Chinese Traditional Medical Journal

Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia borneensis Leaves Infusions

Siti Nur Faezah H. Ajak1, Fairuzeta Ja’afar1, Hartini M. Yasin1,*, Mark I.R. Petalcorin2, Anwar Usman1,** 1Department of Chemistry, Faculty of Science, 2PAPRSB Institute of Health Sciences Universiti Brunei Darussalam, Jalan Tungku Link, Gadong BE1410, Brunei Darussalam

ABSTRACT

Aidia borneensis is an endemic plant to Brunei Darussalam and its leaves are traditionally consumed as herbal medicine as a home-remedy beverage. Until recently, the beverage is conventionally prepared by decoction of the fermented leaves, similar to the preparation of black tea from Camelia sineensis leaves. In this present work, the green tea method, involving the decoction of unfermented leaves, was used to produce A. borneensis leaf infusions. Their antioxidant property was evaluated using electrochemical analyses, including cyclic voltammetry and differential pulse voltammetry, and using chemical assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). Their toxicity was also tested using in vivo Caenorhabditis elegans studies. The green tea infusions of A. borneensis was found to possess significant antioxidant properties with the total antioxidant capacity (TAC) determined in the range of 11.10– 11.99 g gallic acid equivalent antioxidant capacity (GEAC) per 100 g of dry green tea. The TAC of the infusions showed the same trend as their total phenolic and flavonoid contents, suggesting that the antioxidant property might be due to the phenolic and flavonoid compounds. The infused compounds were identified using GC-MS, and their oxidation-reduction reactions were evaluated and quantified using electroanalytical analyses. The green tea infusion was found to be non-toxic to C. elegans after 24 h exposure. These findings suggest that A. borneensis green tea infusions have the potential to be used as a beverage to reduce the risks of oxidation due to free radicals.

Keywords: Antioxidant activity, voltammetry, spectroscopy, chemical composition, Aidia borneensis, green tea

INTRODUCTION there is a general trend to replace these synthetic antioxidants with natural antioxidants found in plants [6,7]. This Free radicals arising from environmental pollutants, growing interest has prompted the evaluation of antioxidant radiations, chemicals, toxins and physical stress can damage capacity of different families of plants [8-12]. In addition the immune system, leading to many diseases and accelerated to the conventional tea from Camellia sinensis (L.) leaves, aging process in the human body [1-2]. Antioxidants, natural antioxidants from wine, fruits, vegetables and including polyphenol derivatives such as phenolic, flavonoid spices have also been exploited, but with limited current and flavanol have the ability to scavenge the reactive oxygen use as additives or supplementary ingredients [13]. To species, which are known to cause many health issues [2-4]. exploit the natural antioxidants from different families of Antioxidants can inhibit the formations of free radicals and plants, intensive research activities have been focused on lipid peroxidations. The presence of antioxidant enzymes maximizing the health benefits of their natural antioxidants such as catalase and phytochemical compounds such as prepared from different parts of the plants into beverage ascorbic acid, tocopherols and carotenoids enhances the total and food. For instance, herbal infusions (also known as antioxidant activity of the polyphenols [3,4]. Some examples tisanes) have been reported to have medicinal effects due of synthetic antioxidants which are currently consumed to their stimulant, relaxant and sedative properties [14,15], are tertiary-butylhydroquinone, butylated hydroxyanisole, provoking new interest in phytomedicine looking at new butylated hydroxytoleune and propyl gallate [5]. However, potential pharmacological uses of plants.

*Corresponding to: One of the plants that has been traditionally used as herbal Hartini M. Yasin and Anwar Usman, Department of infusions in Brunei Darussalam is Aidia borneensis, a Chemistry, Faculty of Science, Brunei Darussalam. perennial evergreen, locally grown plant belonging to the E-mails: hartini.yasin@ ubd.edu.bn; Rubiaceae family [16]. The infusions of A. borneensis [email protected] leaves are usually consumed as tea by brewing the fresh

CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 1 1 Ajak, et al.: Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia borneensis Leaves Infusions leaves in hot water, and it has been reported to relieve body paper (Whatman No. 1) with the pore size of 11 μm, and aches and gastric pains [16]. It has been previously reported the filtrate was allowed to cool down at room temperature. that the antioxidant capacity of the A. borneensis leaves For chemical assays, the infusion was diluted with distilled lies in the middle rank among the twelve commercially water to achieve concentrations within 10–10,000 mg L-1 available teas from C. sinensis (L.) leaves. This is due to depending on each specific assay. its high phenolic content, revealing the significance and feasibility of this new potential plant as a rich source of Chemicals and Reagents natural antioxidants [12]. In this study, the young and mature A. borneensis leaves were treated as green tea (or All chemicals and reagents were of analytical grade and unfermented), hence the herbal infusions can be consumed as they were used without further purifications. 2,2-diphenyl- a beverage [9], and phytochemical and biological activities, 1-picrylhydrazyl radical (DPPH), 2,4,6-tris(2-pyridyl)-s- including antioxidant capacity and toxicity of the green tea triazine (TPTZ), gallic acid monohydrate and trolox were infusions were evaluated. The relationship between the total purchased from Sigma-Aldrich (St. Louis, MO, USA), while antioxidant capacity and total phenolic and flavonoid content hydrochloric acid, iron(III) chloride, aluminium chloride, in the green tea of A. borneensis leaves was also estimated. Folin-Ciocalteu reagent, glacial acetic acid, methanol, The phenolic and flavonoid compounds were identified sodium acetate, sodium carbonate, sodium chloride and using gas chromatography-mass spectroscopy (GC-MS). sodium nitrite were purchased from Merck (Darmstadt- Since the antioxidant capacity of the phenolic and flavonoid Germany). Double distilled water was used throughout the compounds is mainly due to their redox reactions, the redox sample preparations and measurements. The standard stock properties of the green tea were also investigated using solution of gallic acid was prepared in distilled water and electrochemical approach, namely cyclic voltammetry (CV) trolox in methanol. and differential pulse voltammetry (DPV) with polished glassy carbon electrodes. Overall, this study was aimed to explore the green tea of A. borneensis leaves as a healthy The Effects of Brewing Temperature and Brewing Time beverage from the ethno-medicinal plant with a high content of natural antioxidants. To evaluate the effect of brewing temperature and time on the antioxidant capacity, the infusion was prepared by soaking 2 g of the green tea powder directly into 200 mL distilled MATERIALS AND METHODS water. For the effect of brewing temperature, the distilled water was set to be at 20, 40, 60, 80 and 100 ºC for a constant Sample Preparation brewing time at 3 min. To investigate the effect of brewing time, samples were prepared in distilled water at 80 ºC with Fresh A. borneensis leaves were collected from Kampung various brewing times in the range of 1–120 min. Mungkom in the Tutong District of Brunei Darussalam. The young leaves, which have a soft texture and light pinkish red adaxial and abaxial surfaces, were separated from the Determination of Antioxidant Capacity mature leaves, which have a hard texture and are deep green in color. The leaves were washed with distilled water in order DPPH assay to remove any particulates. The green tea of the young and matured A. borneensis leaves was prepared by heating at The antioxidant capacity of aqueous green tea infusions high temperatures to prevent enzymatic degradation, thus was determined using the DPPH free radical scavenging maintaining the polyphenol forms and the native microflora assay based on the standard procedure as reported by in the leaves [17], according to the procedure as described Delgado-Andrade et al. [19]. Briefly, 200 µL of diluted by Lin et al.. [18] with slight modifications. The fresh leaves samples with varying concentrations from 10 to 1000 mg were blanched in hot water at temperatures ranging from 90 L-1 was added to a freshly prepared 1 mL of 50 mg L-1 (or to 100 ºC for about 45 s to 1 min, followed by rolling and 0.127 mM) DPPH dissolved in methanol. The mixture was drying at room temperature. The dried leaves were then kept undisturbed in the dark at room temperature for 30 min. ground into powder and sifted through a 350-mm metal The disappearance of the purple color in the presence of the sieve. The powder was stored in an airtight sealed container antioxidant in the sample causes a decrease in the absorbance at room temperature until further analyses. measurements, which can be measured using a single beam UV-vis spectrophotometer (Optizen 1412V). The absorbance The infusions of the green tea of the young and matured was recorded at 517 nm against 1 mL methanol and 200 A. borneensis leaves were prepared using aqueous extraction mL of water acting as a blank. Radical scavenging activity according to a standard procedure, similarly to typical (RSA) of gallic acid monohydrate was used as the standard. household tea preparation. To prepare the infusion, 2 g of The absorbance of the resultant solution was measured the green tea powder was soaked in 200 mL distilled water using spectrophotometer at 517 nm (denoted as Asample), and while stirring, allowing their constituents to infuse into a methanolic DPPH solution without infusion was used the solution. The infusion was then passed through a filter as a control (denoted as Acontrol). The antioxidant capacity

CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 1 2 Ajak, et al.: Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia borneensis Leaves Infusions of the sample was expressed as a percentage of inhibition of standard catechin hydrate were added into 1.35 mL of of the DPPH radical and calculated using the following distilled water in a sample vial. 50 μL NaNO2 solution equation;[19] (5% w/v) was added into the mixture, followed by 50 μL aluminum chloride solution (10% w/v). The mixture was

AAcontrols- ample vortexed and then left in the dark for 10 min at room RSA (%) = ×100% temperature. The absorbance of the mixture was then A control measured using spectrophotometer at 415 nm. A calibration curve of catechin hydrate was used to determine the TFC The RSA was then plotted against concentration to give a of the green tea infusion, where TFC of the green tea linear plot, and the IC50, which was defined as the green tea infusion was calculated based on the linear regression of concentration required to scavenge 50% initial DPPH was the calibration curve. determined. Based on the IC50 value, the antioxidant capacity of the green tea was estimated and it is expressed in gallic acid equivalent antioxidant capacity (GEAC) per 100 g green tea. Cyclic Voltammetry

Electrochemical analyses of the green tea infusion were Ferric reducing antioxidant power (FRAP) assay performed using eDAQ system (eDAQ Pty Ltd, Australia), which includes an integrated potentiostat type ER466 and For FRAP assay, the antioxidant capacity of the green tea was a potentiostat type EA163. The current as a function of determined based on the procedure as reported by Thaipong applied voltage was plotted as voltammograms using EChem et al.. [20]. Fresh FRAP reagent was prepared by mixing software version ES260. A three-electrode system includes a 100 mL of 300 mM acetate buffer, 10 mL of 10 mM TPTZ 3-mm diameter polished glassy carbon electrode as working solution and 10 mL of 20 mM FeCl .6H O solution and kept 3 2 electrode, a platinum wire counter electrode, and a silver/ warm at 37 ºC until the reagent was used. The pH of the silver chloride (in 2 M NaCl) as reference electrode. The buffer was then measured. The TPTZ solution (10 mM) was green tea infusion (1000 μL) was diluted with 9 mL of 0.1 prepared in 40 mM HCl and 20 mM FeCl .6H O in distilled 3 2 M acetate buffer and placed in an electrochemical cell. The water. A calibration curve of Trolox (6-hydroxy-2,5,7,8- pH of the supporting electrolyte was adjusted to be in the tetramethylchroman-2-carboxylic acid) in the range of 0–250 range of 2.0–7.0 by adding glacial acetic acid or sodium μg/mL was used as standard. Under a dark condition, 150 hydroxide. For a standard reference, an experiment with μL of standard solution or 150 μL of green tea infusion was similar sample conditions and electrochemical analyses for allowed to react with 2850 μL of FRAP reagent in separate gallic acid dissolved in the same supporting electrolyte was test tubes for 30 min. Absorbance of the colored solution performed. (ferrous tripyridyltriazine complex) of the standard and the samples was measured at 593 nm. The concentration of FRAP Typically, the CVs were recorded at a potential range content in the extract was reported as milligrams Trolox between -0.4 V and +1.0 V with a scan rate of 100 mV s-1 equivalent antioxidant capacity (TEAC) per 100 g green tea. at room temperature. To determine the electrochemical mechanisms of the compounds in green tea infusion, the Total Phenolic Content (TPC) scan rate was variably set at 10, 20, 50, 100, and 200 mV s-1. To ensure that the observed peak potentials are consistently Total phenolic content of the green tea infusion was determined presenting the redox reactions of the same compounds in spectroscopically using Folin-Ciocalteu reagent based on the infusion, the concentration of the infusion was in the procedures as described by Chan et al.. [21]. Briefly, 1.2 mL of range from 100 to 1000 mg L-1. The electrodes were gently sodium carbonate 7.5% (w/v) was added into a 300 µL of diluted polished with 0.3-micron alumina and rinsed with double infusion, followed by the addition of ten-fold dilution 1.5 mL distilled water prior to each voltammetric scan. of Folin-Ciocalteu reagent. The color of the sample mixtures turned from yellow, upon addition of sodium carbonate, to Differential Pulse Voltammetry (DPV) blue with the addition of Folin-Ciocalteu reagent. The mixture was incubated in the dark at room temperature for 30 min. DPV was another electrochemical tool used to quantify The absorbance of the solution was measured at 765 nm. the antioxidant capacity of the green tea infusions. DPV A calibration curve of standard gallic acid was established, and employs a series of pulsed potentials with an amplitude the TPC of the green tea infusion was calculated based on the typically 50 mV on a staircase scan mode. In this experiment, linear regression of the calibration curve. 1 mL green tea infusion was spiked into the supporting electrolyte and analyzed. Under optimized conditions, the Total Flavonoid Content (TFC) infusions of young and matured leaves were dissolved in buffer solution at pH 3.7 and 4.2, respectively. To quantify The TFC in the green tea infusion was measured using the total antioxidant activity, a calibration curve was plotted colorimetric assay according to procedure reported by using gallic acid as the standard reference with five different Damiani et al. [22]. 100 μL infusion or ethanolic solution concentrations ranging from 10 to 100 mg L-1. The linear

CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 1 3 Ajak, et al.: Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia borneensis Leaves Infusions regression coefficient of the calibration plot was 0.9862. infusions and control solutions have been performed at The total antioxidant content was expressed as gallic acid least in triplicates and all data have been analyzed. The data equivalent (GEAC). The result was validated by the detection presented in this report represent the mean values. limit of this DPV technique, which was determined based on signal-to-noise ratio for the gallic acid measurement [23]. RESULTS AND DISCUSSION

Gas Chromatography-Mass Spectrometry (GC-MS) Total Antioxidant Capacity (TAC) of the Green Tea Infusion The compounds in the green tea infusion were analyzed through GC-MS analysis using a Shimadzu-2010 GCMS The IC of the green tea infusion of A. borneensis leaves analyser and Agilent J and W GC Capillary DB-5 column 50 was quantified to be 97 to 105 mg L-1 by DPPH free radical (30 m length; 0.25 mm diameter; 0.25 μm thick). Helium was scavenging and FRAP assay, respectively. Based on the used as the carrier gas and the carrier pressure was 100.1 kPa. DPPH assay, TAC of the green tea infusion was 11.10 to The total flow rate was 50.0 mL min-1 while the column flow 11.99 g GEAC per 100 g dry green tea. On the other hand, rate was 1.69 mL min-1. Sample injection was carried out at FRAP assay indicated that TAC of the green tea infusion was 250 ºC. The GC oven temperature was started and held at 54.05 to 119. 35 g TEAC per 100 g dry green tea. Considering 50 ºC for 1 min, then the temperature was raised to 140 ºC at the conversion between TEAC to GEAC being 0.13 [25], a rate of 20 ºC min-1, and again with no holding in between, to using FRAP assay TAC of the infusion was approximately 300 ºC at a rate of 10 ºC min-1 and this temperature was held 7.03 to 15.5 g GEAC per 100 g dry green tea, implying that for 10 min. The ion source temperature was set at 200 ºC, TAC of the green tea infusion evaluated by both methods while the interface temperature was set at 250 ºC. The total within their experimental errors are comparable with each run time was 31.50 min. The compounds detected from the other. The TAC of the green tea infusion depends on the GC-MS analysis were determined by comparing their mass brewing time and temperature, as described below, and both spectra and retention indices with those of the compounds DPPH free radical scavenging and FRAP assays show the stored in the National Institute Standard and Technology same trend, as shown in Figure S1-S2 in Supplementary (NIST version 8) library. Information.

Toxicity Assay The TAC of the A. borneensis green tea infusion detected with both DPPH and FRAP methods continuously increases The assessment of toxicity was performed using with the brewing temperature from 20 to 100 ºC, as shown in synchronously grown C. elegans var. Bristol strain N2 Figure 1. As the brewing temperature reaches above 40 ºC, the (obtained from the Caenorhabditis Genetics Center, TAC of the infusion tends to be saturated. Overall, the trend is CGC) in 96-well plates following the method with slight in line with the literature for C. sineensis green tea [26]. It is modifications as described by Bonamigo et al.. [24] with noteworthy that, unlike black teas, green tea is usually prepared slight modifications. Briefly, 30 nematodes at L4 stages with non-boiling water due to the bitter taste of green tea when were transferred into each well in triplicates containing it is prepared at high temperature. This is the main reason 50 ml of M9 buffer (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, why the brewing temperature is recommended to be lower 0.25 g MgSO4 in 1 L H2O) as control or 50 ml of A. borneensis than that for black tea [27]. As shown in Figure 1, even the green tea infusion at a concentration of 2 g leaf powder per infusion at 40 ºC already has more than 80% of the maximum 200 ml distilled water. value of antioxidant capacity in both the green tea infusions of young and matured A. borneensis leaves. This implies that o After incubation for 24 hours at 20 C, the viability of the the antioxidants of the green tea are readily infused into the nematode was evaluated by assessing its movement through solution even at low brewing temperatures. This suggests repeatedly touching the worms with platinum microspatula. that the water effectively extracts the antioxidants from A. The worm response was monitored using the Olympus SZX18 borneensis green tea. The TAC of the A. borneensis green tea Stereo Microscope. The number of dead and alive nematodes infusions show the same trend as their TPC and TFC. TAC was evaluated based on response. Immotile non-responsive of green tea infusions produced from matured A. borneensis nematodes were considered as dead while those moving were leaves shows lower correlation coefficient compared with that counted as alive. In order to prevent confusion, the dead produced from young A. borneensis leaves, where the latter worms were discarded while performing subsequent counting. has a strong correlation and a linear relationship between TAC The toxicity assessment was measured in terms of percentage with TPC and TFC (R2 = 0.9612 and R2 = 0.9765, respectively). viability and mortality rate curves of the nematode. Nevertheless, this indicates that TAC is correlated to the TPC and TFC of the green tea infusions, suggesting that the TAC Data Analysis of A. borneensis green tea infusions are determined by the phenolic and flavonoid compounds infused into the solution. Appropriate and careful treatments were implemented This notion is supported by chemical identification using GC- throughout all the experiments. All measurements on the MS analyses, which is described in section 3.5. These current

CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 1 4 Ajak, et al.: Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia borneensis Leaves Infusions

Figure 2: Effect of brewing time on antioxidant expressed Figure 1: Effect of brewing temperature on antioxidant in GEAC (white), TPC (black) and TFC (grey) for the expressed in GEAC (white), TPC (black) and TFC (grey) green tea infusion from A. borneensis (A) young and (B) for the green tea infusion from A. borneensis (A) young matured leaves. The bar graphs were overlaid for easy and (B) matured leaves. The bar graphs were overlaid for comparison. easy comparison. related to high TAC and there are other chemical components findings are in agreement with other published reports forC. of the green tea that could contribute to its TAC [30]. One sinensis tea [28,29]. The TPC and TFC have been attributed can consider that the decrease of polyphenol contents with to the antioxidant capacity of the C. sinensis tea [28], and the further increase in brewing times might be due to the high brewing temperature increases the amount of compounds epimerization reactions [31]. From this study, the maximum extracted, particularly polyphenols. This is due to the increased extraction efficiency of polyphenols in the infusion prepared permeability of the cell walls of the leaves, resulting in an from the green tea of matured leaves was found to be 3 min increase in the amount of polyphenols in the infusion [29]. while that prepared from young leaves was 5 min. These results were in contrast with some results reported for green Figure 2 shows a graph of TAC of the A. borneensis green tea tea of C. sinensis where the maximum extraction efficiency infusions as a function of brewing time. As expected, TAC was obtained after 20 min for brewing time at 80 °C or 10 min generally increases gradually with brewing time, as more at 95 °C [31]. These differences may be indicative of the phenolic and flavonoid compounds infused into the solution different nature of green tea prepared fromA. borneensis and over a longer period of time. The antioxidant capacity was C. sinensis leaves, which additionally may contribute to the correlated with the TPC and TFC for both green teas prepared health effect. It is noted that overall results indicate that the from young and matured A. borneensis leaves. As shown infusion of young A. borneensis leaves has higher antioxidant in Figure 2, there is a non-linear correlation between the capacity as compared with that of matured leaves. antioxidant capacity and polyphenols present. It is easier to observe for the infusions prepared from matured A. borneensis Cyclic Voltammetry (CV) leaves, because GEAC, TPC, and TFC of the infusions prepared from young leaves are slightly fluctuated. Thus, it is The CVs of the infusions prepared from young and matured suggested that high TPC and TFC values are not necessarily A. borneensis leaves (pH 3.7 and 4.2, respectively) along

CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 1 5 Ajak, et al.: Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia borneensis Leaves Infusions with that of distilled water are shown in Figure S3. The leaves infusion were observed at +0.77 V (176.6 μA cm-2) oxidative scan of the green tea infusions was recorded and -0.118 V (38.8 μA cm-2). By integrating the current in the in the range between -1.0 V to +1.2 V vs. Ag/AgCl. As a potential range from 0.35 to 1.0 V, an alternative quantification comparison, the CV of the supporting electrolyte is also of the current response, the charge (Qp) below voltammetric depicted in Figure S3. Oxidative peak was readily observed waves was estimated to be 91.55 and 86.44 μC cm-2 for at 1.03 V with a peak current of 82.8 μA for the infusion of the infusion of young and matured A. borneensis leaves, young leaves and at 0.87 V with a peak current of 27.9 μA respectively. This result clearly indicates higher antioxidant for that of matured leaves. Upon reversing the scan direction, capacity of young leaves compared with matured leaves, no obvious reductive peaks were observed for both green tea supporting the quantification using both DPPH free radical infusions. This indicates that the compounds contained in scavenging and FRAP assays in section 3.1. the infusions may undergo an irreversible reaction. Similar behavior of redox reaction, where there were no reductive The effect of scan rate on the CV of the green tea infusions peaks observed, has been observed for the green tea infusion of A. borneensis leaves was recorded in 0.1 M acetate of C. sineensis [32].

When acetate buffer (pH 3.7 and 4.2, respectively) was used as a supporting electrolyte, both CV of infusions prepared from young and matured A. borneensis leaves show the oxidative and reductive peaks, as shown in Figure 3. The existence of the reductive peaks in this condition implies that the acidic environment supported some compounds to undergo reversible redox reaction. Considering that the oxidative and reductive peak intensities are different to each other by at least 3-fold, most of the compounds still undergo an irreversible redox reaction even in the presence of the supporting electrolyte. Thus, the presence of the supporting electrolyte also shifts down the oxidative potential, which can be attributed to electron transfer from the electroactive compounds to the buffer solution. From this observation, CV analyses of the green tea infusions of A. borneensis leaves works better in aqueous media without any supporting Figure 4: The plot of oxidation potential as a function electrolyte. For the infusion prepared from young leaves, after of pH for the infusions of (a) young and (b) matured the subtraction of the background signal from the supporting A. borneensis leaves. electrolyte, the oxidative and reductive peaks were observed at +0.71 and at -0.028 V with the peak current densities of 199.2 and 66.0 μA cm-2, respectively, whereas those of the matured

Figure 5: The differential pulse voltammograms of (a) green tea infusion of young A. borneensis leaves Figure 3: Cyclic voltammogram of (a) buffer solution and (b) green tea infusion of matured A. borneensis (pH 3.7), (b) the green tea infusion from young leaves, measured in 0.1 M acetate buffer (pH 3.7 and A. borneensis leaves, and (c) the green tea infusion from 4.2 respectively), using a 3-mm diameter glassy carbon matured A. borneensis leaves measured at 100 mV/s at electrode with modulation amplitude of 70 mV and scan 3-mm diameter polished glassy carbon electrode. rate of 20 mV/s.

CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 1 6 Ajak, et al.: Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia borneensis Leaves Infusions

Table 1: GC-MS analysis of green tea infusion of young A. borneensis leaves Retention time Compound Name Compound Structure Compound Formula % Peak Area

6.327 Coumaran C8H6O 38.68

7.255 4-Hydroxy-3-methylacetophenone C9H10O2 1.47

8.369 1-(bromomethyl)-3-nitro-benzene C7H6BrNO2 4.06

8.896 Levoglucosan C6H10O5 4.46

10.158 Quinic Acid C7H12O6 32.74

buffer. The concentration of the green tea infusions was The effect of pH on the voltammetric response of the green 10,000 mg L-1, the pH of the supporting electrolyte was tea infusions of A. borneensis leaves (10,000 mg L-1) in 3.7–4.2, and the scan rate was in the range of 10–200 mV acetate buffer was monitored in the pH range of 2.0–7.0, s-1. As shown in Figure S4 for the first scan of CVs, the as shown in Figure S5(A) and S5(B). It is clearly seen that oxidation potential shifted to higher values with the scan rate. for both young and matured leaves, the anodic and cathodic Moreover, distortion of the voltammogram was observed to peak potentials of the CV are shifted towards less positive occur at higher scan rates, indicating the scan rate-dependent values, and their peak currents are strongly pH-dependent, oxidation peak current. To clarify this issue, the insets of where the peak current reduces as the pH increases. This Figure S4 show the oxidative and reductive peak currents of can be rationalized by considering the protonation of the the green tea infusions as a function of the square root of the electroactive compounds in A. borneensis at higher pH, where scan rate (v1/2). It is clearly observed that the peak currents the thermodynamic driving force for the catalysis is modified show a linear relationship with the square root of the scan by the protonation state of the compound, leading to distortion rate, and the intercept was near zero current. The linear of the peak currents and shapes of the voltammogram [33]. relation of the peak currents versus v1/2 indicates directly that the redox process on the electrodes was controlled In order to evaluate the number of electrons involved in the by diffusion. This suggests that the redox process takes a redox reaction of the electroactive compounds in the green shorter time as the scan rate is increased, as the flux of ions teas of A. borneensis, the plot of the formal potential as a to the electrode surface becomes higher at a faster scan rate function of pH is shown in Figure 4, demonstrating that the leading to higher oxidative and reductive peak currents due peak potential decreases linearly with the pH. The slope of to the linear proportion of the current with the flux towards the linear relationship was estimated to be 68 mV per unit the electrode. pH for the green tea infusion prepared from young leaves,

CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 1 7 Ajak, et al.: Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia borneensis Leaves Infusions

Table 2: GC-MS analysis of green tea infusion of matured A. borneensis leaves Retention Time Compound name Compound structure Compound formula % Peak area 6.139 Pyrocatechol C6H6O2 5.75

6.325 Coumaran C8H6O 29.99

6.743 Hydroquinone C6H6O2 1.95

7.252 2-Methoxy-4-vinylphenol C9H10O2 1.66

8.368 1,3,2-Benzodioxaborol-2-ol C6H5BO3 5.32

10.24 Quinic acid C7H12O6 55.33

which is close to the Nernstian value (59.2 mV per unit concentrations between 10 to 100 mg L-1 was obtained. pH), indicating that the redox reaction of the electroactive Figure 5 shows the DPVs with oxidation peak approximately compounds involves a two-electron and two-proton at +0.40V and the calibration curve of the gallic acid with R2 process [34]. On the other hand, the slope is 32 mV per unit coefficient of the linear regression being 0.986. With the same pH for the green tea infusion prepared from matured leaves, experimental conditions, the DPV of the A. borneensis green which suggests a one-electron and one-proton process was tea infusions (10,000 mg L-1) was measured. The DPV shows involved in the redox reaction. This is in agreement with the an oxidation peak at +0.59 for the green tea infusion of young higher charge density for the infusion of young A. borneensis leaves and +0.46 V for that of matured leaves. From the peak leaves compared with that of matured leaves. current at +0.13 V, the TAC of the green tea infusion prepared from young and matured leaves was estimated to be 0.431 and Different Pulse Voltammetry (DPV) 0.261 g GEAC per 100 g dry green tea, respectively. These values were approximately one order of magnitude lower than Based on the electrochemical oxidation of the green tea those estimated using the chemical assays. There are several infusions of A. borneensis leaves, DPV method has also plausible reasons for the low estimation of TAC of the green tea been employed to quantify the TAC of the infusions. Under infusions using the electrochemical assay. First, as discussed the optimized conditions, a linear response for gallic acid in section 3.1, the supporting electrolyte has suppressed the

CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 1 8 Ajak, et al.: Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia borneensis Leaves Infusions electron transfer and therefore gives low peak current intensity. 24 hours. This observation of non-toxic or lethal effects was Second, the sizes of the electroactive compounds should be comparable with those of the control group incubated in M9 considered, where the large sized compounds have a slow buffer Fig( ure S6) that all yielded 100% viability. diffusion and the peak current of the DPV signal is low. Third, the TAC determined by chemical assays is enhanced CONCLUSIONS due to the synergetic effect of compounds in the infusions, rather than just due to their phenolic and flavonoid contents. In this paper, the antioxidant activity of green tea infusions The non-linear relation between antioxidant capacity and the of young and matured A. borneensis leaves, an endemic and polyphenols present in the infusions supports the latter notion. traditional folk medicinal plant of Brunei Darussalam, have To clarify this issue, the compounds infused from the green been investigated. The antioxidant capacity of the green tea tea infusions of A. borneensis leaves were then identified and infusions was evaluated using chemical and electrochemical discussed in section 3.4. assays. Using different chemical assays, the total antioxidant capacity of the green tea infusions was found to be in the Identified Compounds in the Green Tea Infusions range of 11.10–11.99 g GEAC per 100 g dry green tea. The total antioxidant capacity was underestimated when analyzed The chemical constituents in the green tea infusions were using the electrochemical assay due to the suppression of identified using GC-MS, where the compounds were electron transfer by the supporting electrolyte. The total identified by comparing their mass spectra, molecular antioxidant capacity of the green tea infusions varied formula, and accurate mass of molecular ions with those in depending on the brewing time and temperature and it NIST database library and published literature data. Tentative shows the same trend compared to their TPC and TFC, chemical composition, retention time, and respective suggesting that the antioxidant property of the infusions percentage are summarized in Table 1 and 2. From the GC might be due to the phenolic and flavonoid contents. peaks, a total of 5 and 6 main compounds were identified from Interestingly, the electrochemical analyses suggested that the green tea infusions of young and matured A. borneensis there were two electrons and two protons involved in the leaves, respectively. The remainder are unidentified redox reaction of the compounds in the infusion of young compounds, which are low intensity peaks having an area leaves, whereas it was only one electron and one proton for of less than 0.5% of the total area. It is noteworthy that two the infusion of matured leaves. This notion was supported of the compounds found in the infusions, namely coumaran by GC-MS data, where there is an additional compound and quinic acid are identical. The coumaran has similar identified in the infusion of young leaves. The toxicity structures found in flavonoid compounds, whereas quinic tests of the A. borneensis green tea infusions on C. elegans acid was suspected to be a derivative of hydroquinone. The worms indicated that the green tea is nontoxic. Based on the latter compound is identified in the matured A. borneensis high antioxidant capacities and non-toxicity, A. borneensis leaves, in addition to the phenolic compounds. Thus, this green tea infusions shows promise as a natural source of finding suggested that these compounds in their natural forms antioxidants and could be consumed as a beverage to reduce should be responsible for the antioxidant activity detected by the risks of oxidation due to free radicals. Further studies DPPH and FRAP assays and the electrochemical analyses. focusing on their bioactivities are needed to further explore their pharmacological applications. Interestingly, pyrocatechol which is a derivative of catechin was also identified in the matured A. borneensis leaves. Conflict of Interest Statement The phenolic, flavonoid, and catechins in C. sineensis green tea have been identified and quantified, and the green We declare that we have no conflict of interest. tea composition has been associated with TAC [26,35]. Catechins and phenolic compounds have also been confirmed to be responsible for the astringency and bitterness of green ACKNOWLEDGEMENTS tea [36]. The additional compound found in the green tea This publication has emanated from research conducted with infusion of young A. borneensis leaves is levoglucosan, the financial support of Universiti Brunei Darussalam. The which may be related to the carbohydrates present in the authors also would like to thank Hj Amran Hj Johari (Deputy young leaves. This may explain why most of the young Chairman of the 3MPK Tea Factory) and Hj Mohd Saiful leaves were found to be partially eaten by insects during bin Abdullah Anja (the Village Head of Kampong Kiudang, sample collection. Tutong, Brunei Darussalam) for providing fresh samples of Aidia borneensis leaves and plants. The authors declare that In Vivo Toxicity they have no conflict of interest.

The green tea infusions prepared at a concentration of 2 g Supporting Information leaf powder per 200 ml water using either young and mature leaves of A. borneensis were not toxic to the nematode The Supplementary Information consisting of the total C. elegans (for both L1 and L4 stages) after incubation for antioxidant at different brewing times and temperatures of

CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 1 9 Ajak, et al.: Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia borneensis Leaves Infusions the green tea infusion from young and matured A. borneensis 11. Ariffin F, Chew SH, Bhupinder K,et al.. Antioxidant capacity leaves, cyclic voltammogram as prepared and at different and phenolic composition of fermented Centella asiatica scan rates and pHs and toxicity assessment of the green herbal teas. J Sci Food Agric, 91: 2731–2739 (2011). tea infusions associated with this manuscript is available 12. Metussin N, Mohamed H, Ahmad N, et al.. Evaluation of antioxidant capacity of Aidia borneensis leaf infusion, an at the website. endemic plant in Brunei Darussalam. Food Res, 2: 12– 19 (2018). Author Contribution Statement 13. Halliwell B. Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional All of authors, Siti Nur Faezah H. 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