Reactive oxygen species are reactive atoms that have unpaired electrons. They can cause oxidative stress in DNA, lipids, proteins, or carbohydrates, eventually leading to neurodegenerative disease and cancer (Halliwell, 2012). The source of free radicals may come from external factors, such as junk food, alcohol, drugs, tobacco, environmental pollution, and heavy metals. However, free radicals can also be produced in the human body from endogenous reactions, such as phagocytosis, mitochondria by producing adenosine triphosphate, and β-oxidation of fatty acids (Jakubczyk et al., 2020; Liguori et al., 2018; Namkoong et al., 2018; Petruk et al., 2018). Therefore, an antioxidant that can slow or inhibit cellular damage in the human body is urgently needed (Lobo et al., 2010). Antioxidants may vary in the mechanism of actions and composition, which can scavenge free radicals, chelate metals, and inhibit enzymes (Gioti et al., 2009; González-Palma et al., 2016).
Plants are potential sources of antioxidants. Phenolic compounds, vitamins, and carotenoids have been reported as the primary contributors to antioxidant activities (Indrianingsih et al., 2015b; Olugbami et al., 2015; Rodríguez-García et al., 2019). The utilization of plants includes different parts of them, such as flowers, leaves, bark, fruits, and roots. Several customers and manufacturers use plant extracts for medicinal use and cosmetic products (Sitarek et al., 2020). Antioxidants from plants are preferred over synthetic antioxidants because of their safety and nutritional benefits (Deng et al., 2011).
Diabetes mellitus (DM) is a disease diagnosed with hyperglycemia. It is caused by either insulin resistance or an insulin production problem, and the symptoms include weight loss, thirst, and frequent urination (Klein et al., 2007). DM complications could damage the kidneys, eyes, nerves, and heart (Sukardiman and Ervina, 2020). According to the WHO, DM patients are estimated to reach approximately 342 million by 2030 and will be a burden for developing countries (Sukardiman and Ervina, 2020). One treatment to overcome DM, primarily type 2, is consuming an α-glucosidase inhibitor. It can postpone glucose absorption and prevent increasing blood glucose levels (Sy et al., 2005). Plants are a potential alternative to treat DM. Besides managing stable blood glucose levels, it can also prevent complications, an advantage over synthetic drugs (Sukardiman and Ervina, 2020). Approximately 1,200 plants were found to have an ability to lower blood glucose levels in ethnopharmacological surveys (Pandey et al., 2011).
Quercus gilva Blume is an evergreen tree grown in Japan and South Korea (Kim and You, 2012; Noshiro and Sasaki, 2011). In Japan, Q. gilva was used as a raw material for agriculture tools, such as spades, axe handles, and hoes. The previous study has reported the bioactivity of phenolic compounds from the bark of Q. gilva, such as anti-inflammatory, antiurolithiasis, and antioxidative agents (Youn et al., 2017). Several studies also identified terpenes from the fruit of Q. gilva and antioxidative agents from the branches of Q. gilva (Itokawa et al., 1978; Moon et al., 2009). The bioactive compounds from Quercus species leaves were phenolic compounds, vitamins, aliphatic alcohols, fatty acids, and sterol (Lämke and Unsicker, 2018; Vinha et al., 2016). Several phenolic constituents in the Quercus species leaves are tannins, flavonoids (epicatechin, quercetin, rutin, kaempferol, and naringin), and phenolic acids (ellagic acid, gallic acid, gentisic acid, p-coumaric acid, vanillic acid, caffeic acid, and ferulic acid) (Brossa et al., 2009; Cantos-Villar et al., 2003; Jong et al., 2012). Our previous study isolated three compounds, namely, catechin, epicatechin, and tiliroside, from Q. gilva leaves (Indrianingsih et al., 2015). However, the isolation of polyphenol and study of its bioactivity on the Q. gilva leaves have not yet been done. Polyphenols such as procyanidins were often obtained as a mixture constituent, such as oligomeric and stereochemical mixtures. It was hard to isolate it as a pure compound (Oizumi et al., 2010). The synthesis of procyanidin itself also had several problems, such as the needed large amount of nucleophile at low temperature and the formation of side products in oligomeric form (Kozikowski et al., 2001).
In the present study, we isolated the polyphenol compound from methanolic extract of Q. silva leaves and studied its bioactivity, such as antioxidant and antidiabetic activity.
MATERIALS AND METHODS
Plant materials, reagents, and general instrumentation
Quercus gilva leaves were harvested from Ehime University Garden, Matsuyama, Japan. Samples had been saved in the Faculty of Agriculture, Ehime University, Japan. The α-glucosidase enzyme, 1,1-diphenyl-2-picrylhydrazyl (DPPH), β-carotene, p-nitrophenyl α-D-glucopyranoside (p-NPG), potassium ferricyanide [K3Fe(CN)6], hydrogen peroxide (H2O2), ferric chloride (FeCl3), trichloroacetic acid, n-hexane, chloroform, toluene, methanol, acetone ethanol, and ethyl acetate were obtained from Wako, Ltd. (Japan). Gallic acid, quercetin, and Tween 40 were obtained from Sigma-Aldrich, Ltd. (Japan). Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL JNM-AL 500 spectrometer (Tokyo, Japan) using tetramethylsilane as the internal standard. Gas chromatography-mass spectrometry and fast atom bombardment-mass spectroscopy (FAB-MS) were performed using equipment from Shimadzu, Japan.
Compound isolation from Q. gilva
Leaves powder of Q. gilva (1.1 kg) was macerated with 8.8 l of methanol at room temperature. After 2 days of immersion, the filtrate was evaporated, separated, and isolated using bioassay-guided isolation. The solvent for silica column chromatography was started from n-hexane (100%), followed by n-hexane:ethyl acetate (50:50) and ethyl acetate:methanol (50:50), and finally methanol (100%). Five fractions F1–F5 were obtained, and further repeated silica column chromatography of F5 resulted in F51 to F53. Compound 1 was isolated as a light brown powder (20 mg) from silica column chromatography of F53.
Compound 1 (light brown powder): Catechin (4α→8)-Catechin (Procyanidin B3). UV, λmax 280.5 nm; Electrospray ionization–mass spectrometry (ESI-MS, positive ion mode) m/z 579 [M + H]+. 13C-NMR (125 MHz, CD3OD) δ 157.9 (C-5u), 157.2 (C-5t), 156.7 (C-7u), 156.4 (C-7t), 146.9 (C-3u?), 146.7 (C-3?t), 146.6 (C-4?u), 146.4 (C-4?t), 133.1 (C-1?u, C-1?t), 120.6 (C-6?u), 120.2 (C-6?t), 116.8 (C-2?u), 116.4 (C-2?t), 116.3 (C-5?u), 116.1 (C-5?t), 108.0 (C-8t), 98.4 (C-10u; C-10t), 96.9 (C-6t), 96.8 (C-6u), 96.7 (C-8u), 83.2 (C-3u), 74.5 (C-2u), 69.7 (C-2t), 68.2 (C-3t), 39.3 (C-4t), 29.6 (C-4u).
HRFAB-MS: [M + H]+: m/z 579 for C30H26O12.
The antioxidant activity of the isolated compound was performed by the DPPH assay (Indrianingsih et al., 2021). The sample was diluted in methanol in several concentrations, reacted with DPPH (1.01 mM) at room temperature for 30 minutes in dark conditions. The absorbance of the final solution was measured using a UV spectrophotometer at 517 nm. The radical scavenging activity of compound 1 was calculated using
where A0 is the absorbance of the control and A1 is the absorbance of the sample. The measurement was conducted in triplicate. Quercetin was used as the positive standard in the DPPH assay.
The capability of compound 1 in the H2O2 radical scavenging assay was evaluated according to the literature (Indrianingsih et al., 2015) with slight adjustments. H2O2 was diluted in phosphate buffer solution (PBS) (pH 7.4) to obtain the concentration of 40 mmol/l. Compound 1 (4 ml in Aquades) was reacted for 10 minutes with 0.6 ml of H2O2. The absorbance of the final solution was measured at 230 nm using a UV spectrophotometer.
Reducing power assay
According to the literature, the reducing power assay was conducted (Indrianingsih et al., 2015). Compound 1 (20 g/ml) in PBS (2.5 ml) was reacted with K3Fe(CN)6 (2.5 ml), left to stand for 20 minutes at 50°C, and added with trichloroacetic acid (2.5 ml). After centrifugation for approximately 8 minutes, the upper layer (2.5 ml) was reacted with Aquades and FeCl3 (0.5 ml). The final solution was evaluated at 700 nm using a spectrophotometer.
β-Carotene bleaching assay
According to a previous study, the capability of compound 1 in preventing β-carotene bleaching was performed (Indrianingsih et al., 2015). β-carotene (0.2 mg/ml) in CHCl3 was reacted with linoleic acid (20 mg) and Tween 40 (200 mg). After evaporation, distilled water (50 ml) was added, and 4.8 ml of the solution was reacted with 0.2 ml of compound 1 solution in methanol. This solution was incubated at 50C. The absorbance of the solution was evaluated at 20 minutes intervals at 470 nm.
α-Glucosidase inhibitory assay
The α-glucosidase assay was conducted according to the literature (Indrianingsih et al., 2015). Several concentrations of compound 1 in dimethyl sulfoxide (10 µl) were mixed with PBS (pH 7, 490 µl) and 250 µl of p-NPG (3 mmol/l). After being reacted for 5 minutes at 37°C, 250 µl of the α-glucosidase enzyme (0.065 IU/ml) was added and left to stand for 15 minutes. One milliliter of Na2CO3 (0.2 mol/l) was added to stop the reaction, and the absorbance of the final solution was evaluated at 400 nm.
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