carrier gas was used at a rate of 0.53ml/min. Statistical methods Data were analyzed statistically using SAS version 6.12. All samples were prepared and analyzed in triplicates except for FAME which is in duplicate. To verify the statistical significance of all parameters, the values of means ± SD were calculated. Analysis of variance (ANOVA) was used to compare several groups. Probability value of p < 0.05 was adopted as the criteria for significance differences. Determination of pitaya seeds as a natural antioxidant and source of essential fatty acids 1005 International Food Research Journal 17: 1003-1010 Results and Discussion Proximate composition Seeds constitute about 2-5 % dry weight of the whole fruit and are usually discarded in juice or wine processing. The proximate composition of pitaya seeds (Hylocereus polyrhizus, Hylocereus undatus and Selenicereus megalanthus) are showed in Table 1. The moisture content of the seeds were between 6.9% to 7.2%. The results in Table 1 showed that protein and fat were high. Protein content was between 21.5 to 26.6% dry weight. These result are higher than the protein content of another cactus namely Opuntia ficus indica which contained 1.5 % dry weight (Habibi et al., 2008). However, the protein range were suitable for seeds of legumes type such as soybean which has 24.5 % of protein (JeffAgboola and Oguntuase, 2006), 39.5% in chinese bottle Lagenaria sciceraria (Olaofe et al., 2009) and 35 % in Piliostigma thonningii seeds (Jimoh and seeds with 14-16% (Kamer et al., 1985). Apple seeds contained very high protein ~34% content however grape seeds was low with 7 to10%. Ash content of pitaya seeds in this study resulted with 3.1 to 6.1 % and was between the apple seeds ~4.1% as reported in Yu et al. (2007) and grape seeds with 2.2% (Kamer et al., 1985). Phenolic and flavonoid content Phenolic and flavonoid content of pitaya seeds are showed in Table 2. Polyphenolic are found abundant in fruits and vegetables and it is one of the major parameters to determine the significance of the type of fuits and vegetables. Although it is a general method, Folin-Ciocalteu assay is one of the most commonly used method to determine total phenolic in fruits and vegetables. Ethanolic extracts of H.polyrhizus seeds showed significantly higher (p<0.05) phenolic content (43.9 mg GAE/100g dry weight) . The seed of H.polyrhizus had higher phenolic (p<0.05) content than the other Parameter H. polyrhizus H. undatus S. megalanthus Protein 26.3 ± 0.2b 23.1 ± 0.1c 28.6 ± 0.4a Oil 22.8 ± 0.5b 27.5 ± 1.0a 18.8 ± 0.8c Ash 6.1 ± 0.0a 3.1 ± 0.1c 3.8 ± 0.1b Carbohydrate 44.8 ± 0.3b 46.3 ± 1.1b 48.7 ± 1.1a Olidiji, 2005). The oil content of H.polyrhizus, H.undatus and S.megalanthus in this study was 22.8%, 27.5% and 18.8% respectively. These values were higher than the crude oil from another type of cactus i.e. Opuntia ficus which contained 10.9 to 11.1% oil (Ennouri et al., 2006). Additionally, Habibi et al. (2008) found the fat/wax content is 8% in Opuntia ficus indica. As for the ash content in pitaya seeds, the results were in the range of 2.9 to 5.7% and this result is in agreement with Habibi et al. (2008). In summary the proximate composition of pitaya seeds were comparable to the apple seeds with high protein and oil contents. Pitaya seeds contained oil at the range between 18.8 27.5% as compared to apple seeds ~27.7% (Yu et al., 2007) but higher than grape two type of seeds. Generally the ethanolic extracts showed better extraction recovery. The recovery of polyphenols from plant materials is influenced by the solubility of the phenolic compounds in the solvent used for the extraction process (Alothman et al., 2009). In addition, the polarity of the solvent will play a key role in increasing phenolic solubility (Naczk and Shahidi, 2006).However when compared with seeds of another cactus Opuntia dillenii Haw, the latter had almost 10 fold higher phenolic and flavonoid content than the pitayas (Chang et al., 2008). The phenolic contents of pitaya seeds were comparable to the caneberry seed composition (44.6 58.2 mg GAE/g seeds) as reported by Bushman et al. (2004). Fruits and vegetable contained phenolics and flavonoids, however its content varies between Table 1. Proximate composition of pitaya seeds (g/100 g) Each value is mean of three determinations (n=3) p < 0.05. Carbohydrate were determined by difference. 1006 Chemah, T. C., Aminah, A., Noriham, A. and Wan Aida, W. M. International Food Research Journal 17: 1003-1010 types and varieties. Antioxidant activity of pitaya seeds extracts The antioxidant assays as determine by DPPH scavenging abilities of the extracts showed that H.polyrhizus ethanolic seed extracts had higher capacity (p<0.05). Ethanolic extracts showed significantly higher DPPH scavenging capacity compared to the water extracts. The results indicated that the antioxidant effectiveness decreases in the following order H.polyrhizus seeds ethanolic extracts (46.6%), H.undatus (44.5%) and S.megalanthus (44.6%). The FRAP results showed similar trend, the ethanolic extracts had better antioxidant capacity than the water extracts. H. polyrhizus seeds resulted with 59.1mg Trolox equivalent/100g dry weight of seeds showed significantly higher (p<0.05) antioxidant capacity as compared to the other two types of pitaya seeds. Grape seeds exhibited high antioxidant capacity when tested under FRAP assay (Guo et al., 2003). While, Yilmaz and Toledo (2006) found that the grape seeds exhibited better antioxidant capacity than the peel. The study by Yilmaz and Toledo (2006) also found that aqueous mixtures of either ethanol, methanol or acetone were better than a monocomponent solvent for the extraction of phenolics from the grape seeds. Our results were in agreement with Yilmaz and Toledo (2006), which reported that the 50% ethanol extracts showed higher antioxidant capacity. There are several methods being used to measure antioxidant capacity of fruits such as direct competition methods and indirect methods (Roginsky and Lissi, 2005). But, there are also many different antioxidants contained in fruits and thus were difficult to measure each antioxidant individually (Guo et al., 2003). However, DPPH and FRAP assay were common, simple to standardized and reproducible (Thaipong et al., 2006). Eventhough the method used is the same in other study, the results sometimes could not correlate, therefore, difficult to compare between study (Guo et al., 2003). Fatty acid composition of pitaya seeds Pitaya seeds oil is rich in linoleic (C18:2) and oleic (C18:1) acids. S. megalanthus showed significantly high (p<0.05) linoleic acid (65.4%) content as compared to H. undatus (53.8%) and H. polyrhizus (48.7%). Pitaya seeds contained high linoleic acid when compared to caneberry seeds which contained only 54.2% in the red raspberry seeds and 62.7% for marion blackberry (Bushman et al., 2004). Results from this study showed that pitaya seeds contained higher oleic acid when compared to Bushman et al. (2004). Oleic acid in H. polyrhizus (25.5%) were significantly higher (p<0.05) than the two types of pitaya seeds, 23.3 % for H. undatus and 13.92% for S. megalanthus. Results showed that the lipid pattern of pitaya seeds oil are comparable to that of cactus pear seed oil (Ramadan and Morsel, 2003; Ennouri et al., 2006); also in sunflower and grapeseed seed oils (Tan and Yaacob, 2000). Linoleic acid is a polyunsaturated omega-6 fatty acid and it is essential in human diet. Linoleic acid consumption were important to counter the cholesterol increase effect of the carbon 12-16 saturated fatty acid. Although pitaya seeds contained lauric, myristic and