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Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia Borneensis Leaves Infusions Chinese Traditional Medica

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Electrochemistry, Antioxidant Activity, and Toxicity of Unfermented Aidia Borneensis Leaves Infusions Chinese Traditional Medica 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 mm, 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 mL 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
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