Antioxidant activities of Rambutan (Nephelium lappaceum L) peel in vitro

Rambutan, Nephelium lappaceum L, peel due to consumption of fresh rambutan fruit is taken into account as waste, therefore the exploration of rambutan peel as a natural antioxidant is highly needed. The aim of this study is to investigate the antioxidant activity of rambutan peel from two cultivars (Aceh and Binjai) using ABTS radical assay of and ferric reducing activity power (FRAP) and to correlate with total phenolics and flavonoids. The powdered rambutan peel is extracted using maceration technique using methanol as extracting solvent. The methanolic extract is added with warm water and fractionated using petroleum ether, chloroform, and ethyl acetate to get corresponding fractions. Rambutan cultivar Binjai revealed the higher ABTS antiradical activity than that of cultivar Aceh. Furthermore, among methanolic extract and its fraction, ethyl acetate fraction exhibited the highest antiradical activity using ABTS radical with IC50 values of 3.10 μg/mL and 0.77 μg/mL for Aceh and Binjai, respectively. The ethyl acetate fraction also revealed the highest FRAP values of 1424.897 ± 28.56 μg/mg fraction sample (Aceh) and 968.57± 7.48 μg/mg fraction sample (Binjai). These activities were correlated with phenolics and flavonoid contents. Rambutan peel exhibited strong antioxidant activities, contained high amounts of phenolics and flavonoid and is potential to be developed as a functional food.


Introduction
Rambutan (Nephelium lappaceum L.), family of Sapindaceae is important tropical fruit, widely cultivated in south East Asian such as Malaysia, Thailand, and Indonesia.In Indonesia, some cultivars of Rambutan existed with different characteristics, namely Narmada, Rapiah, Sinyonya, garuda binjai, Kapulasan, lebak bulus, Si Batuk Ganal, Tangkue, Antalagi, Lebak, Bahrang Simacan, and Sibongkok.Two cultivars commonly available are Aceh and Binjai.In most Indonesian communities, Rambutan is consumed freshly and as consequence, it produces a lot of wastes from peel and seed, therefore some scientist develops rambutan peel and seed as functional food via assessing it with several activities, including antioxidant (Thitilertdecha et al., 2008;Saeed et al., 2012).
The community awareness toward the safety of synthetic antioxidant has attracted a scientist of antioxidant coming from plants (Thitilertdecha et al., 2010).The antioxidant can be defined as any substances in low concentrations, derived from natural or synthetic, capable of delaying or inhibiting oxidation reaction significantly (Antolovich et al., 2002).Based on reaction mechanism, antioxidant assays can be group into five categories, namely radical scavenging assay, reducing power, chelating agent, lipid peroxidation reaction and synergist.These mechanisms are commonly exploited for assessment of antioxidant from natural sources.
Several biological activities of rambutan fruit and its part (peel and seed) along with chemical composition are reported such as antioxidant, antibacterial and antiinflammation activities due to ellagic acid, corilagin and geraniin contained (Thitilertdecha et al., 2008;Palanisamy et al., 2011;Fidrianny et al., 2015a), hypoglycaemic (Soeng et al., 2015), antidiabetic (Soeng et al., 2015), and anticancer (Khonkarn et al., 2010).Therefore, the objective of this research is to evaluate the antioxidant activities of rambutan peel from two cultivars (Aceh and Binjai) using ABTS radical assay of and FRAP and to correlate with total phenolics and flavonoids.

Preparation of methanolic extract and its fraction of Rambutan peel
Preparation of extract and fraction was done according to Permatasari and Rohman (2016).The peel of Rambutan (cultivars Aceh and Binjai) was washed, cut into small and dried under sun drying.The dried peel was powdered using powdering machine and subjected to extraction with methanol using maceration process.The macerate was filtered and evaporated using vacuum rotary evaporator.The methanolic extract was added with warm distilled water and is then fractionated using petroleum ether.The residue of methanol extract was then fractionated again using chloroform and ethyl acetate to get the fractions of PE, chloroform, ethyl acetate, and water.The initial methanolic extract and the fractions were used for antioxidant assay as well as for determination of phenolics and flavonoid.

Antiradical assay using ABTS radical
The ABTS radical assay of extract and fractions of rambutan peel was carried out using the decolourization assay with ABTS + radical cation according to Arnao et al. (2001).The solution of ABTS 7 mM and potassium persulphate 2.45 mM were mixed in ratio 1:1 and allowed to stand in the dark for 12-16 h to produce stock solution of ABTS radical cation (ABTS + ).This solution was further diluted with methanol to attain absorbance of 0.600-0.800at 734 nm.The ABTS + working solution (3 mL) and 30 μL of blank, standard or sample were mixed and the absorbance was measured at 734 nm after 6 min using a spectrophotometer.The blank was run with methanol.

Determination of total phenolics content
The levels of total phenolics in samples was analysed using Colorimetric Method (Folin-Ciocalteau method) according to Chun et al. (2003), while flavonoid contents of extract and fractions were determined using aluminium chloride colorimetric method according to Zou et al. (2004).

Data analysis
All data were analysed in triplicate and expressed as mean ± standard deviation using Excel (Microsoft Inc., USA).

Results and discussion
Among antioxidant mechanisms radical scavengers and ferric reducing activity power (FRAP) are the most reported ones for the evaluation of antioxidant capacity derived from plants.The radical of 2,2′-azinobis(3ethylbenzothiazoline-6-sulphonic acid) diammonium salt cation (ABTS + ) is widely used to screen antioxidant activities of plants (Zheng et al., 2016).In this assay, ABTS radical is generated by oxidation of ABTS with potassium persulfate to form the blue/green colour.When anti-radical is present, the ABTS radical is reduced, and the extent of decolourization is measured at a fixed operating time at a wavelength of 734 nm (Re et al., 1999).
Table 1 listed the IC 50 values of methanolic extract and its fraction using ABTS radical assay.In general, Rambutan cultivar Binjai exhibited the higher ABTS antiradical activity than that of cultivar Aceh due to high FULL PAPER phenolics contents present in cultivar Binjai.This can be seen from the lower values of IC 50 in cultivar Binjai than those in Aceh from the same extract and fractions.The lower value of IC 50 , the higher antiradical activity is.Furthermore, among methanolic extract and its fraction, ethyl acetate fraction exhibited the highest antiradical activity using ABTS radical.These results are in agreement with those using DPPH as radical sources (Rohman et al., 2016).Therefore, it is suggested that ethyl acetate fraction is sub-fractionated further to obtain active isolate as anti-radical.Figure 1 revealed the relationship between the concentration of ethyl acetate fraction (x-axis) and ABTS antiradical activity of rambutan peel cultivar Aceh and Binjai (y-axis).The equation for such correlation is also included in such figure .The ferric reducing antioxidant power (FRAP) method of methanolic extract and its fraction of rambutan is relied on the reduction of Fe 3+tripyridyltriazinetripyridyltriazine (Fe(TPTZ) 3+ ) by antioxidant in acidic medium to produce complex Fe 2+- Fe(TPTZ) 2+ having intense blue color, which can be measured at wavelength 593 nm.Results are obtained as absorbance increases at 593 nm and can be expressed as mg Fe 2+ equivalents (Ozgen et al., 2006).Chemically, the reaction which takes place can described as: Table 2 compiled antioxidant activity of methanol extract and its fraction using FRAP method.The results showed that among evaluated samples of rambutan cultivar Aceh, ethyl acetate fraction revealed the highest FRAP values of 1424.897±28.56µg/mg samples.In addition, ethyl acetate fraction also exhibited the highest FRAP in rambutan cultivar Binjai with a value of 968.57± 7.48 µg/mg samples.
A group of phenolics and flavonoids are deduced to be responsible for the antioxidant activities of plants (fruits and vegetables) due to its capability to provide radical hydrogen in ABTS assay and due to its properties as reducing agents (Prior et al., 1998), therefore the FULL PAPER Fe 3+ + 2,3,5-triphenyl-1,3,4-triaza-2-azoniacyclopenta-1,4-diene chloride (TPTZ) complex Fe 3+ -TPTZ (yellow)  antioxidants activities are frequently correlated with the contents of phenolics and flavonoid (Javanmardi et al., 2003;Rohman et al., 2006).Figure 1 and Figure 2 revealed the correlation between (A) phenolic contents and (B) flavonoid contents (x-axis) with IC 50 values (yaxis) of extracts and fractions of Rambutan peel cultivar Aceh and Binjai using ABTS assay (Figure 2) and FRAP (Figure 3).The coefficient of determination (R 2 ) is used as a means for relationship assessment.The R2 values obtained using ABTS assay are 0.0353 (phenolics) and 0.2009 (flavonoids), indicating that phenolics and flavonoids contributed to ABTS radical scavenging of 3.53% and 20.09%, respectively.In terms of FRAP, phenolics and flavonoids contributed 18.52% and 19.17%, respectively.From these results, it can be stated that ABTS radical scavenging activity and FRAP of extract and fractions of rambutan peel is not limited to phenolics and flavonoids.Antiradical activity may also come from the presence of other anti-radical components such as alkaloids, vitamins, carotenoids, and lignans.

Conclusion
Rambutan peel exhibited ABTS antiradical activity and reducing power, as determined using FRAP method.Ethyl acetate fraction showed the highest antiradical activity using ABTS radical and FRAP values.The correlation of phenolics and flavonoid contents toward antioxidant activities are relatively weak, meaning that other compounds are also responsible for those antioxidants.Rambutan peel is potential to be developed as a functional food.

Figure 2 .
Figure 2. The correlation between (A) phenolic contents and (B) flavonoid contents (x-axis) with IC 50 values (y-axis) of extracts and fractions of Rambutan peel.

Figure 3 .
Figure 3.The correlation between (A) phenolic contents and (B) flavonoid contents (x-axis) with FRAP values (y-axis) of extracts and fractions of Rambutan peel.

Table 1 .
The antiradical activity of methanol extract and its fraction using ABTS radical assay.

Table 2 .
The antioxidant activity of methanol extract and its fraction using FRAP assay.