Subscribe Now Subscribe Today
Abstract
Fulltext PDF
References
Research Article
 

Antioxidative Chemical Constituents from the Stems of Cleyera japonica Thunberg



Jung Eun Kim, Sang Suk Kim, Chang-Gu Hyun and Nam Ho Lee
 
ABSTRACT

Chemical investigation to identify the antioxidative constituents from the stems of a tree Cleyera japonica Thunb. resulted in the isolation of seven compounds: catechin (1), catechin 3-O-α-L-rhamnopyranoside (2), epicatechin (3), taxifolin (4), taxifolin 3-O-α-L-arabinopyranoside (5), taxifolin 3-O-α-L-rhamnopyranoside (6) and proanthocyanidin A-1 (7). These isolates were studied for their scavenging activities against DPPH radical, hydroxyl radical and superoxide anion radicals using spectrophotometry and/or electron spin resonance. All isolates 1-7 exhibited more potent DPPH radical inhibition activities than the positive control, ascorbic acid. In the hydroxyl radical scavenging test, compound 7 (SC50 301.6 μM) showed potent activity higher than ascorbic acid (SC50 859.7 μM). All of the compounds 1-7 exhibited comparable activities to ascorbic acid for superoxide anion radical scavenging. These results demonstrated that C. japonica stem extracts could be potentially used as antioxidative agents in food or cosmetic applications.

Services
Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Jung Eun Kim, Sang Suk Kim, Chang-Gu Hyun and Nam Ho Lee, 2012. Antioxidative Chemical Constituents from the Stems of Cleyera japonica Thunberg. International Journal of Pharmacology, 8: 410-415.

DOI: 10.3923/ijp.2012.410.415

URL: https://scialert.net/abstract/?doi=ijp.2012.410.415
 
Received: December 17, 2011; Accepted: April 23, 2012; Published: June 13, 2012

INTRODUCTION

Reactive Oxygen Species (ROS) include oxygen-centered free radicals such as superoxide (O2•-), hydroxyl radical (HO), alkoxy radical (RO) and peroxyl radical (ROO) as well as nonradical species such as singlet oxygen (1O2) and hydrogen peroxide (H2O2). These radical and nonradical ROS are formed in living organisms during the normal metabolic processes. ROS, especially free radicals are chemically very reactive and can attack molecules in cells or tissues. For example, ROS can react with lipids in cell membranes, proteins in tissues or enzymes and bases in DNA (Denisov and Afanas’ve, 2005). This oxidative damage is believed to be a primary factor not only in various diseases but also in the normal process of aging (Valko et al., 2007). Humans have evolved with antioxidant systems to protect against free radicals, which include enzymatic defense such as superoxide dismutase, catalase and glutathione peroxidase. Through these enzymes, superoxide and hydrogen peroxides are metabolized and therefore production of detrimental hydroxyl radical is prevented. Even though this endogenous defense system is provided, under some physiopathological situations such as air pollutants, UV radiation and inflammation, ROS is produced in excess. In order to diminish the cumulative effects of oxidative damage, exogenous antioxidants are needed. Vitamins (A, C and E) and flavonoids from plant sources are antioxidants in diet (Pietta, 2000). Antioxidants are applied in the food industry as well as in the cosmetic industry as the functional ingredient to prevent oxidative damages (Elzaawely and Tawata, 2012; Gajula et al., 2009; Ham et al., 2010). Since application of antioxidants become broad in various areas, it is necessary to develop different type of novel antioxidative agents. Especially, natural antioxidants from plant sources are more favorable in the industry due to their environmentally friendly properties (Adisa et al., 2011; Chanda et al., 2011; Hajimahmoodi et al., 2008; Oboh and Ademosun, 2006).

We are continuously conducting phytochemical studies on plants growing in Jeju, the largest island located at the southernmost part in Korea (Kim et al., 2010a, b; Kim et al., 2011; Ko et al., 2011). In the course of our investigation for the biologically active natural products, we observed antioxidative activities in the ethanol extract prepared from the stems of Cleyera japonica, which led us to identify the active constituents.

C. japonica Thunb. (Theaceae family) is an evergreen tree of height up to 10 m distributed over Korea, Japan and Taiwan. This tree has usually been used as lumber for household furniture in Korea. The acetone extract from the leaves of C. japonica var. morii collected in Taiwan showed a strong free radical scavenging activities (Hou et al., 2003). However, no chemical constituents have been presented responsible for the activities. We herein described the antioxidative constituents from the ethanol extract of C. japonica stems and their scavenging activities against DPPH, hydroxyl and superoxide anion radicals.

MATERIALS AND METHODS

Reagent and equipment: All solvents used in this experiment were of analytical grade. 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a JEOL (JNM-ECX 400) instrument with chemical shift data reported in ppm relative to the solvent used. JES-FA200 (JEOL) Electron Spin Resonance (ESR) spectrometer was used for the radical scavenge tests. Merck silica gel (0.063-0.2 mm) was used for normal phase column chromatography. Silica gel 60 F254 coated on aluminum plates by Merck were used for Thin Layer Chromatography (TLC). Gel Filtration Chromatography (GFC) was performed using Sephadex LH-20 (25–100 μm) from Fluka. DPPH (2,2-diphenyl-1-picrylhydrazyl) was purchased from Aldrich. Xanthine, xanthine oxidase and DMPO (5,5-dimethyl-1-pyrroline N-oxide) were purchased from Sigma.

Plant material: The stems of C. japonica Thunb. were collected from the Halla Botanical Garden, Jeju Island in Korea. A voucher specimen (No. 124) was prepared and deposited at the laboratory of Natural Product Chemistry, Department of Chemistry, Jeju National University.

Extraction and isolation: The dried stems of C. japonica (1.1 kg) were extracted three times with 70% ethanol by stirring with a magnetic stirrer at room temperature each time for 24 h. The combined solutions were filtered and the filtrate was concentrated in a vacuum rotary evaporator at a maximum temperature of 40°C to afford a gummy extract (116.6 g). A portion of the extract (14.2 g) was suspended in water (1 L) and partitioned into n-hexane, ethyl acetate (EtOAc) and n-butanol soluble fractions. The EtOAc-soluble fraction (3.9 g) was subjected to Vacuum Liquid Chromatography (VLC) over silica gel with elution (each 300 mL) of n-hexane-EtOAc (0~100%) and EtOAc-methanol (0~50%) gradients to afford 18 fractions (frs. 1-18). Fraction 6 (138.7 mg) was further purified by silica gel Column Chromatography (CC) with chloroform-methanol (5:1) to provide compounds 1 (55.9 mg) and 4 (5.9 mg). Fraction 7 (144.5 mg) was purified by CC over Sephadex LH-20 with chloroform-methanol (2.5:1) to give the compounds 1 (2.3 mg), 3 (2.6 mg), 4 (3.0 mg) and 7 (11.5 mg). Fraction 8 (306.3 mg) was subjected to Sephadex LH-20 CC with chloroform-methanol (3:1) to give compounds 5 (16.3 mg) and 6 (39.2 mg). Fraction 9 (520.9 mg) was also purified by Sephadex LH-20 CC with chloroform-methanol (3:1) to provide compound 5 (264.5 mg). Fraction 10 (953.1 mg) was subjected to silica gel CC with chloroform-EtOAc-methanol (2:2:1) to yield five fractions (frs. 10-1 to 10-5). Subfraction 10-2 (356.0 mg) was further purified over Sephadex LH-20 CC with chloroform-methanol (3:1) to afford compounds 2 (11.9 mg) and 5 (191.9 mg). Fraction 11 (313.3 mg) was purified by Sephadex LH-20 CC with chloroform-methanol (3:1) to give compounds 2 (9.6 mg) and 5 (80.5 mg). Fraction 12 was also purified by Sephadex LH-20 CC with chloroform-methanol (4:1) to give compound 5 (9.4 mg).

Catechin (1): 1H-NMR (400 MHz, CD3OD) δ 6.83 (1H, d, J = 1.8 Hz, H-2’), 6.76 (1H, d, J = 8.2 Hz, H-5’), 6.71 (1H, dd, J = 1.8, 8.2 Hz, H-6’), 5.92 (1H, d, J = 2.3 Hz, H-6), 5.85 (1H, d, J = 2.3 Hz, H-8), 4.56 (1H, d, J = 7.6 Hz, H-2), 3.97 (1H, m, H-3), 2.85 (1H, dd, J = 5.3, 16.0 Hz, H-4), 2.50 (1H, dd, J = 8.2, 16.0 Hz, H-4); 13C-NMR (100 MHz, CD3OD) δ 158.0 (C-9), 157.8 (C-5), 157.1 (C-7), 146.5 (C-4’), 146.4 (C-3’), 132.3 (C-1’), 120.2 (C-6’), 116.2 (C-5’), 115.4 (C-2’), 100.9 (C-10), 96.4 (C-6), 95.6 (C-8), 83.0 (C-2), 69.0 (C-3), 28.7 (C-4).

Catechin 3-O-α-L-rhamnopyranoside (2): 1H-NMR (400 MHz, CD3OD) δ 6.84 (1H, d, J = 1.8 Hz, H-2’), 6.77 (1H, d, J = 8.0 Hz, H-5’), 6.72 (1H, dd, J = 1.8, 8.0 Hz, H-6’), 5.94 (1H, d, J = 2.3 Hz, H-6), 5.86 (1H, d, J = 2.3 Hz, H-8), 4.62 (1H, d, J = 8.0 Hz, H-2), 4.29 (1H, d, J = 1.4 Hz, H-1”), 3.93 (1H, m, H-3), 3.68 (1H, m, H-5”), 3.57 (1H, dd, J = 3.2, 9.6 Hz, H-3”), 3.51 (1H, dd, J = 1.8, 3.2 Hz, H-2”), 3.31 (overlapped with CD3OD, H-4”), 2.88 (1H, dd, J = 5.5, 16.0 Hz, H-4), 2.64 (1H, dd, J = 8.5, 16.0 Hz, H-4), 1.25 (3H, d, J = 6.2, H-6”); 13C-NMR (100 MHz, CD3OD) δ 158.1 (C-9), 157.7 (C-5), 157.0 (C-7), 146.5 (C-4’), 146.4 (C-3’), 132.1 (C-1’), 120.0 (C-6’), 116.2 (C-5’), 115.2 (C-2’), 102.3 (C-1”), 100.8 (C-10), 96.5 (C-6), 95.6 (C-8), 81.3 (C-2), 76.1 (C-3), 74.1 (C-4”), 72.4 (C-3”), 72.1 (C-2”), 70.5 (C-5”), 28.1 (C-4), 18.1 (C-6”).

Epicatechin (3): 1H-NMR (400 MHz, CD3OD) δ 6.97 (1H, d, J = 2.1 Hz, H-2’), 6.80 (1H, dd, J = 2.1, 8.0 Hz, H-6’), 6.76 (1H, d, J = 8.0 Hz, H- 5’), 5.94 (1H, d, J = 2.3 Hz, H-6), 5.91 (1H, d, J = 2.3 Hz, H-8), 4.82 (1H, s, H-2), 4.18 (1H, m, H-3), 2.86 (1H, dd, J = 4.6, 16.7 Hz, H-4), 2.73 (1H, dd, J = 2.8, 16.7 Hz, H-4); 13C-NMR (100 MHz, CD3OD) δ 158.2 (C-5), 157.8 (C-7), 157.5 (C-9), 146.1 (C-3’), 145.9 (C-4’), 132.4 (C-1’), 119.5 (C-6’), 116.0 (C-5’), 115.5 (C-2’), 100.2 (C-10), 96.5 (C-6), 96.0 (C-8), 80.0 (C-2), 67.6 (C-3), 29.4 (C-4).

Taxifolin (4): 1H-NMR (400 MHz, CD3OD) δ 6.96 (1H, d, J = 1.8 Hz, H-2’), 6.85 (1H, dd, J = 1.8, 8.0 Hz, H-6’), 6.80 (1H, d, J = 8.0 Hz, H-5’), 5.92 (1H, d, J = 2.1 Hz, H-6), 5.88 (1H, d, J = 2.1 Hz, H-8), 4.91 (1H, d, J = 11.5 Hz, H-2), 4.50 (1H, d, J = 11.5 Hz, H-3); 13C-NMR (100 MHz, CD3OD) δ 198.6 (C-4), 168.9 (C-5), 165.5 (C-7), 164.7 (C-9), 147.3 (C-3’), 146.5 (C-4’), 130.0 (C-1’), 121.0 (C-6’), 116.2 (C-5’), 116.0 (C-2’), 102.0 (C-10), 97.4 (C-6), 96.4 (C-8), 85.3 (C-2), 73.8 (C-3).

Taxifolin 3-O-α-L-arabinopyranoside (5): 1H-NMR (400 MHz, CD3OD) δ 6.97 (1H, d, J = 2.1 Hz, H-2’), 6.85 (1H, dd, J = 2.1, 8.2 Hz, H-6’), 6.79 (1H, d, J = 8.2 Hz, H-5’), 5.92 (1H, d, J = 2.1 Hz, H-6), 5.90 (1H, d, J = 2.1 Hz, H-8), 5.13 (1H, d, J = 10.5 Hz, H-2), 4.80 (1H, d, J = 10.5 Hz, H-3), 3.92 (1H, dd, J = 7.6, 11.7 Hz, H-5”), 3.84 (1H, d, J = 3.9 Hz, H-1”), 3.80 (1H, m, H-4”), 3.59 (1H, dd, J = 3.9, 6.0 Hz, H-2”), 3.55 (1H, dd, J = 3.2, 6.0 Hz, H-3”), 3.38 (1H, dd, J = 3.7, 11.7 Hz, H-5”); 13C-NMR (100 MHz, CD3OD) δ 196.1 (C-4), 169.6 (C-7), 165.6 (C-5), 164.4 (C-9), 147.6 (C-4’), 146.7 (C-3’), 129.1 (C-1’), 120.9 (C-6’), 116.4 (C-5’), 115.8 (C-2’), 102.4 (C-1”), 101.5 (C-10), 97.6 (C-6), 96.7 (C-8), 83.9 (C-2), 76.4 (C-3), 73.2 (C-2”), 71.2 (C-4”), 66.9 (C-3”), 63.5 (C-5”).

Taxifolin 3-O-α-L-rhamnopyranoside (6): 1H-NMR (400 MHz, CD3OD) δ 6.96 (1H, d, J = 1.8 Hz, H-2’), 6.84 (1H, dd, J = 1.8, 8.2 Hz, H-6’), 6.81 (1H, d, J = 8.2 Hz, H-5’), 5.92 (1H, d, J = 2.1 Hz, H-6), 5.90 (1H, d, J = 2.1 Hz, H-8), 5.07 (1H, d, J = 10.5 Hz, H-2), 4.57 (1H, d, J = 10.5 Hz, H-3), 4.25 (1H, m, H-5”), 4.05 (1H, d, J = 1.4 Hz, H-1”), 3.66 (1H, dd, J = 3.2, 9.6 Hz, H-4”), 3.54 (1H, dd, J = 1.6, 3.2 Hz, H-3”), 3.31 (overlapped with CD2HOD, H-2”), 1.19 (3H, d, J = 6.2 Hz, H-6”); 13C-NMR (100 MHz, CD3OD) δ 196.1 (C-4), 169.0 (C-7), 165.7 (C-5), 164.2 (C-9), 147.5 (C-4’), 146.7 (C-3’), 129.3 (C-1’), 120.6 (C-6’), 116.4 (C-5’), 115.6 (C-2’), 102.6 (C-10), 102.3 (C-1’=), 97.6 (C-6), 96.5 (C-8), 84.1 (C-2), 78.7 (C-3), 73.9 (C-4”), 72.3 (C-3”), 71.9 (C-2”), 70.6 (C-5”), 18.0(C-6”).

Proanthocyanidin A-1 (7): 1H-NMR (400 MHz, CD3OD) δ 7.13 (1H, d, J = 2.1 Hz, H-12), 7.02 (1H, dd, J = 2.1, 8.2 Hz, H-16), 6.92 (1H, d, J = 2.0 Hz, H-12’), 6.81 (3H, overlapped, H-15, 15’, 16’), 6.09 (1H, s, H-6’), 6.07 (1H, d, J = 2.3 Hz, H-8), 5.96 (1H, d, J = 2.3 Hz, H-6), 4.73 (1H, d, J = 7.8 Hz, H-2’), 4.23 (1H, d, J = 3.4 Hz, H-4), 4.15 (1H, m, H-3’), 4.07 (1H, d, J = 3.2 Hz, H-4’=), 2.94 (1H, dd, J = 5.5, 16.5 Hz, H-4’), 2.57 (1H, dd, J = 8.5, 16.5 Hz, H-4’); 13C-NMR (100 MHz, CD3OD) δ 158.3 (C-7), 156.9 (C-5), 156.3 (C-5’), 154.4 (C-9), 152.4 (C-7’), 151.6 (C-9’), 146.9 (C-13), 146.9 (C-13’), 146.5 (C-14’), 145.8 (C-14), 132.4 (C-11), 130.7 (C-11’), 120.8 (C-16’), 120.0 (C-16), 116.5 (C-15), 115.9 (C-12’), 115.8 (C-12), 115.8 (C-15’), 106.9 (C-10), 104.2 (C-8’), 103.3 (C-10’), 98.3 (C-6), 96.7 (C-8), 96.7 (C-6’), 84.7 (C-2’), 68.3 (C-3’), 68.0 (C-3), 29.4 (C-4), 29.2 (C-4’).

DPPH radical scavenging activity: Sample solutions (20 μL) of different concentrations (100, 50, 25, 12.5, 6.25 and 3.125 μg mL-1 in DMSO) were added to a 0.2 mM DPPH ethanol solution (180 μL) and allowed to react at room temperature. The absorbance values were measured after 10 min at 515 nm with a UV/Vis spectrophotometer. The DPPH radical scavenging activities of samples were calculated according to the formula:

where, Abssample is the absorbance of the experimental sample, Absblank is the absorbance of the blank, Abscontrol is the absorbance of the control. Ascorbic acid (vitamin C) was used as a positive control. Each treatment was replicated thrice.

DPPH radical scavenging activity with ESR: Test solution was prepared by mixing 10 μL of sample and 90 μL of DPPH (0.2 mM) in a methanol solution. After mixing vigorously for 10 sec, the solution was then transferred into a 100 μL Teflon capillary tube fitted into the cavity of the ESR spectrometer. The ESR spectrum was recorded 2 min after test solution preparation. ESR spectrometer parameters were set as follows: magnetic field of 337 mT, power of 1.00 mW, frequency of 9.4375 GHz, modulation amplitude of 0.8 mT, gain of 500, scan time of 0.5 min, scan width of 10 mT and time constant of 0.03 sec at room temperature (Kim et al., 2010c). Ascorbic acid was used as a positive control and each treatment was replicated thrice.

Hydroxyl radical scavenging activity: Hydroxyl radicals generated by the Fenton reaction (H2O2 plus FeSO4) were reacted with a radical spin trap, DMPO. The resulting DMPO-OH radicals were detected by using an ESR spectrometer. Briefly, ESR signaling was recorded after 2.5 min of test solution preparation by mixing of sample (10 μL) with 0.3 M DMPO (30 μL), 10 mM FeSO4 (30 μL) and 10 mM H2O2 (30 μL). ESR spectrometer parameters were set as follows: magnetic field of 337 mT, power of 1.00 mW, frequency of 9.4354 GHz, modulation amplitude of 0.2 mT, gain of 200, scan time of 0.5 min, scan width of 10 mT and time constant of 0.03 sec at room temperature (Kim et al., 2010c). Ascorbic acid was used as a positive control and each treatment was replicated thrice.

Superoxide anion radical scavenging activity: Superoxide anion radicals produced by a xanthine/xanthine oxidase system were reacted with the spin trap agent, DMPO. The generated DMPO-OOH radicals were detected using ESR spectrometry. Briefly, ESR signaling was recorded 5 min after of test solution preparation by mixing of sample (10 μL) with 1.5 M DMPO (30 μL), 5 mM xanthine (30 μL) and 0.25 U mL-1 xanthine oxidase (30 μL). The parameters of the ESR spectrometer were: magnetic field of 337 mT, power of 5.00 mW, frequency of 9.4374 GHz, modulation amplitude of 0.2 mT, gain of 700, scan time of 0.5 min, scan width of 10 mT, time constant of 0.03 sec and a temperature of 25°C (Kim et al., 2010c). The ascorbic acid was used as a positive control and each treatment was replicated thrice.

Statistical analysis: Means±SEM of the data were calculated; statistical analysis of the results was performed by Student's t-test for independent samples. Values of p<0.05 were considered significant.

RESULTS AND DISCUSSION

In the course of preliminary screenings, the ethanol extract of C. japonica stems was found to have significant radical scavenging activities. These antioxidative activities were measured against DPPH and hydroxyl radicals by using Electron Spin Resonance (ESR) spectrometry. ESR spectroscopy, whose signal intensities depend on the concentrations of free radicals, is a sensitive and specific method for the detection of radical species in chemical and biological systems. Direct measurement of radical species with ESR in the reaction mixture has advantages over traditional spectrophotometric method leading to high reliability and precision. This technique has been successfully applied for systematic studies on the evaluation of antioxidant capacity of natural extracts (Jiang et al., 2010). Upon the DPPH radical inhibition activities, the ethanol extract exhibited SC50 13.6 μg mL-1 which was comparable to ascorbic acid (SC50 4.6 μg mL-1; positive control). In addition, the extract was comparable (SC50 442.5 μg mL-1) to ascorbic acid (SC50 153.6 μg mL-1) in the hydroxyl radical inhibition tests.

As the ethanol extract exhibited considerable free radical inhibition properties, a phytochemical study was conducted in order to identify the active constituents. The extract was partitioned successively into n-hexane, ethyl acetate (EtOAc), n-butanol and water soluble fractions. The EtOAc fraction was chosen and subjected to repeated column chromatography over silica gel and Sephadex LH-20.

Fig. 1: Structures of isolated compounds 1-7 from C. japonica

Table 1: DPPH radical scavenging activities for compounds 1-7 determined by UV-VIS spectrophotometry
SC50: Concentration (μM) at which radical scavenging activity is 50%

Compounds 1-7 (Fig. 1) were isolated from these purification procedures. These isolates were identified as catechin (1) (Martin et al., 2000), catechin 3-O-α-L-rhamnopyranoside (2) (Bonefelda et al., 1986; Ishimarua et al., 1987), epicatechin (3) (Martin et al., 2000), taxifolin (4) (Han et al., 2007), taxifolin 3-O-α-L-arabinopyranoside (5) (Chosson et al., 1998), taxifolin 3-O-α-L-rhamnopyranoside (6) (Lucas-Filbh et al., 2001) and proanthocyanidin A-1 (7) (Lou et al., 1999). The chemical structures of these compounds were elucidated based on the spectroscopic data, including 1D and 2D NMR spectra, as well as comparison to those in the literature. As far as we know, all of the compounds 1-7 were isolated for the first time from the plant C. japonica.

The antioxidant properties for compounds (1-7) were examined in radical inhibition assays. DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activities can be assayed by a spectrophotometer and it forms a stable radical species with a strong absorption at 515 nm bearing purple color; its degradation by test sample can be monitored by disappearance of absorption. Using the DPPH radical inhibition assay, all isolates 1-7 showed more potent activities than the positive control ascorbic acid (SC50 44.9 μM) (Table 1). Proanthocyanidin A-1 (7) displayed the most potent activity. The disappearance of DPPH radical species can also be monitored by an ESR spectrum.

Fig. 2: DPPH and hydroxyl radical ESR spectra for the compound 7

Table 2: DPPH radical scavenging activities for compounds 1-7 determined by ESR spectrometry
SC50: Concentration (μM) at which radical scavenging activity is 50%

As shown in Table 2, this assay also showed that compound 7 was the most potent inhibitor with a SC50 of 9.4 μM, indicating better activity than ascorbic acid (SC50 23.3 μM). The ESR spectrum for compound 7 is shown in Fig. 2.

As hydroxyl radical is the most reactive chemical species among ROS, this species produces the most deleterious effect on cells in living organisms (Denisov and Afanas’ve, 2005). In the scavenging activity assays, hydroxyl radical was generated by the reaction of hydrogen peroxide and Fe2+ ion known as the Fenton reaction. The hydroxyl radical inhibition was verified using ESR by monitoring the DMPO-OH radical peak. In this experiment, the reactive hydroxyl radical was trapped by a nitrogen N-oxide, DMPO to yield a relatively stable radical DMPO-OH, which can be detected by ESR. Using this hydroxyl radical inhibition assay, compound 7 exhibited more potent activity (SC50 301.6 μM) than ascorbic acid (SC50 859.7 μM) (Table 3). Figure 2 indicated that hydroxyl radical was scavenged by compound 7 in a dose-dependent manner. The other compounds appeared to have relatively lower activities.

Superoxide anion is a ROS generated during the respiratory metabolic process in mitochondria. Even though this radical anion is not as reactive as hydroxyl radicals, it can initiate a cascade of ROS.

Table 3: Hydroxyl radical scavenging activities for compounds 1-7 determined by ESR spectrometry
SC50: Concentration (μM) at which radical scavenging activity is 50%

Table 4: Superoxide anion radical scavenging activities for compounds 1-7 determined by ESR spectrophotometry

Therefore, inhibition of the superoxide reduces ROS production. In the scavenging activity assay, superoxide was generated by the xanthine and xanthine oxidase system. At the concentration of 100 μg mL-1, compounds 1-7 showed comparable scavenging activities to ascorbic acid (Table 4).

In conclusion, phytochemical investigation of the stems of C. japonica led to the isolation of seven compounds. We demonstrated that compounds 1-7 possessed relatively potent inhibition activities against DPPH, hydroxyl and superoxide anion radicals. Natural antioxidants may be responsible for the protective effects against the risk of many physiological and pathological processes. Based on these results, it is suggested that the extract of C. japonica stems containing potent antioxidative constituents could have potential in many industrial applications.

ACKNOWLEDGMENT

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0007254).

REFERENCES
Adisa, R.A., K. Abas, I.A. Oladosu, A. Ajaz, M.I. Choudhary, O.O. Olorunsogo and A. Rahman, 2011. Purification and characterization of phenolic compounds from the leaves of Cnestis feruginea: Investigation of antioxidant activity. Res. J. Phytochem., 5: 177-189.

Bonefelda, M., H. Friedricha and H. Kolodziej, 1986. (+)-catechin 3-rhamnoside from Erythroxylum novogranatense. Phytochemistry, 25: 1205-1207.

Chanda, S., R. Dave and M. Kaneria, 2011. In vitro antioxidant property of some Indian medicinal plants. Res. J. Med. Plant, 5: 169-179.
CrossRef  |  Direct Link  |  

Chosson, E., A. Chaboud, A.J. Chulia and J. Raynaud, 1998. Dihydroflavonol glycoside from Rhododendron ferrugineum. Phytochemistry, 49: 1431-1433.
Direct Link  |  

Denisov, E.T. and I.B. Afanas've, 2005. Oxidation and antioxidants in organic chemistry and biology. Taylor and Francis, New York.

Elzaawely, A.A. and S. Tawata, 2012. Antioxidant activity of phenolic rich fraction obtained from Convolvulus arvensis L. leaves grown in Egypt. Asian J. Crop Sci., 4: 32-40.
CrossRef  |  Direct Link  |  

Gajula, D., M. Verghese, J. Boateng, L.T. Walker, L. Shackelford, S.R. Mentreddy and S. Cedric, 2009. Determination of total phenolics, flavonoids and antioxidant and chemopreventive potential of basil (Ocimum basilicum L. and Ocimum tenuiflorum L.). Int. J. Cancer Res., 5: 130-143.
CrossRef  |  Direct Link  |  

Hajimahmoodi, M., N. Sadeghi, B. Jannat, M.R. Oveisi, S. Madani and M. Kiayi, 2008. Antioxidant activity, reducing power and total phenolic content of iranian olive cultivar. J. Boil. Sci., 8: 779-783.
CrossRef  |  Direct Link  |  

Ham, Y.M., K.N. Kim, W.J. Lee, N.H. Lee and C.G. Hyun, 2010. Chemical constituents from Sargassum micracanthum and antioxidant activity. Int. J. Pharmacol., 6: 147-151.
CrossRef  |  Direct Link  |  

Han, X.H., S.S. Hong, J.S. Hwang, M.K. Lee, B.Y. Hwang and J.S. Ro, 2007. Monoamine oxidase inhibitory components from Cayratia japonica. Arch. Pharm. Res., 30: 13-17.
PubMed  |  

Hou, W.C., R.D. Lin, K.T. Cheng, Y.T. Hung and C.H. Cho et al., 2003. Free radical-scavenging activity of Taiwanese native plants. Phytomedicine, 10: 170-175.
CrossRef  |  PubMed  |  Direct Link  |  

Ishimarua, K., G.I. Nonakaa and I. Nishioka, 1987. Flavan-3-ol and procyanidin glycosides from Quercus miyagii. Phytochemistry, 26: 1167-1170.
Direct Link  |  

Jiang, L.Y., S. He, Y.J. Pan and C.R. Sun, 2010. Bioassay-guided isolation and EPR-assisted antioxidant evaluation of two valuable compounds from mango peels. Food Chem., 119: 1285-1292.

Kim, A.D., K.A. Kang, R. Zhang, C.M. Lim and Y. Jee et al., 2010. Reactive oxygen species scavenging effects of Jeju water containing vanadium components. Cancer Prev. Res., 15: 111-117.

Kim, K.N., Y.M. Ham, M.S. Yang, D.S. Kim, W.J. Lee, N.H. Lee and C.G. Hyun, 2010. Molecular mechanisms of apoptosis induced by Scytosiphon gracilis kogame in HL-60 cells. Int. J. Pharmacol., 6: 249-256.
CrossRef  |  Direct Link  |  

Kim, S.S., J.E. Kim, C.G. Hyun and N.H. Lee, 2011. Neolitsea aciculate essential oil inhibits drug-resistant skin pathogen growth and Propionibacterium acnes-induced inflammatory effects of human monocyte leukemia. Natural Prod. Commun., 6: 1193-1198.
PubMed  |  

Kim, S.S., W.J. Yoon, C.G. Hyun and N.H. Lee, 2010. Down-regulation of tyrosinase, TRP-2 and MITF expressions by Neolitsea aciculata extract in murine B16 F10 melanoma. Int. J. Pharmacol., 6: 290-295.
CrossRef  |  Direct Link  |  

Ko R.K., G.O. Kim, C.G. Hyun, D.S. Jung and N.H. Lee, 2011. Compounds with tyrosinase inhibition, elastase inhibition and DPPH radical scavenging activities from the branches of Distylium racemosum Sieb. Et Zucc. Phytotherapy Res., 25: 1451-1456.
CrossRef  |  

Lou, H., Y. Yamazaki, T. Sasaki, M. Uchida, H. Tanaka and S. Oka, 1999. A-type proanthocyanidins from peanut skins. Phytochemistry, 51: 297-308.
CrossRef  |  

Lucas-Filho, M.D., G.C. Silva, S.F. Cortes, T.R. Mares-Guia, V.P. Ferraz, C.P. Serra and F.C. Braga, 2010. ACE inhibition by astilbin isolated from Erythroxylum gonocladum (Mart.) O.E. Schulz. Phytomedicine, 17: 383-387.
CrossRef  |  Direct Link  |  

Martin, T.S., H. Kiduzaki, M. Hisamoto and N. Nakatani, 2000. Constituents of Amomum tsao-ko and their radical scavenging and antioxidant activities. J. Am. Oil Chem. Soc., 77: 667-673.
Direct Link  |  

Oboh, G. and A.O. Ademosun, 2006. Comparative studies on the ability of crude polyphenols from some nigerian citrus peels to prevent lipid peroxidation-in vitro. Asian J. Biochem., 1: 169-177.
CrossRef  |  Direct Link  |  

Pietta, P.G., 2000. Flavonoids as antioxidants. J. Nat. Prod., 63: 1035-1042.
CrossRef  |  PubMed  |  Direct Link  |  

Valko, M., D. Leibfritz, J. Moncol, M.T.D. Cronin, M. Mazur and J. Telser, 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol., 39: 44-84.
CrossRef  |  PubMed  |  Direct Link  |  

©  2019 Science Alert. All Rights Reserved
Fulltext PDF References Abstract