Physicochemical and antioxidant characteristics of Baccaurea angulata fruit juice extract

1 Department of Nutrition Science, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia, Jalan Istana, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia. 2 Department of Biotechnology, Kuliyyah of Science, International Islamic University Malaysia, Jalan Istana, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia. 3 Malaysian Agriculture Research and Development Institute (MARDI), Sarawak, Malaysia.


INTRODUCTION
Reactive oxygen species (ROS) is as important as antioxidant to human health.However, due to imbalance of free radicals and antioxidant in our body, development of chronic diseases such as hyperlipidemia, cancer, diabe-tes, and many others may occur (Liu et al., 2010).There are many conventional therapies and pharmacological drugs available to combat these diseases.Despite this, the treatment is quite expensive; taking synthetic drugs in *Corresponding author.E-mail: abumaisarah@iium.edu.my.Tel: 013-9234998.
Epidemiological studies showed that there is an inverse association between fruit and vegetable consumption and the risk of developing degenerative diseases (Oliveira et al., 2009).This is because fruits and vegetables contain antioxidant compounds, such as flavonoids, carotenoid, and polyphenols, which have protective effects to human body (Ashraf et al., 2010).These compounds help our body to balance out the ratio with free radical by scavenging free radicals, decomposing peroxides, and making complex of redox-catalytic metal ions (Zibadi et al., 2007).
Malaysia has plenty of nutritious fruits and medicinal plants due to constant exposure to 6-8 h of daily sunlight throughout the year.Therefore, tropical fruits in Malaysia are rich in antioxidants.Interestingly, there are some fruits which are commonly consumed by the locals but yet to be discovered by researchers in terms of nutritional as well as health benefits.Baccaurea angulata, locally known as 'belimbing dayak' or 'belimbing hutan', is one of the underutilized fruits from Borneo island of Malaysia.B. angulata belongs to the Euphorbiaceae family.The tree is about 8-10 m tall.It has thick and broad leaves, and the leaves are ellipse in shape.This fruit is seasonal, and the fruit grows on stems and branches of the tree (Rukayah, 2002).The review of literature showed that very limited studies have been conducted using this fruit in terms of physical and chemical characteristics.In view of this, the determination of antioxidant content and antioxidant activities of this fruit is carried out in this work.

Sample preparation
Fresh B. angulata fruits were collected from Bau, Sarawak, Malaysia.These fruits were separated into three portions, whole fruit, fruit skin, and berries (edible portion).The fruits were rinsed with distilled water, sliced, and blended with ratio of 1:1 (v/v) of edible portion to water.For berries, they were blended with ratio of 1:3 (v/v) of edible portion to water.These juices were filtered and transferred into separated containers.The samples were freezedried at -52°C, 0.63 mbar (Freezone 18 Liter Console Freeze Dry System, Labconco, USA).Freeze-dried powder retrieved was stored in an air-tight container at 4°C for further analysis.

Sample extraction of freeze-dried Baccaurea angulata
Extraction of powdered sample was carried according to a method by Velioglu et al. (1998) with slight modification.About 200 mg of sample was extracted with 2 mL of methanol at room temperature for 2 h with continuous stirring.Then, the mixture was centrifuged at 1000 g for 15 min.The supernatant was collected and kept at -20°C before the determination of total phenolic content, total flavonoid, total anthocyanin, and antioxidant capacity study.

Physical characteristics of B. angulata fruits
The fruit was randomly selected for physical measurement which includes average fruit, skin, and berry weight (g), fruit and berry length (cm), fruit and berry width (cm).Fruit weight was measured using calibrated weighing scale.The length and width of the fruit were measured using vernier caliper.All the measurements were done in triplicate and the average of the reading was reported.

Nutritional composition of B. angulata freeze-dried extract
Analysis of the moisture, ash, protein, total fat, crude fiber, dietary fiber, and carbohydrate contents were carried out according to the Association of Official Analytical Chemist (AOAC) (2003) method.Gross energy was calculated according to FAO/WHO/UNU (1981) method.Water activity was measured using AquaLab CX-2 water activity equipment.

Determination total phenolic content
Total phenolic content was determined according to the method of Lim et al. (2006) with slight modifications.About 1 mL of extract was mixed with 5 mL of Folin-Ciocalteu reagent and allowed to stand at room temperature for 5 min.4 mL of 7.5% sodium carbonate solution was then added into the mixture.The solution was incubated for 40 min.The absorbance was recorded at 765 nm.Calibration curve was constructed using different concentration of gallic acid range between 0.001 to 0.02 mg/mL.Results were expressed as mg gallic acid equivalent in 1 g of dried sample (mg GAE/g).

Determination of total flavonoid content
Total flavonoid content was determined by colorimetric method as described by Abu Bakar et al. (2009).Briefly, 0.5 mL of extract was mixed with 2.25 mL of distilled water and 0.15 mL of 5% NaNO 2 solution.The mixture was allowed to stand for 6 min before 0.3 mL of 10% AlCl 3 .H 2 O solution was added and incubated for another 5 min. 1 mL of NaOH was then added into the mixture, vortexed, and the absorbance was immediately measured at 510 nm.A linear standard calibration curve was constructed using quercetin range from 0.01-0.1 mg/mL.Results were expressed as mg quercetin equivalents per gram of dried sample (mg QE/g).

Determination of total anthocyanin content
Determination of total anthocyanin content was based on pH differential method according to the modified method by Lee et al. (2005) and Abu Bakar et al. (2009).About 3.5 mL of 0.025 M potassium chloride buffer pH 1.0 was added to 0.5 mL of the sample extract and mixed thoroughly using vortex.The mixture was allowed to stand for 15 min before the absorbance was read at 515 and 700 nm.The extract was then treated similarly with 0.4 M sodium acetate buffer pH 4.5 and the absorbance was measured at the same wavelengths.The total anthocyanin concentration was calculated using the following equation and expressed as mg of cyanidine-3glucoside equivalent in 100 g of dried sample (mg c-3-gE/ 100g dried sample).

Xanthine oxidase dismutase (XOD)/superoxide scavenging assay (SOD)
Xanthine oxidase dismutase (XOD) assay was conducted according to the method of Vimala et al. (2003) with slight modification.About 4.1 mM/l 4-nitroblue tetrazolium chloride (NBT) solution was prepared by adding 3.15 g TrisHCl, 0.1 g MgCl 2 , 15.0 mg 5-bromo-4chloro-3-indolyl phosphate and 34.0 mg NBT to 100 mL of distilled water.Reaction mixture was prepared by dissolving 0.53 g of Na 2 CO 3 (pH10.2),4.0 mg ethylenediaminetetra acetate (EDTA), and 5.0 mg xanthine before the addition of 10 mL of NBT solution prior to use and kept at 4°C. 5 µL of the sample extract, 995 µL of the reaction mixture and 0.1 µL of XO were added into a cuvette, mixed thoroughly and the absorbance was taken at 560 nm for 120 s.Absorbance of 1000 µL of the reaction mixture and 0.1 µL XO of the same spectrophotometer condition was considered as control.

1,1-Diphenyl, 2-picryl hydrazyl (DPPH) free radical scavenging assay
Radical scavenging activity of Freeze-dried whole fruit (FDWF), freeze-dried berry (FDB), and freeze-dried skin (FDS) was determined according to the method described by Molyneux (2004) with slight modification.About 1 mL of sample was mixed with 2 mL of DPPH solution.The mixture was shaken vigorously and left to stand in a dark room for 30 min.After incubation, the absorbance of the mixture was measured using UV/Vis spectrophotometer at 517 nm.
A linear standard calibration curve was constructed using ascorbic acid range from 0.001 -0.007 mg/mL (r 2 = 0.996).Results were expressed as mg ascorbic acid equivalents per 100 g sample (mg AA/100 g).
To prepare the working solution, both solutions were mixed in equal quantities and left to stand in a dark room for 12-16 h.Then, about 1 mL from the working solution was diluted with 60 mL methanol to obtain an absorbance of ±0.70 at 734 nm using spectrophotometer.For every assay, fresh ABTS· + solution was prepared.About 150 µL of the methanol extract sample was mixed with 2850 µL of ABTS· + and left to react for 6 min.A linear standard calibration curve was constructed using ascorbic acid range from 0.5 -18 µM/mL (r 2 = 0.998).Results were expressed as mg trolox equivalents per 100 g sample (mg TE/100g).

Ferric reducing /antioxidant power (FRAP) assay
FRAP assay was conducted based on Benzie and Strain (1996) with slight modification.The fresh working solution of FRAP reagent was prepared by mixing 300 mM acetate buffer pH 3.6, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ), and 10 mM FeCl 3 .6H 2 O in a 10:1:1 ratio and warmed to 37°C in water bath prior to use.Briefly, 3 mL of FRAP reagent was taken into a cuvette and a blank reading was measured at 593 nm and considered as blank reading.Then, 100 µL of the sample extract and 300 µL of distilled water were added into the cuvette and the second reading was performed after 4 min incubation in dark condition at the same wavelength.The change in absorbance was compared with a standard curve.The standard curve was linear between 0.002 to 0.2 µM trolox (r 2 = 0.993).Results were expressed in micromolar trolox equivalent per gram of dried sample (µM TE/g dried sample).

Physical characteristics of B. angulata
Physical characteristics of B. angulata which include weight of the whole fruit, berry, and skin, the length and perimeter of whole fruit are shown in Table 1.The shape of this fruit is like star fruit (Averhhoa carambola), from which the name "belimbing" was derived.The ripe fruit is bright red in colour.It is round at the base of the fruit and pointed at the end.Each fruit contains 1 -4 cloves of berry which are white in colour and sticky.
The average length and width of B. angulata was 4.61±0.59cm and 3.02±0.52cm compared to diameter of B. ramiflora (Burmese grape) which was 2.5 -3 cm (Singh, 2007).About 70.3% of whole fruit weight of B. angulata was contributed by fruit skin.The edible portion was about 34.1% fraction of fruit weight which is lesser than B. ramiflora by 12% (Haque et al., 2009).Furthermore, the average weight of berry of B. angulata was 7.73±4.55g which is heavier when compared to B. ramiflora berry (2 g) (Singh, 2007).pH of edible portion of B. ramiflora was more acidic (pH 2.4) than B. angulata (pH 4.05) (Haque et al, 2009).Values are presented in mean ± standard deviations of three replicates analyses of freeze-dried samples.

Nutritional and chemical composition of B. angulata
Nutritional and chemical compositions of freeze-dried samples are shown in Table 2. From the results, almost 20% of freeze-dried powder from all samples contained moisture.Furthermore, water activity (a w ) recorded for these samples ranges from 0.2 to 0.5.Moisture and water activity are essential properties as they can determine the shelf life of food (Ruzainah et al, 2009).Presence of high moisture content can facilitate microbial growth in food products.However, in food with water activity that ranges from 0.2 to 0.7 a w , microbial proliferation will not be observed (Fontana and Campbell, 2004).Percentage of ash represents the mineral content that is present in the sample.Therefore, FDS recorded the highest ash content (10.04 %), protein content (2.12 %) and total fat (2.09%) compared to FDB (5.04%; 1.28%; 1.73%) and FDWF (4.74%; 1.33%; 0.11%), respectively.Furthermore, FDB has higher crude fiber content (0.36%) compared to FDS and FDWF with 0.13 and 0.07%, respectively.Carbohydrate contributed the major composition for FDWF, FDB and FDS which was about 65 -75% and gross energy for all three samples were about 300 kcal/100 g.FDS and FDWF contained about 6.3% of total dietary fiber which is higher than orange juice (NDB No: 09206) and apple juice (NDB No: 09016) by 6.1% (USDA National Nutrient Database for Standard Reference, 2011).Dietary fiber further adds nutritional benefits to FDS because it is known to have many positive effects such as lowering the risk of cardiovascular disease, obesity, and gastrointestinal diseases (Ibrahim et al., 2010;Rosamond, 2002).

Antioxidant constituent and capacity of samples
From the results (Table 3), FDS showed the highest total phenolic content (TPC) of 16.58 mg GAE/g, compared to FDB (4.78 mg GAE/g) and FDWF (7.91 mg GAE/g).The result was expressed in gallic acid equivalent because, in plant, gallic acid is considered as one of the major phenolic compounds (Norshazila et al., 2010).
Natural phenolic compounds are largely found in teas, fruits, and vegetables.The most common and widely distributed plant phenolic compounds are flavonoids that are characterized by benzo-y-pyrone structure (Abu Bakar et al., 2009).From the results, FDS contained flavonoid compound of 31.05 mg QE/g and anthocyanin compound, which is higher compared to other samples.Skin of freeze-dried B. angulata was richer in total flavonoid compared to total phenol and anthocyanin.Therefore, flavonoids probably are the most attributable scavenger in it and it has high pharmacological activity as radical scavengers (Abu Bakar et al., 2009).Polyphenols are able to scavenge free radicals in human body such as superoxide anion radicals that cause oxidative stress (Ajila et al., 2007).Thus, phenolic substances in freezedried skin of B. angulata may exert an antioxidant effect, thus, may prevent the development of atherosclerosis (Gorinstein et al., 1999).
XOD, DPPH, TEAC and FRAP assays were selected to measure the capacity of polyphenols in B. angulata.Xanthine oxidase dismutase assay measure the ability of sample to scavenge superoxide free radical anions.From the result, superoxide scavenging (%) by XOD was as follows: FDWF>FDB>FDS.DPPH assay measures the ability of B. angulata extract to donate hydrogen to the radicals (Lim et al., 2006).The result for DPPH was in the order of FDS>FDWF>FDB.Reduction capability of DPPH is determined by the degree of discoloration at 517 nm.This indicates the reaction of antioxidant with DPPH by converting it to 2,2-diphenyl-1-picrylhydrazine (Ajila et al., 2007).Ascorbic acid equivalent capacities (AEAC) for FDWF and FDB are less than 70 mg AA eq /100g (Leong and Shui, 2002).These results indicate that FDWF and FDB fall into low antioxidant capacity category.While, FDS, having more than 70 mg AA eq /100g, but less than 200 mg AA eq /100g, falls into moderate antioxidant capacity.Nevertheless, the reducing ability of FDWF, FDB and FDS is strongly correlated with total phenol (r = 0.979, p<0.01) and total flavonoid (r = 0.987, p<0.01), while anthocyanin constituent has no correlation with DPPH values.
ABTS scavenging assay is widely adopted to study food substances because this intensely-coloured protonated radical has maximum absorbance at 734 nm and mostly coloured food do not absorb light at 734 nm.ABTS + is a cation radical that will decrease as the protons are scavenged.From the result, trolox equivalent antioxidant capacities are as follows: FDS > FDWF > FDB.Pearson correlation shows strong correlation between trolox value in studied extract and total phenol (r = 0.948, p<0.01) and total flavonoid (r = 0.960, p<0.01), while there is no correlation between anthocyanin and trolox value.
The freeze-dried samples were further evaluated for antioxidant capacities through FRAP assay.This assay was adopted in order to measure the ability of B. angulata to reduce Fe 3+ complex of tripyridyl triazine Fe(TPTZ) 3+ to the intensely blue coloured Fe 2+ complex Fe(TPTZ) 2+ in acidic medium (Lim et al, 2006).FRAP value was highest in FDS, followed by FDWF and FDB.A strong correlation was observed between all samples with total phenol (r = 0.997, p<0.01) and total flavonoid (r = 0.992, p<0.01).However, correlation was found moderate with total anthocyanin (r = 0.734, p<0.05).The presence of polyphenol compounds may contribute to the high antioxidant activities via hydrogen donation or electron donation (Ajila et al, 2007).

Conclusion
The result of this investigation shows that FDWF, FDB, and FDS contain the essential nutrients including dietary fiber that are good in promoting human health.Besides, Ibrahim et al. 5337 all freeze-dried powder showed presence of essential amount of antioxidant constituents as confirmed by antioxidant activities.Comparing all the samples, it was noted that FDS has the best potential as nutrient supplement due to its higher antioxidant activity contributed by polyphenol compounds.Therefore, FDS derived from B. angulata has the potential to be promoted as functional food and drinks.Further study is suggested to isolate and identify phenolic compounds present in the B. angulata, as well as identifying other biomarker compounds such as ascorbic acid and beta-carotene.

Table 1 .
Physical characteristics of whole fruit, berries, and skin of Baccaurea angulate.
Values were the means standard deviations of three replicates analysis.

Table 3 .
Antioxidant content and antioxidant capacities of FDWF, FDB, and FDS.