Isolation of active compound from Nephelium lappaceum L. rind as an antioxidant

Isolation of active compound from Nephelium lappaceum L. rind as an antioxidant Nurani, L.H., Edityaningrum, C.A., Suhaera, Windarsih, A., Riyanto, S. and Rohman, A. Faculty of Pharmacy, Universitas Ahmad Dahlan, Yogyakarta 55164, Indonesia Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia Study Program of Pharmacy, Health Institute of Mitra Bunda, Batam, 29444, Riau Island, Indonesia Research Center for Food Technology and Processing (PRTPP), National Research and Innovation Agency (BRIN), Yogyakarta, 55861, Indonesia Institute of Halal Industry and Systems (IHIS), Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia


Introduction
The imbalance between reactive oxygen species (ROS) and reactive nitrogen species (RNS) with endogenous antioxidants has led to the formation of oxidative stress. ROS and RNS have been associated with some degenerative diseases including diabetes, cancer, ageing, neurodegenerative and cardiovascular diseases (White et al., 2014). Some efforts have been driven by a group of researchers to explore exogenous antioxidants either natural or synthetic antioxidants (Palanisamy et al., 2008). The antioxidant can be defined as any compounds or materials capable of delaying or inhibiting the oxidation reactions of lipids, proteins and other molecules by terminating free radical reaction (Pavithra and Banu, 2017). Natural antioxidants derived from fruits and vegetables have been explored and commercialized such as grape seed extracts. One of the potential natural antiradical derivatives is from Rambutan (Nephelium lappaceum L.).
Rambutan (king of fruits) with the scientific name of Nephelium lappaceum L. (Family of Sapindaceae) is a tropical fruit commonly found in South East Asian FULL PAPER regions like Indonesia, Thailand Malaysia, and Vietnam (Fidrianny et al., 2015). Rambutan is most commonly consumed in cultivars Aceh, Binjai and Rapiah (Rohman et al., 2016). The consumption of Rambutan fruit has resulted in a vast amount of waste, including rind and peel. Therefore, it is very interesting to explore of underutilized part of rambutan fruit as a natural antioxidant and identify the active components contributing to antioxidant activities.
Several biological activities of Nephelium lappaceum L. fruit including its rind and seed have been reported, namely antiradical due to its phenolic compounds (Thitilertdecha and Rakariyatham, 2011), in vitro and in vivo antioxidant activities using different standardized methods (Pavithra and Banu, 2017;Iman Kamaludin et al., 2016;Mistriyani et al., 2018), lipid peroxidation inhibition (Setyawati et al., 2015) antiinflammatory effects (Chingsuwanrote et al., 2016) antihypercholesterolemia activities (Muhtadi et al., 2016) antibacterial activities against some pathogenic bacterial strains (Thitilertdecha et al., 2008), inhibitors of alphaamylase and alpha-glucosidase activities in vitro (Thinkratok et al., 2014) hypoglycemic effects (Soeng et al., 2015), anti-hyperglycemic activity (Palanisamy et al., 2011), α-and β-glucosidases inhibition , and anti-diabetic activity on rats induced by alloxan (Muhtadi et al., 2016) Some active compounds have been identified in rambutan including geraniin, corilagin and ellagic acid (Hernández et al., 2017). A total of 39 compounds were also identified in rambutan rind, including 1 simple phenolic acid, 1 flavone, 5 hydrolyzable tannins, 5 hydroxybenzoic acids, 10 flavonols, 11 flavonols, 6 ellagic acid and conjugates (Zhuang et al., 2017). However, the study on the antioxidant activities of Nephelium lappaceum L. rind fraction is still limited. To the best of our knowledge, the isolation of active antioxidant components from Nephelium lappaceum L. rind chloroform fraction has not been reported. Mistriyani et al. (2021) have carried out the isolation of antioxidant components in the ethyl acetate fraction of Rambutan peel. In this study, the chloroform fraction of Rambutan rind was chosen for further fractionation to investigate the active antioxidant compound because the chloroform fraction and subfraction showed good antioxidant activities. Furthermore, the active component contributing to antioxidant activity was identified.

Materials
Rambutan fruit was obtained from Bantul, Yogyakarta, Indonesia. The authentication of rambutan samples was performed in the Department of Pharmaceutical Biology, Faculty of Pharmacy with assistance from Dr Djoko Santosa. The chemical reagents and solvents used were of pro-analytical grade.

Preparation of chloroform extract of rambutan
The chloroform fraction was prepared according to Rohman et al. (2016). Rambutan rind was cleaned, cut into small pieces using a commercial cutter and dried in a conventional oven at 65 o C for 2 days then grounded into a powder. The powder was subjected to extraction using the maceration technique using methanol as extracting solvent and occasionally shaken. The solvent was evaporated using a vacuum rotary evaporator to obtain the methanolic extract. The extract was then added with aquadest and subjected to partition using petroleum ether, chloroform, and ethyl acetate to get corresponding fractions, namely petroleum ether (PE), chloroform (CH) and ethyl acetate (EA) fractions. CH fraction was then further sub-fractionated to get subfractions. The most active sub-fraction as an antioxidant was subjected to isolation to identify the compounds responsible for antioxidant activity. The methanol extracts and their fractions (water, PE, CL, and EA) were evaluated by determining antiradical activities using 2 2'-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzo thiazoline-6-sulphonic acid) diammonium salt (ABTS), reducing power, and metal chelating activity.

Evaluation of radical scavenging activity using DPPH radical
Scavenging activity of DPPH radicals was performed according to (Khalil et al., 2018) with slight modification. A-50 μL of extract and fraction samples with different concentrations was added to 1.0 mL DPPH 0.4 mM and 3.950 mL methanol. The mixture was allowed to react at room temperature for 20 mins. Then, the absorbance was measured at 515 nm with a calibrated UV-visible spectrophotometer. All measurements were done in triplicate.
Where A o is the absorbance of the control (without samples) and A 1 is the absorbance of samples.

Radical scavenging activity using ABTS technique
The ABTS radical assay of extract and fraction samples was performed according to Fidrianny et al. (2015). The mixture of potassium persulphate 2.45 mM and ABTS 7 mM (1:1) was allowed to stand in the dark for 12-16 hrs at room temperature to produce ABTS radical cation (ABTS + ). This ABTS + solution was eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER diluted with methanol to obtain absorbance values of 0.600-0.800 at 734 nm. The ABTS + working solution (3 mL) and 30 μL of blank (methanol), standard or sample were mixed and the absorbance was measured at 734 nm after 6 mins using a spectrophotometer.

Metal chelating activity
Metal chelating activity of extract and fractions was measured as described by (Wong et al., 2014). A certain amount (mL) of samples was added with 0.1 mM FeSO 4 (0.2 mL) and 0.25 mM ferrozine (0.4 mL). After incubating at room temperature for 10 min, the absorbance of the mixture was recorded at 562 nm. The chelating activity was calculated using the following formula: Where A o is the absorbance of the control reaction (without any samples) and A 1 is the absorbance in the presence of extracts or fractions.

Determination of total phenolics and flavonoid contents
Total phenolics contents of extracts and fractions were determined according to (Kovarovič et al., 2019)., while total flavonoids were determined using the spectroscopic method according to Rohman et al. (2010). For phenolics content analysis, Folin Ciocalteu reagent (FCR) was used. Briefly, extracts and fractions in methanol were mixed with 0.4 mL of FCR. The solution was allowed to stand at room temperature for 8-10 mins, added with 4 mL Na 2 CO 3 7% and made to 10.0 mL with bidistilled water. The mixture was allowed to stand for 2 hrs and subsequently measured at 725 nm. The phenolic contents were expressed as mg gallic acid equivalents (GAE) per gram of sample (mg/g). For total flavonoid analysis, samples in methanol were added with 4 mL of distilled water and 0.3 mL of NaNO 2 5%. After 5 mins, 0.3 mL AlCl 3 10% was added to the mixture. At 6 mins, 2 mL NaOH 1 M was added to the mixture. Immediately, the mixture was diluted to a volume of 2.4 mL distilled water and thoroughly mixed and its absorbance was measured at 510 nm versus a blank containing all reagents except samples of extracts or fractions. The total flavonoid content of the extracts and fractions were expressed as mg rutin equivalents (RE) per gram of sample (mg/g).

Fractionation of chloroform extract
Chloroform fraction has good antioxidant activities, therefore, it was further subjected to fractionation using vacuum liquid using silica gel G 60 GF 254 as in (Rohman et al., 2010). The chloroform fraction was dissolved in chloroform, then anhydrous sodium sulphate was added that was previously heated for 2 hrs at 110°C, and allowed to stand for one night and filtered using a filter paper. The solvent was concentrated using a vacuum rotary evaporator. Samples were added to a column that has been let stand overnight. The chloroform fraction was eluted using the solvents of chloroform, ethyl acetate, and methanol in various compositions. A volume of 50 mL of eluates each was collected for each and evaporated using a vacuum rotary evaporator.

Fractionation of active fraction
The sub-fractions were loaded into TLC using the stationary phase of GF254 with suitable eluting solvents. The plate was sprayed with DPPH 0.02% in methanol. Sub-fraction capable of bleaching the DPPH colour was subjected to further fractionation using a gravimetric column. The sub-fraction was loaded into a column using silica gel 230-400 mesh (0.040-0.063 mm) and isocratically eluted with n-hexane: acetone (2:2 v/v) and with petroleum ether: ethyl acetate (1:1 v/v), 5 mL of each elute was then collected.

Isolate purity identification
The purity of the isolate was identified using two methods, melting point and thin-layer chromatography (TLC). The melting point of the crystal was identified using the Buchi Melting Point B-450. Temperatures were recorded at the time the crystal began to melt and the temperature at which the crystal turns into liquid. The test was repeated by measuring the temperature of ±10°C below the melting point obtained, ramped at 1°C/min. For the purity test using TLC, the isolate was eluted using three eluent systems with a mobile phase with different polarity index, acetone: ethyl acetate: chloroform with a ratio of 4: 3: 3 v/v.

Isolate identification
The chemical structure of the isolate was determined using FTIR spectroscopic method (FTIR 100 PERKIN ELMER) and gas chromatography-mass spectrometry (GCMS-QP2010S SHIMADZU, Japan).

Results and discussion
Antioxidant activities of Nephelium lappaceum L. rind were evaluated using radical scavenging DPPH and ABTS, ferric reducing activity power (FRAP) and metal chelating activity, which can be differentiated as Hydrogen Atom Transfer (HAT) and Single Electron Transfer (SET) (Prior et al., 2005;Rohman et al., 2020).

Antiradical scavenging activities
DPPH is a synthetic radical which is stable and soluble in polar organic solvents such as methanol and ethanol. The principle of the DPPH radical scavenging test was based on the reduction of radical DPPH (violet in colour) into non-radical DPPH (colourless). Thus, the antiradical compounds were able to reduce DPPH radical intensity at wavelength 517 nm, with the absorptivity molar decreased from 9660 M -1 cm -1 (radical DPPH) into 1640 M -1 cm -1 (non-radical DPPH). When antiradical such as phenolics and flavonoids donate their hydrogen radical into DPPH radical, the colour of radical DPPH decreases. The proposed reaction between antiradical compounds (antioxidants) with DPPH radical could be seen in Figure 1. The parameter used for describing DPPH radical activity was IC 50 , the concentration of the samples necessary to cause 50% scavenging of DPPH radical, calculated from a linear regression equation (Rohman et al., 2016). The higher the antiradical activities, the lower the IC 50 values. Table 1 compile the IC 50 values of methanol extract and its fraction of Nephelium lappaceum L. rind using DPPH radical with vitamin C (ascorbic acid) as the positive control as it has the strongest anti-radical activities (the lowest IC 50 ). In addition, ethyl acetate fractions showed antiradical activities among extract and fractions evaluated followed by methanol and chloroform (which are not statistically different or P > 0.05) and petroleum ether. It was in accordance with the study conducted by Mistriyani et al. (2021). The antioxidant activity of the ethyl acetate fraction of Nephelium lappaceum L. peel was the lowest compared to other fractions (22.6 μg/mL). Samples with IC 50 values of 10-50 μg/mL were considered to have strong antioxidant activity. This can be explained that the presence of compounds capable of donating hydrogen radicals present in the EA (ethyl acetate) fraction was effective to decolourize radical DPPH. The antiradical activity of samples measured by the ABTS method using Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid) as a positive control was compiled in Table 2. Among extracts, fractions and positive control evaluated, Trolox exhibited the highest antiradical activities using ABTS radical followed by methanol extract, petroleum ether, ethyl acetate and chloroform fractions.

Reducing power activities
The reducing power of Fe(III) into Fe(II), known as ferric reducing activity power (FRAP), differs from radical scavenging activities because there are no free radicals, but involved the reduction of ferric ion (Fe 3+ ) from potassium ferricyanide into Ferro ion (Fe 2+ ). The Ferro ion can be monitored by measuring the intensity of Prussian blue colour at a wavelength of 700 nm, and the higher the absorbance at 700 nm indicated the higher the reduction power. The antioxidant activity based on the reducing power of Fe 3+ into Fe 2+ was expressed as mg equivalent of vitamin C in one (1) gram sample. The linear equation describing the relationship between concentration of vitamin C (x-axis) and absorbance of Fe 2+ due to reduction of Fe 3+ with vitamin C (y-axis) was: y = 10.771x + 14.878 (R² = 0.9891). Table 3 compiled the reducing power of extract and fractions (calculated as mg equivalent vitamin C/g sample). The methanol extract exhibited the strongest reducing power with a FRAP value of 14.446±0.161 mg equivalent vitamin C/gram sample followed by ethyl acetate fraction, chloroform fraction, and petroleum ether fraction. This indicated that reducing compounds present in methanol extract were active and may be present in a high amount.

Metal chelating activities
The metal chelating activity of extract and fractions was performed in a slightly acidic medium (pH 6.0). The phenolic compounds can bind to Fe 2+ and the remaining Fe 2+ could react with ferrozine to form blue-coloured complexes which can be monitored spectrophotometrically at 562 nm. The absorbance of this complex could be reduced by antioxidants such as phenolic compounds, due to its capability to bind to metal (Fe 2+ ). Therefore, any compounds capable of reducing the complex Fe 2+ -ferrozine could be considered an antioxidant through the mechanism of metal chelating. As a positive control, ethylene diamine tetraacetic (EDTA) was used, as a consequence, the metal chelating activity of extract and fractions were expressed as mg Na. EDTA/gram sample. Table 3 displays the metal chelating activity of methanol extract and its fractions. The chloroform extract revealed the highest metal activity compared to other fractions and methanol extract. Previous research also reported high metal chelating activity of chloroform fraction of Nephelium lappaceum L. peel, however, the highest metal chelating activity was found in the water fraction (Mistriyani et al., 2021).

Phenolics and flavonoid contents
Due to its capability to provide hydrogen radicals, reducing Fe 3+ and binding metals catalyzing oxidation reactions, phenolic and flavonoids contents were correlated with these antioxidants. Total phenolic contents were determined using Folin-Ciocalteau (F-C) reagent and gallic acid was used as standard, therefore phenolic contents were expressed as mg gallic acid equivalent/gram sample (mg GAE/g). The linear regression describing the relationship between gallic acid (x-axis) and its absorbance after reaction with F-C reagent (y-axis) was expressed as y = 1.338x -0.0068 (R 2 = 0.998). In addition, flavonoid contents were determined after being reacted with NaNO 2 , AlCl 3 and NaOH to form a red-coloured complex which can be measured spectrophotometrically at 510 nm. Rutin was used as standard during quantitative analysis of flavonoids, therefore, the flavonoid contents were expressed as mg rutin equivalent/g sample (mg RE/g). The linear regression describing the correlation between rutin (x-axis) and its absorbance (y-axis) was expressed as y = 0.1438x -0.0365 (R 2 = 0.995).

Isolation and identification of active compound
The chloroform fraction was then fractionated to sub -fractions to obtain isolates with good antiradical activities. Isolate 1 has been isolated from chloroform fraction and subjected to a purity test using TLC and melting point test. In addition, structure identification was performed using infrared spectroscopy and mass spectrometry. The purity test performed by TLC using three different solvent systems indicated that isolate 1 was TLC pure because only one spot was observed. Melting point analysis showed that isolate 1 had a sharp melting point of 62-64 o C. Based on TLC and melting point results, isolate 1 can be considered pure and can be continued to be identified. Identification of isolate 1 using FTIR spectroscopy resulted in IR spectra with several peaks. The peak at 2925 cm -1 corresponded to the stretching vibration of C-H. The peak at 1741 cm -1 originated from the stretching vibration of the carbonyl group of C=O, while the peak at 1458 cm -1 was corresponding to the stretching vibration of C=C (benzene), while peaks at 989cm -1 and 722 cm -1 came from C-H bending vibration. Using mass spectrometry, the molecular ion (M + ) appeared at m/z of 390 amu with a base peak at m/z 279 amu. The fragment ions appeared at m/z of 279 (M-CHCH 3 C 2 H 5 O(C 2 H 5 ) 2 (CH 2 ) 3 )OCH 3 ), 167, 149(M-CH 2 CH 3 (CH 2 ) 2 C(O)CHCH 3 CHCH), 132,113,93,83,71,57,43. Based on IR and mass spectra, the compound was tentatively identified as 1,2benzenedicarboxylic acid, bis(2-ethylhexyl) ester (CAS) bis(2-ethylhexyl) phthalate (Figure 2). This compound can be found in some plant extracts such as Podophyllum hexandrum rhizome, the stem of Hugonia mystax L., and endophytic fungi and it has been reported to have strong antioxidant activity (Li et al., 2012;Vimalavady and Kadavul, 2013;Govindappa et al., 2014).

Conclusion
Nephelium lappaceum L. (rambutan) rind showed good antioxidant activities determined using either DPPH or ABTS method. The chloroform fraction demonstrated IC50 of 49.77±0.22 for DPPH and 45.36±0.27 for ABTS. Moreover, it also demonstrated good reducing power (34.478±0.245 as mg vitamin C equivalent/g sample) and metal chelating capacity (332.753±0.695 as mg Na EDTA equivalent/g sample). The content of phenolic and flavonoid compounds was correlated to the antioxidant and metal chelating activities. Identification from the most active fraction found that the compound of 1,2-benzenedicarboxylic acid is the active compound responsible for antioxidant activities. This result could be further explored the potential use of rambutan rind compound as a food supplement of antioxidant activities.

Conflict of interest
The authors declare no conflict of interest.  Table 5. The correlation between antioxidant activities with phenolics and flavonoid contents of methanol extract and its fraction of rambutan rind