Abstract
Antiradical and antioxidant activities of ethanolic extract and its fractions from Calocedrus formosana Florin heartwood were investigated, and active compounds were identified by spectral analyses. Among extract and three fractions, the ethyl acetate soluble fraction had a high total phenolic content and exhibited the desirable reducing power, antiradical activity, and antioxidant activity. The compounds haplomyrfolin, O-ethyl-α-conidendral, matairesinol, 7,8-dehydro-4-O-methyl-thujaplicatin, and 5-methoxy-isosalicifoline were isolated from antioxidative activity guided fractionation of the ethyl acetate soluble fraction. Haplomyrfolin and O-ethyl-α-conidendral were for the first time isolated and identified from C. formosana. Matairesinol and 5-methoxy-isosalicifoline exhibited superior DPPH radical scavenging ability and antioxidant activity, as evaluated by TEAC assay. Results obtained in the present study revealed that ethyl acetate soluble fraction from C. formosana heartwood extract and its active compounds, matairesinol and 5-methoxy-isosalicifoline, exhibit antioxidant potential as natural nutraceuticals.
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Antioxidative Lignans from Phytochemical Extract of Calocedrus formosana Florin
Pei-Ling Yen, a Chi-Lin Wu, a Shang-Tzen Chang, a Shou-Ling Huang, b and Hui-Ting Chang a,*
Antiradical and antioxidant activities of ethanolic extract and its fractions from Calocedrus formosana Florin heartwood were investigated, and active compounds were identified by spectral analyses. Among extract and three fractions, the ethyl acetate soluble fraction had a high total phenolic content and exhibited the desirable reducing power, antiradical activity, and antioxidant activity. The compounds haplomyrfolin, O-ethyl-α-conidendral, matairesinol, 7,8-dehydro-4-O-methyl-thujaplicatin, and 5-methoxy-isosalicifoline were isolated from antioxidative activity guided fractionation of the ethyl acetate soluble fraction. Haplomyrfolin and O-ethyl-α-conidendral were for the first time isolated and identified from C. formosana. Matairesinol and 5-methoxy-isosalicifoline exhibited superior DPPH radical scavenging ability and antioxidant activity, as evaluated by TEAC assay. Results obtained in the present study revealed that ethyl acetate soluble fraction from C. formosana heartwood extract and its active compounds, matairesinol and 5-methoxy-isosalicifoline, exhibit antioxidant potential as natural nutraceuticals.
Keywords: Calocedrus formosana; Antioxidant activity; Lignan; Matairesinol; 5-Methoxy-isosalicifoline
Contact information: a: School of Forestry and Resource Conservation, National Taiwan University, No. 1 Section 4, Roosevelt Road, Taipei 106, Taiwan; b: Instrumentation Center, National Taiwan University, No. 1 Section 4, Roosevelt Road, Taipei 106, Taiwan. * Corresponding author: chtchang@ntu.edu.tw
INTRODUCTION
Natural products present in numerous plants are of great potential as nutraceuticals or pharmaceuticals (Clark 1996; Chan et al. 2007; Mittal et al. 2009; Garzón et al. 2010). Kuti and Konuru (2004) reported that antioxidant activity of leaf extract from Cnidoscolus spp. were highly correlated with total phenolic contents, and Cnidoscolus spp. leaves are recommended to be the source of natural antioxidant supplements. Many studies also have revealed that phenolic constituents such as flavonoids, lignans, etc., from higher plants possess marvelous antiradical and antioxidant activities (Sultana et al. 2007; Singh et al. 2009). Leaf extract (EGb 761) from Ginkgo biloba contains bioactive constituents, mainly flavonoids and terpenoids, and is one of the famous commercial complementary nutraceuticals with antiradical, antioxidant, antiplatelet, and anti-aging effects (Chan et al. 2007). Using natural nutraceuticals and alternative medicines to benefit human health is an increasing trend throughout the globe (Holst and Williamson 2008; Joseph et al. 2009).
Species of Calocedrus belonging to Cupressaceae are living fossil plants and are distributed in eastern Asia and western North America; only four species are recognized in this genus (Chen et al. 2009). Calocedrus formosana (Taiwan incense cedar) is one of the precious endemic woods in Taiwan. Ichikawa (1932) first reported that the main constituents of C. formosana heartwood oil were acidic components. Part constituents, including terpenoids, lignans, and steroids, have been isolated and identified from C. formosana wood, bark, and leaf (Lin et al. 1956; Kuo et al. 1975; Fang et al. 1985, 1990; Chiang et al. 2003; Hsieh et al. 2006). Bioactivities of natural products from C. formosana were reported in recent years, including antitumor, antioxidant, anti-inflammatory, antibacterial, antitermitic, and antifungal activities (Chao et al. 2005; Cheng et al. 2004; Chiang et al. 2003; Wang et al. 2004; Hsieh et al. 2006).
In the present study, the content of antioxidants of ethanolic extract from C. formosana heartwood and its fractions were analyzed, and the antiradical and antioxidant activities of ethanolic extract and its fractions were evaluated. In addition, the antioxi-dative lignans were isolated and identified from active fraction by chromatographic and spectroscopic methods.
EXPERIMENTAL
Plant Materials and Extraction
The heartwood of Calocedrus formosana Florin was collected from 36-year-old trees at the Experimental Forest of National Taiwan University (located in Nan-Tou county, Taiwan). The species was identified, and a voucher specimen (CF92) was deposited in National Taiwan University. Specimens were extracted twice with 95% ethanol at ambient temperature. Then, the crude extract (200 g) was further separated into an n-hexane-soluble fraction (HEXF, 42.5%), an ethyl acetate-soluble fraction (EAF, 41.9%), and a water-soluble fraction (WF, 12.4%) by liquid-liquid partition. Antioxidant activities of three fractions were evaluated to screen the active one for further separation. The active fraction, EAF, was coated onto silica gel (Silica-60, Merck 7734); the eluted solvents were n-hexane, ethyl acetate, and ethanol with different proportions. EAF (43.7 g) was separated into 11 subfractions by open column chromatography (CC) and thin layer chromatography (TLC). In order to obtain pure active compounds, high performance liquid chromatography (HPLC) was used to isolate the compounds from the active subfractions based on an activity-guided fractionation procedure.
HPLC Analysis
Active subfractions were analyzed by HPLC (Hitachi L-2130) fitted with a C18 column (Phenomenex, Luna 5u, 250 x 10 mm). The gradient mobile phase consisted of water (A) and acetonitrile (B). The flow rate was 3 mL/min. The elution program involved a linear gradient from 40% B in A for 0 to 15 min, 40 to 55% B in A by 35 min, and 55 to 100% B in A by 30 min followed by 5 min of equilibrium with 100% B. The elution profile was detected by UV detector at 254 nm. Isolated and purified compounds (ca. 10 to 25 mg) were obtained for spectral analyses and antioxidant assays.
Spectral Analyses
Structures of compounds were identified by spectroscopic methods including NMR (Nuclear magnetic resonance spectroscopy, Bruker Avanace 500 MHz), FTIR (Fourier transform infrared spectroscopy, Bio-rad FTS-40), UV/VIS (Ultraviolet and visible absorption spectroscopy, Jasco V-550), and MS (Mass spectroscopy, Finnigan MAT-958).
Determination of Total Phenolics Content
The total phenolics contents in tested specimens were determined according to the Folin-Ciocalteu procedure and expressed as milligrams per gram gallic acid equivalents (GAE). The Folin-Ciocalteu phenol reagent (0.5 mL) was mixed with 0.5 mL of a diluted sample solution. After an interval of 5 min, 1 mL of 10% aqueous sodium carbonate was added to the vial, and the mixture was allowed to stand for 8 min at ambient temperature; then the mixture was centrifuged at 12,000 rpm for 10 minutes. The absorbance of the solution was read at 730 nm. The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram sample. The R-squared value of the calibration curve of gallic acid (y = 27.637x – 0.1362) was 0.9962.
DPPH Free Radical Scavenging Assay
Different concentrations of specimens (0.5 mL) were added to an ethanol solution of 1 mL of 0.15 mM DPPH (1,1-diphenyl-2-picrylhydrazyl) in test tubes. The mixture was shaken and left to stand for 30 min at room temperature. The absorbance of the solution was then measured at 517 nm with a spectrophotometer (Hitachi U-3900). IC50 values are the concentrations of compounds required for a 50% decrease of the DPPH radicals (Bamoniri et al. 2010).
Reducing Power Assay
The reducing power of specimen was determined according to the method of Kapoor et al. (2009). Specimen (0.5 mL) was mixed with phosphate buffer (0.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (0.5 mL, 1%). The mixture was incubated at 50°C for 20 min. After cooling by ice water, 0.5 mL trichloroacetic acid (10%) was added to the mixture, and then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (0.5 mL) was mixed with distilled 0.5 mL water and 0.1 mL FeCl3 (0.1%). The absorbance of the solution was measured at 700 nm.
Total Antioxidant Activity
The ability of the specimen to scavenge ABTS·+ radical cation was compared to trolox standard (Garzón et al. 2010). The ABTS·+ radical cation was generated by mixing 7 mM ABTS·+stock solution with 2.45 mM potassium persulfate and incubating for 12 to 16 h in the dark at room temperature until the reaction was complete and the absorbance was stable.
The absorbance of the ABTS·+ solution was diluted to 0.70 (± 0.02) with water at room temperature, then 1.485 mL was mixed with 15 μL of the specimens, and the absorbance was measured at 734 nm after 6 min. All experiments were repeated three times. The percentage inhibition of absorbance was calculated and plotted as a function of the concentration of standard and specimen to determine the trolox equivalent antioxidant concentration (TEAC).
Statistical Analyses
Mean values (n ≥ 3) and standard deviations of specimens examined were calculated. The data were grouped by Scheffe’s test of the SAS system (at a level of significance of p < 0.05).
RESULTS AND DISCUSSION
Plant extracts with high phenolic phytochemicals exhibit superior antioxidant potency as the sources of nutraceuticals (Sultana et al. 2007; Enayat and Banerjee 2009). The total phenolic contents in the heartwood extract and its fractions of C. formosana were expressed as gallic acid equivalents (GAE) as shown in Table 1. Total phenolic contents of the specimens fell within the range of 161.4 to 238.5 mg of GAE/g, except for the n-hexane soluble fraction (HEXF, 33.3 mg of GAE/g). Heartwood extract (187.1 mg of GAE/g) and ethyl acetate soluble fraction (EAF, 238.5 mg of GAE/g) contained more phenolic constituents.
Table 1. Antioxidant Content and Antioxidative Activities of Ethanolic Extract of Calocedrus formosana Heartwood and Its Fractions