Antioxidant activity of jambu bol [ Syzygium malaccense (L.) Merr. and Perry] leaf extract

Jambu bol [ Syzygium malaccense (L.) Merr. and Perry] is well known to be used in folk medicine and reports are proving the biological activity of each part of this plant. This study aimed to determine the antioxidant activity of S. malaccense L. leaf extract . The methods used in this study were total phenolic content (TPC) assay, 2,2 - Diphenyl - 1 - picrylhydrazyl (DPPH) assay, 2,2' - azino - bis (3 - ethylbenzothiazoline - 6 - sulfonic acid) (ABTS •) radical scavenging activity assay and ferric reducing antioxidant power (FRAP) assay. The results showed that the extract contained TPC of 44.10±0.06 mg GAE/g. The IC 50 scavenging activities of DPPH and ABTS • were 0.0334 mg of Ascorbic acid E/g and 0.1352 mg of TE/g, respectively, while the antioxidant reducing power was 1013.50±0.07 mM Fe 2+ / g. In conclusion, S. malaccense L. leaf extract was found to possess antioxidant activity, and this extract has potential to be developed to be used in food as a natural antioxidant agent.


Introduction
Antioxidants play a pivotal role in our lives nowadays because they defuse or destroy "reactive oxygen species" (ROS), also known as free radicals before they hurt living cells.Cell membrane degradation, membrane protein deterioration and DNA mutations are all results of oxidation reactions that are induced by ROS.The relationship between ROS and some common illnesses such as ageing, cancer, inflammation, skin damage, liver injuries, diabetes mellitus, inflammatory diseases, cardiovascular diseases, arteriosclerosis, coronary heart diseases, and arthritis is the oxidation reaction that ROS is responsible for its activation (Dontha, 2016).Recent reports have illustrated the benefits of phytochemicals that possess antioxidant activities in terms of eliminating the occurrence of some dangerous diseases such as cancer through frequent consumption of high-quality sources of antioxidants such as fruits.The importance of fruits is due to their richness in antioxidants such as vitamins C and E, carotenoids, flavonoids, and other phenolic compounds (Saura-Calixto and Goni, 2006;Lim and Rabeta, 2013).
Plants are known to have primary and secondary metabolites, where phenolic compounds represent a large portion of the secondary metabolites.Interestingly, phenolic compounds have been shown to have a wide range of biological activities, including antimicrobial, antioxidant, and anti-diabetic properties (Rohman et al., 2010;Shahraki, 2013).According to some studies, each part of the plant (stems, fruits, roots, flowers, seeds, barks, and pulps) possesses a wide range of metabolites (Loganathan et al., 2017;Jamshidi-Kia et al., 2018).Plants can be classified based on their characteristics into families where each family may contain many genera that also have thousands of species, such as the Myrtaceae family, which is considered the eighth-largest family with around 140 species and 3,800-5,800 species (Ranghoo-Sanmukhiya et al., 2019).Syzygium is the 16 th largest genus of flowering plants in Myrtaceae family (Soh, 2017).This includes high diversity cultivated for many purposes such as colourful, edible and fleshy fruits (Rabeta et al., 2013;Wardana et al., 2018).Syzygium malaccense L. Merr.and Perry is one of the most popular plants in Malaysia and Indonesia and it is well known to be used in folk medicine.Different parts of S.
RESEARCH PAPER malaccense L. extracts have been reported to have antioxidant activity (Savitha et al., 2011;Arumugam et al., 2014;Nunes et al., 2016;Batista et al., 2017).Hence, this study aimed to demonstrate the total phenolic content and the antioxidant activity of S. malaccense L. ethanolic leaf extract.

Plant sampling and extraction
Syzygium malaccense L. Leaves were collected in February 2020 from Taman Pertanian, Universiti Putra Malaysia (UPM).The chosen leaves were uniform in shape, fresh, healthy, and mature.Samples were directly cleaned under running tap water to clean them up from dust and dirt and then washed again with distilled water.The extraction method was performed using the maceration technique as described by Rukayadi et al. (2013), with slight modifications.Cleaned leaves were dried in the shade for 2 days and then complete drying was done in a hot-air oven (Euroasaia, Malaysia) at 43±2°C temperature for 3 hrs.Dried leaves were crushed to a fine powder using stainless steel grinder (sieving was done after each grinding process to avoid the existence of big particles).A hundred grammes of powdered leaves were soaked in 400 mL of absolute ethanol (RandM Chemicals, UK) in a universal bottle and incubated for 24 hrs in a water bath incubator shaker (Heidolph, Germany) at 40±3°C with 110 revolutions per minute (rpm).The ethanolic solution was filtered using Whatman filter paper size No. 2 (Whatman International Ltd., England) by using a continuous vacuum filtration machine, and then it was concentrated using a rotary evaporator (Heidolph, Germany) at 40±3°C and a speed of 110 rpm.The crude extract was diluted in DMSO to obtain 100 mg/mL and then further diluted in 1:10 (v/v) distilled water to get 10 mg/mL stock solutions.

Total phenolic content and antioxidant activity determination 2.2.1 Total phenolic content
The total phenolic content (TPC) in S. malaccense L. leaf extract was determined by the Folin-Ciocalteu spectrophotometric method as described by Oki et al. (2002), with slight modifications.Briefly, different concentrations of the extract were prepared in a range from 5 -0.001 mg/mL.Folin-Ciocalteu (RandM Chemicals, Essex, UK) reagent (FCR) was prepared by dissolving 1 mL of the reagent in 9 mL of distilled water to get a 10% concentration of FCR.A 7.5% sodium carbonate (Na 2 CO 3 ) solution (Firma Chempur, Piekary lskie, Poland) was prepared by dissolving 0.75 g of Na 2 CO 3 in 10 mL of distilled water.20 µL of the extract was transferred into a sterile 96-well microtiter flat bottom plate.Then, 100 µL of FCR was added to the extract and homogenised properly, and then left for 5 mins.Then, 80 µL of Na₂CO₃ was added to the solution, bringing the total volume to 200 µL.The microtiter plate was wrapped in aluminium foil and left in the dark for 30 mins.The absorbance was read at a 750 nm wavelength using the BIO-RAD 170-6930 Benchmark Plus Microplate Spectrophotometer (BIO-RAD, California, United States) and the results was obtained using Microplate Manager Software.The same protocol was applied to gallic acid (Acros Organics, New Jersey, United States) at a range of 0.100-0.006mL to create a calibration standard curve.Results were presented as gallic acid in milligram equivalents per mL (mg GAE/ mL).The following equation was obtained from the calibration standard curve (y = 0.011x + 0.0308, R² = 1)

DPPH radical scavenging activity assay
The antioxidant activity of S. malaccense L. leaf extract was performed in vitro by a method described by Gerhäuser et al. (2003) with some modification, and it was measured by the free radical 2,2-Diphenyl-1picrylhydrazyl (DPPH) assay.Briefly, a range of 5-0.001 mg/mL of S. malaccense L. leaf extract concentrations was prepared by dissolving the extract in methanol.Ascorbic acid (RandM Chemicals, Essex, UK) as a positive control was prepared in different concentrations from 0.1-0.001mg/mL.The DPPH radical (Alfa Aesar, Massachusetts, UK) was prepared by dissolving 3.9 g of DPPH in 100 mL of methanol where the last adjusted optical density (OD) using BIO-RAD was 0.93±0.02at 517 nm wavelength.An amount of 100 μL of the extract concentrations was transferred into a sterile 96-well microtiter and then mixed with 100 μL of prepared DPPH radicals.The same steps were carried out for the ascorbic acid, and then the microtiter plate was kept in the dark for 30 mins.The plate was then applied to the 170-6930 Benchmark Plus Microplate Spectrophotometer to get the OD values of absorbance at 517 nm wavelength while the results were obtained using Microplate Manager Software.The results were measured as the percentage of scavenging activity by using the following equation: Where A 0 represents the absorbance of the negative control and A 1 represents the absorbance of the sample.The results were plotted as IC 50 values and ascorbic acid equivalents (AAE, mg/mL) of S. malaccense L. leaves extract.The ABTS• assay, also known as the 2,2′-azinobis (3 -ethylbenzthiazoline-6-sulphonic acid) assay, was used to determine the scavenging activity of S. malaccense L. leaf extract, with some modifications to the protocol described by Kanagasabapathy et al. (2011).In brief, the ABTS• radical solution was prepared by reacting 1 mL of ABTS (Sigma-Aldrich, Missouri, United States) (7 mM) with 1 mL of potassium persulfate (Emsure, Darmstadt, Germany) (K 2 S 2 O 8 -2.45 mM).The solution was kept in a universal bottle wrapped with aluminium foil for 12 hrs to produce a dark solution of the ABTS• radical.In the preliminary experiment, the ABTS• radical solution was diluted with 50 mL of distilled water and then adjusted to an absorbance of 0.70±0.02at 734 nm using the BIO-RAD 170-6930 Benchmark Plus Microplate Spectrophotometer.In the experiment, 20 µL of different prepared concentrations of the extract were transferred into a 96-well microtiter plate with a flat bottom.Following that, 200 L of the adjusted ABTS• radical was added to the extract, and the 96-microtiter plate was placed in the dark for 10 mins.The absorbance was recorded at a wavelength of 734 nm.The same procedures for the ABTS• radical were performed with Trolox (Sigma-Aldrich, Missouri, United States), and the scavenging activity was determined as the following equation: Where A 0 represents the absorbance of the negative control and A 1 represents the absorbance of the sample.The results were plotted as IC 50 value which is the concentration of the extract that is necessary for 50% reduction of ABTS•.

Ferric reducing antioxidant power
The antioxidant activity of S. malaccense L. leaf extract was determined by a ferric reducing antioxidant power (FRAP) assay according to the method described by Benzie and Strain (1996) with some modifications.Briefly, the FRAP reagent was freshly prepared by mixing the following solutions together and they were 300 mM acetate buffer (pH 3.6), 10 mM 4,6-tripryridylstriazine (TPTZ) (Sigma-Aldrich, Missouri, United States) solution, 40 mM HCl (RandM Chemicals, Essex, UK) and 20 mM ferric chloride (Sigma-Aldrich, Missouri, United States) in the ratio of 10:1:1 (v: v: v).Before the beginning of the experiment, the temperature of FRAP reagent was increased up to 37℃ using the calibrated oven.20 µL of S. malaccense L. leaves extract (different concentrations) were transferred into a 96-microtiter plate followed by 180 µL of FRAP reagent.A 180 µL of FRAP reagent was added to 20 µL of distilled water to represent the reagent blank.After incubating the microtiter plate at 37℃ for 5 mins, BIO-RAD 170-6930 Benchmark Plus Microplate Spectrophotometer was used to measure the absorbance at 593 nm wavelength.To generate a standard curve, the same procedures were applied to different concentrations of ferrous sulphate heptahydrate (Sigma-Aldrich, Missouri, United States) (FeSO 4 •7H 2 O) aqueous solution.The absorbance value of the extract tested after subtraction of reagent blank and blank sample was translated into FRAP value (mmol of FeSO 4 •7H 2 O equivalents) using the FeSO 4 •7H 2 O calibration plot with the following equation formula: y = 0.0002x + 0.1853, R² = 0.9804.

Statistical analysis
The results of total phenolic content, ferric reducing power, DPPH, and ABTS• scavenging activities were calculated using Microsoft Excel version 16.0 and presented as mean ± standard deviation (SD) of triplicate experiments.

Results and discussion
The total phenolic content and the antioxidant activity of S. malaccense leaf extract were determined using TPC, FRAP, DPPH, and ABTS• assays.In this study, it was found that the increases in the antioxidant activity of the ethanolic extract were relatively related to the increases in the concentrations of the extract.

Total phenolic content
Plants contain antioxidant compounds that are important with redox properties that are responsible for antioxidant activity.In the present study, the determined TPC amount in S. malaccense L. leaf extract was 44.10±5.26mg GAE/g (Table 1), which is less than what has been reported in previous studies.Arumugam et al. (2014), reported that the TPC of S. malaccense L. leaf extract was 125.81 mg GAE/g which is higher results from this current study.This difference may be contributed to the pre-extraction and the extraction method where they used an air circulation oven at 40 o C for the drying process, ethanol as a solvent with ratio 1:20 (w/v), and then kept the solution in an incubator shaker at 37℃ for 24 hrs which were all factors influenced the TPC content in the extract.According to other studies, the TPC of methanolic S. malaccense L. leaf extract was 53.77 and 108.08 mg GAE/g, while the aquatic extract was 82.07 mg GAE/g (Savitha et al., 2011;Batista et al., 2017).Although the variation among the results from different studies is small, leaves of S. malaccense L. seem to contain more TPC compared RESEARCH PAPER with the other portions of the plant, such as peels and fruits, which contain 12.58 and 8.03 mg GAE/g of TPC, respectively (Nunes et al., 2016).The values of phenolic content in the current study varied compared to those in the other studies.This variation may be due to the presence of different amounts of phytochemicals or the duration, geographical variation, or methods of extraction, which may alter the amounts of phenolics (Aryal et al., 2019).However, phenolic compounds play a major role in plant self-defence mechanisms against incursive microorganisms or against any other threats as well as environmental stress, such as wounding and excessive light or ultraviolet radiation (Harborne et al., 1994;Wallace and Fry, 1994).These compounds can act as antioxidants by chelating metal ions, preventing radical formation and improving the antioxidant endogenous system (Al-Azzawie and Alhamdani, 2006).

DPPH radical scavenging activity assay
Antioxidants upon interaction with DPPH either transfer an electron or hydrogen atom to DPPH, thus neutralizing its free radical character.The colour of the reaction mixture changes from purple to yellow, and the degree of discoloration indicates the scavenging potential of the antioxidants (Foti et al., 2004;Villaño et al., 2007).Table 2 shows the half-maximal inhibitory concentration (IC 50 ) of S. malaccense L. leave extract that inhibited 50% of the DPPH radical and was compared with ascorbic acid.The IC 50 values of S. malaccense L. leaf extract and ascorbic acid were 0.0334 and 0.0037 mg/mL, respectively.The scavenging activity was increased to 92.29±4.12%at a higher concentration of the extract (0.078 mg/mL).Ascorbic acid as a positive control was more effective as a pure antioxidant than the extract, where its highest scavenging activity was 90.37±6.11%at 0.0125 mg/mL.According to the study by Arumugam et al. (2014), the ethanolic S. malaccense L. leaf extract had a lower IC 50 value to scavenge DPPH radicals (0.016 mg/mL).Savitha et al. (2011) determined the IC 50 of the methanolic extract of S. malaccense L. leaves against DPPH radicals at 0.025 mg/mL.Another study mentioned that the IC 50 of S. malaccense L. leaf extract using hexane, methanol, and ethyl acetate as solvents was 0.510, 0.010, and 0.031 mg/ mL, respectively (Ramadhania et al., 2017).From the literature, it was found that methanolic leaf extract of S. malaccense L. has higher activity in terms of DPPH radical scavenging.Syzygium aqueum fruits, also known as water apple, were reported to have powerful scavenging activity in terms of the DPPH radical scavenging activity assay where the recorder IC 50 was 0.004 mg/mL (Saptarini and Herawati, 2017).Furthermore, S. aqueum methanolic leaf extract was found to have high DPPH radical scavenging activity, as S. aqueum fruits have an IC 50 of 0.006 mg/mL (Sobeh et al., 2018).Syzygium samarangense, which is known as wax apple, had reordered antioxidant activity where the IC 50 of methanolic pulp and seed power in scavenging DPPH radicals were 0.072 mg/mL and 0.078 mg/mL, respectively (Simirgiotis et al., 2008).Similarly, the DPPH radical scavenging activity of S. samarangense leaf extracts was reported to have an IC 50 of 0.094 mg/ mL, 0.074 mg/mL, and 0.929 mg/mL using n-Hexane, athel acetate, and methanol as solvents, respectively (Budiono et al., 2019).From the given data, the ethanolic leaf extract of S. malaccense L. was found to have less powerful DPPH scavenging activity than S. aqueum fruits and leaf extracts but better than that reported for S. samarangense pulp, seeds, and leaf extracts.However, S. malaccense L. leaves extract had powerful antioxidant activity because the IC 50 value was less than 50 μg/mL (Saptarini and Herawati, 2017).

ABTS• radical scavenging activity assay
The determination of the scavenged ABTS• radicals was established using S. malaccense L. leaf extract in order to evaluate the extract's antioxidant activity by delocalization of the ABTS• through measuring the reduction of the radical cation as the percentage inhibition of absorbance at 734 nm (Abdullah and Jamil, 2019).Trolox was used as a positive standard control.As shown in Table 2, the highest scavenging activities of the extract and Trolox were observed at 0.625 mg/mL and 3.8 mg/mL, with scavenging powers of 98.60% and 99.90%, respectively.The IC 50 of the extract and the control were 0.135 mg/mL and 0.547 mg/mL, respectively.The extract exhibits more antioxidant activity when compared to Trolox.Arumugam et al. (2014) reported that S. malaccense L. leaf extract has powerful antioxidant activity and estimated the IC 50 of ethanolic leaf extract of S. malaccense L. at 0.047 mg/ mL, which is less than the results obtained from the present study.Leaves methanolic extract of S. aqueum was investigated by Osman et al. (2009) to determine its scavenging activity against ABTS• radicals and it was observed that 0.035 mg/mL had almost 65% of the scavenging power, which is higher scavenging  Stalin (2018), the methanolic leaf extract of Syzygium caryophyllatum had high antioxidant activity and the IC 50 of scavenging ABTS• radicals was 0.013 mg/mL.Generally, methanol is known to be evaporated at 64.7 °C which is far less than the boiling point of ethanol, this difference makes the plant extraction process more convenient with methanol as less temperature is needed to complete the extraction process and less damage to the phytochemical compounds occurred.Furthermore, methanol has been generally found to be more efficient in the extraction of lower molecular weight polyphenols than other solvents which is suggested to be the reason methanolic plant extracts have higher antioxidant activity than other extracts produced by other solvents (Do et al., 2014).Ethanolic leaves extract of Syzygium jambos had its antioxidant activity evaluated and it found that the concentration of 0.057 mg/mL was the IC 50 that scavenged 50% of ABTS• radicals (Hossain et al., 2018).These studies revealed that the ethanolic leaf extract of S. malaccense L. has less scavenging activity for ABTS• radicals than the other extracts from S. aqueum, S. polyanthum, S. caryophyllatum, and S. jambos.

Ferric reducing antioxidant power
The reducing power assay measures the electrondonating ability of antioxidants using the potassium ferricyanide reduction method.Antioxidants cause the reduction of the ferricyanide complex to its ferrous form.The reducing power of S. malaccense L. leaf extract at a 0.312 mg/mL concentration was 1013.54±61.37 mM Fe 2+ /g (Table 1).According to Arumugam et al. (2019), the ethanolic extract of S. malaccense L. had ferric reducing power equal to 847.24 mM Fe +2 /g sample.This revealed the current excrement presented higher activity in ferric reducing power than in the previous report.However, the presence results from reducing Fe 3+ to Fe 2+ by using the leaf extract of S. malaccense L. had better activity than the peels and the edible part of the same plant (Nunes et al., 2016).The present FRAP results were found to be comparable with a study by Abdullah and Jamil (2019) in which they investigated the antioxidant activities of eight different species of the Syzygium genus.According to Lim and Rabeta (2013), solvents that were used in the extraction process had a high impact factor on the antioxidant activity of the produced extracts.However, using acetone as a solvent in the extraction process to extract bioactive compounds from S. malaccense L. leaves and S. aqueum leaves had significantly higher ferric reducing power than the extracts extracted by using water (Lim and Rabeta, 2013).The ferric reducing power of acetonic leaves of S. malaccense L. and S. aqueum extracts were 2062.78 and 3545.56M Fe (II) per g of freeze-dried sample, respectively, while water had 378.33 and 847.22 M Fe (II) per g of freeze-dried sample, respectively.From this comparison, it was remarkable that the ethanolic extract that was used in the present study had higher ferric reducing power than both acetonic extract and waterbased extracts.Generally, in the presence of 2,4,6tripyridyl-s-triazine, phenolic compounds are responsible for electron transfer, leading to a reduction of Fe 3+ to Fe 2+ .This reduction reaction appears and can be observed by the formation of a coloured complex with Fe 2+ absorption at 593 nm (Romes et al., 2019).

Conclusion
The present study showed that the ethanolic leaf extract of S. malaccense L. had less total phenolic content than the other reported results.The reason has been suggested to be the steps of the pre-extraction

Table 1 .
TPC value and antioxidant activity as determined using FRAP assay in S. malaccense L. leaves extract.

Table 2 .
Antioxidant activity of S. malaccense L. leaves extract as determined using DPPH and ABTS• assays.Values are presented as mean±SD, n = 3. , such as the drying process or the size of powdered particles during the grinding step, the extraction process itself, and the solvent used in the extraction process.Antioxidant activity of S. malaccense L. leaf extract showed better activity than some of the other species of Syzygium plants, and that was in terms of DPPH, ABTS•, and FRAP assays.This study concluded that S. malaccense L. leaf extract had noteworthy antioxidant activity and that it may be developed to be used alone or in combination with other extracts as food additives for antioxidant purposes.