Antioxidant Activity and Delayed Aging Effects of Hot Water Extract

Article pubs.acs.org/JAFC

Antioxidant Activity and Delayed Aging Effects of Hot Water Extract from Chamaecyparis obtusa var. formosana Leaves Szu-Chin Cheng,† Wen-Hsuan Li,§ Yeu-Ching Shi,§ Pei-Ling Yen,† Huan-You Lin,† Vivian Hsiu-Chuan Liao,*,§ and Shang-Tzen Chang*,† †

School of Forest and Resource Conservation, National Taiwan University, Taipei 10617, Taiwan Department of Bioenvironmental Systems Engineering, National Taiwan University, Tapei 10617, Taiwan

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S Supporting Information *

ABSTRACT: The antioxidant activity and delayed aging effects of hot water extracts from leaves of Chamaecyparis obtusa var. formosana were investigated. Free radical, superoxide radical scavenging, and total phenolic content assays were employed to evaluate the in vitro activities of the extracts. In addition, in vivo assays using the nematode Caenorhabditis elegans were also performed in this study. The results showed that among all soluble fractions obtained from the extracts, the ethyl acetate-soluble fraction has the best in vitro and in vivo antioxidant activities. Moreover, it decreased significantly the deposition of lipofuscin (aging pigment) and extended the lifespan of C. elegans. Bioactivity-guided fractionation yielded six potent antioxidant constituents from the ethyl acetate-soluble fraction, namely, catechin, quercetin, quercetin-3-O-α-rhamnoyranoside, myricetin-3O-α-rhamnoyranoside, vanillic acid, and 4-hydroxybenzoic acid. Quercetin-3-O-α-rhamnoyranoside pretreatment showed the highest survival of C. elegans upon juglone exposure. Taken together, the results revealed that hot water extracts from C. obtusa var. formosana leaves have the potential to be used as a source for antioxidant or delayed aging health food. KEYWORDS: antioxidant activity, Chamaecyparis obtusa var. formosana, delayed aging effect, Caenorhabditis elegans

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INTRODUCTION Chamaecyparis obtusa var. formosana is a conifer species indigenous to Taiwan and is commonly called “Taiwan Hinoki”. It can be found in mountains at elevations from 1800 to 2600 m. Several studies have revealed that C. obtusa var. formosana exerts various biological activities, including cytotoxic, allopathic, antioxidant, and antitermite activities.1−5 However, to the best of our knowledge there is no prior report on the antioxidant activities of hot water extracts from C. obtusa var. formosana leaves. Thus, this study was the first to investigate the antioxidant compounds of C. obtusa var. formosana leaves. Oxidative stress has been linked to the development of various diseases, including inflammation, diabetes, cancers, aging, and Alzheimer’s disease.6−9 Oxidative stress reflects an imbalance between the production of free radicals and reactive metabolites, also known as oxidants or reactive oxygen species (ROS) in organisms. Intracellular ROS are considered harmful because they react with proteins, lipids, DNA, and other important biomolecules, thus leading to progressive loss of cellular integrity and functionality.10 In particular, an increase in intracellular oxidative stress has been linked to play a vital role in aging and age-related diseases.11,12 The soil nematode Caenorhabditis elegans has become a prominent model for studying stress resistance, aging, and agerelated diseases13 due to its relatively short lifespan and developmental cycle and the existence of mutants and genetic and environmental manipulations that influence its lifespan.13,14 This study evaluated the antioxidant activities of hot water extracts and their constituents from leaves of C. obtusa var. formosana both in vitro and in vivo. The nematode C. elegans was © 2014 American Chemical Society

used as an in vivo model to examine their protective potential including oxidative stress resistance and longevity. Our findings showed that a hot water extract of C. obtusa var. formosana leaves provides resistance against oxidative stress and delayed aging effects in an intact organism.

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MATERIALS AND METHODS

Plant Materials. The fresh, mature leaves of Chamaecyparis obtusa var. formosana were sampled from Mt. Chilan in Ilan County, Taiwan. The species was identified by Yen-Ray Hsui at the Taiwan Forestry Research Institute, and a voucher specimen (ChObvFoL31) was deposited in the laboratory of wood chemistry, School of Forestry and Resource Conservation, National Taiwan University. The materials were air-dried at ambient temperature (25 °C). Chemicals. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), Folin−Ciocalteu reagent, quercetin, nitroblue tetrazolium chloride (NBT), xanthine oxidase, and (+)-catechin were purchased from Sigma Chemical Co. (MO, USA). HPLC grade methanol, Na2CO3, and KH2PO4 were purchased from Merck (Darmstadt, Germany). Ethyl acetate and nbutanol were purchased from ECHO Chemical Co. (Miaoli, Taiwan). Ethanol was purchased from Shimakyu Co. (Osaka, Japan). Gallic acid, Na2EDTA, KOH, and NMR solvent were purchased from Acros (Geel, Belgium). Extraction and Identification of Compounds. Fresh leaves were added to boiling double-distilled water and allowed to infuse for 6 h. The extract was decanted, filtered under vacuum, concentrated in a rotary evaporator, and then lyophilized. The resulting crude extract (579 g) was fractionated successively with ethyl acetate, n-butanol, and Received: Revised: Accepted: Published: 4159

February 17, 2014 April 9, 2014 April 15, 2014 April 28, 2014 dx.doi.org/10.1021/jf500842v | J. Agric. Food Chem. 2014, 62, 4159−4165

Journal of Agricultural and Food Chemistry

Article

Figure 1. Structures of six phytochemicals in the ethyl acetate-soluble fraction of a hot water extract from C. obtusa var. formosana leaves: catechin (1); quercetin (2); quercetin-3-O-α-rhamnopyranoside (3); myricetin-3-O-α-rhamnopyranoside (4); vanillic acid (5); 4-hydroxybenzoic acid (6). water to yield soluble fractions of ethyl acetate (46.7 g), n-butanol (138.5 g), and water (377.3 g). The phytochemicals from the ethyl acetate-soluble fraction were separated and purified by semipreparative HPLC on a Agilent model 1100 pump equipped with a UV detector (Agilent 1100) and a 250 mm × 10.0 mm i.d., 5 μm Luna RP-18 column (Phenomenex, CA, USA). The mobile phase used was solvent A (100% methanol) and solvent B (ultrapure water). Elution conditions were 0−40 min of 25−60% A to B (linear gradient); 40−60 min of 60−100% A to B (linear gradient). ESI-MS data were collected using a Finnigan MAT-95S mass spectrometer, and NMR spectra were recorded by a Bruker Avance 500 MHz FT-NMR spectrometer. The structures of chemical compounds 1−6 (as shown in Figure 1) were identified by ESI-MS and NMR, and all spectral data were consistent with those reported in the literature.15−19 1,1-Diphenyl-2-picrylhydrazyl Assay. The DPPH radical scavenging activity of the test extracts or compounds from C. obtusa var. formosana leaves was examined according to the method previously reported.20 Briefly, 10 μL of test samples in dimethyl sulfoxide (DMSO) were mixed with 90 μL of 50 mM Tris-HCl buffer (pH 7.4) and 200 μL of 0.1 mM DPPH−ethanol solution. After 30 min of incubation at ambient temperature, the reduction of the DPPH radical was measured by reading the absorbance at 517 nm using an ELISA reader (SpectraMax 190, Molecular Devices, CA, USA). (+)-Catechin was used as a positive control. Three replicates were made for each test sample. The inhibition percentage was calculated according to the following equation:

% inhibition = [1 − (rate of sample reaction /rate of control reaction)] × 100 Total Phenolic Content Measurement. The amount of total phenolics was measured by the Folin−Ciocalteu method according to Chang et al.,20 using gallic acid as a standard, and a calibration curve was obtained with solutions of 0.08, 0.04, 0.02, 0.01, and 0.005 mg/mL of this compound. A 0.4 mL aliquot of diluted extract (all fractions were diluted with methanol to adjust the absorbance within the calibration limits), 0.4 mL of 1 mol/L Folin−Ciocalteu reagent, and 0.8 mL of Na2CO3 (20%, w/v) were mixed. After 8 min, the mixture was centrifuged at 15300g for 10 min. Then the absorbance of the supernatant solution was measured at 730 nm by using ELISA and against a blank prepared similarly but containing distilled water instead of extract. The concentration of phenolics thus obtained was multiplied by the dilution factor, and the results were expressed as the equivalent to milligrams of gallic acid per gram of extract (mg GAE/g). C. elegans Handling. The strain used in this study was Bristol N2 (wild-type), which was obtained from the Caenorhabditis Genetics Center (CGC; University of Minnesota). C. elegans was maintained and assayed at 20 °C on nematode growth medium (NGM) agar plates carrying a lawn of Escherichia coli (E. coli.) OP50. Synchronization of nematode cultures was performed by hypochlorite treatment of gravid hermaphrodites.21 C. elegans Oxidative Stress Resistance Assays. Synchronized wild-type L1 larvae were incubated in liquid S-basal medium containing E. coli OP50 bacteria at 109 cells/mL with extract or 0.1% DMSO (Wako, Saitama, Japan) as the solvent control for 72 h. Subsequently, adult nematodes were immediately subjected to the oxidative stress assay. For this assay, juglone (5-hydroxy-1,4naphthoquinone) (Sigma, MO, USA), an ROS-generating compound, was employed to induce oxidative stress in C. elegans. Extract-treated and control adult nematodes were transferred to S-basal medium containing 250 μM juglone for 2.5 h22,23 and then scored for viability. The survival of C. elegans was determined by touch-provoked movement.24 Nematodes were scored as dead when they failed to respond to repeated touching with a platinum wire pick. The test was performed independently at least three times. C. elegans Intracellular Reactive Oxygen Species Measurement. Wild-type nematodes were raised from L1 larvae as described in the oxidative stress resistance assays. Subsequently, intracellular ROS in C. elegans were measured using 2′,7′-dichlorodihydrofluoroscein diacetate (H2DCFDA) (Sigma, MO, USA). Approximately 100 nematodes were sonicated after extract treatment, and the lysates were

% inhibition = [(absorbance of control − absorbance of sample) /absorbance of control] × 100 Superoxide Radical Scavenging Assay (NBT Assay). Measurement of superoxide radical scavenging activity was carried out according to the method of Chang et al.20 Briefly, 20 μL of 15 mM Na2EDTA in buffer (50 mM KH2PO4/KOH, pH 7.4), 50 μL of 0.6 mM nitroblue tetrazolium chloride in buffer, 30 μL of 3 mM hypoxanthine in 50 mM KOH, 5 μL of the test extracts or compounds in methanol, and 145 μL of buffer were mixed in 96-well microplates. The reaction was started by adding 50 μL of xanthine oxidase in buffer (1 unit in 10 mL of buffer) to the mixture. The reaction mixture was incubated at ambient temperature, and the absorbance at 570 nm was determined every 1 min up to 8 min using the ELISA reader. (+)-Catechin and quercetin, well-known antioxidants, were used as positive controls. Three replicates were made for each test sample. The percent inhibition ratio was calculated according to the following equation: 4160

dx.doi.org/10.1021/jf500842v | J. Agric. Food Chem. 2014, 62, 4159−4165


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ability of the hot water extract is 8.5 ± 0.2 μg/mL, and the IC50 values of three fractions increased as follows: ethyl acetate (6.3 ± 0.2 μg/mL), n-butanol (7.2 ± 0.1 μg/mL), and water (30.2 ± 3.1 μg/mL). In addition, the scavenging ability of superoxide, the most common radical in our organisms, was also tested. Their IC50 values decreased in the order ethyl acetate-soluble fraction (1.6 ± 0.0 μg/mL), crude extract (2.6 ± 0.2 μg/mL), n-butanol-soluble fraction (3.1 ± 0.1 μg/mL), and watersoluble fraction (8.4 ± 0.1 μg/mL). These results demonstrated that the hot water extract has potential as an antioxidant source. Among all soluble fractions, the ethyl acetate-soluble fraction has the best inhibitory effect on free radical scavenging ability. Total Phenolic Contents of a Hot Water Extract from C. obtusa var. formosana Leaves and Its Soluble Fractions. Phenolic compounds are well-known radical scavengers in the plant kingdom, and they have been proved to have various biological effects.29−32 Table 1 shows the total phenolic content of the crude extract and its derived fractions calculated as gallic acid equivalent (GAE) in milligrams per gram of sample. Accordingly, the total phenolic content of the ethyl acetate-soluble fraction (649.8 ± 9.6 mg GAE/g) was significantly higher than that of the n-butanol-soluble fraction (368.2 ± 12.1 mg GAE/g), crude extracts (282.4 ± 7.3 mg GAE/g), and water fraction (147.7 ± 5.2 mg GAE/g). These results indicated that the total phenolic content of the hot water extract from C. obtusa var. formosana leaves could be effectively enriched in the ethyl acetate-soluble fraction. Enhanced Oxidative Stress Resistance of Wild-Type C. elegans by a Hot Water Extract from C. obtusa var. formosana Leaves. According to the above-mentioned results, the hot water extract of C. obtusa var. formosana leaves, especially the ethyl acetate-soluble fraction, has an excellent in vitro antioxidant activity. To further investigate the antioxidant activity of the hot water extract and different fractions in living organisms, the C. elegans model was employed. Wild-type L1 larvae were pretreated with the hot water extracts (2 and 20 μg/mL) and 0.1% DMSO as the solvent control for 72 h before immediate exposure to juglone (250 μM), a redox cycler that generates intracellular oxidative stress, and then incubated for 2.5 h.22,23 During pretreatment with the extracts, no adverse effects on the worms including survival, growth rate, and morphological changes were observed (data not shown). Pretreatment with 2 and 20 μg/mL extracts enhanced the worms’ resistance to juglone-induced oxidative stress (survival of 2 μg/mL- and 20 μg/mL-treated worms was 69.3 ± 6.0% and 88.1 ± 3.2%, respectively). Since as low as 2 μg/mL extract was able to significantly defend against the juglone-induced oxidative stress, 2 μg/mL extract was chosen as the working concentration for further experiments. Different soluble fractions derived from hot water extracts were further examined. As shown in Figure 2, both the hot water crude extract and ethyl acetate-soluble fraction increased significantly the survival of C. elegans compared with the control (0.1% DMSO). In addition, the ethyl acetate-soluble fraction exhibited higher survival against juglone-induced oxidative stress (Figure 2). The increase in the worms’ survival may be attributed to the chemical components in extracts with abilities for scavenging free radicals to reduce the juglone-induced oxidative stress in C. elegans, which was in good agreement with the in vitro antioxidant data (Table 1). Influence of Hot Water Extracts from C. obtusa var. formosana Leaves on Intracellular ROS Levels in C.

then collected for ROS measurement.23,25,26 Worm samples were incubated with H2DCFDA (at a final concentration of 50 μM in phosphate-buffered saline) in an FLx800 microplate fluorescent reader (Bio-Tek Instruments, VT, USA) for quantification of fluorescence with excitation at 485 nm and emission at 530 nm. Samples were read after 3 h. C. elegans Lifespan Assays. C. elegans lifespan analyses were performed in the same manner for all treatments at 20 °C. Synchronized L1 larvae were transferred to NGM plates in the absence or presence of 2 or 20 μg/mL ethyl acetate-soluble fraction, and C. elegans was allowed to develop to adulthood. Surviving and dead animals were counted daily (starting on the first day of adulthood) until all nematodes died. Animals that did not move when gently prodded (with a platinum wire) were scored as dead. Nematodes suffering from internal hatch (a defect in egg laying) and those that crawled off the NGM plate were not included in the lifespan counts. During the reproductive period, adult nematodes were transferred to fresh NGM plates every day during the progeny production period and then every other day thereafter. To avoid antibacterial effects of extract, lifespan assays were conducted with UVkilled E. coli OP50 according to Gruber et al.27 The lifespan assays were performed at least three times. C. elegans Intracellular Lipofuscin Assays. Wild-type animals were raised from L1 larvae as in the lifespan assays. On the fourth and eighth days of adulthood, the intestinal autofluorescence of lipofuscin was analyzed according to Liao et al.28 Randomly selected worms from each set of experiments were mounted onto microscope slides coated with 3% agarose, anesthetized with 2% sodium azide, and capped with coverslips. The autofluorescence of lipofuscin was captured with a Leica epifluorescence microscope (Leica, Wetzlar, Germany) using the DAPI filter set (with excitation at 340−380 nm and emission at 435− 485 nm) and a cooled charge-coupled device camera. The lipofuscin level for each worm was quantified by determining the average pixel intensity in each animal’s intestine using Image-Pro Plus software (Media Cybernetics, MD, USA). Statistical Analyses. For the lifespan assays, survival of C. elegans was plotted using Kaplan−Meier survival curves and analyzed by the log-rank test using GraphPad Prism (GraphPad Software, Inc., CA, USA). Other data were expressed as mean ± SE (n = 3). The significance of difference was calculated by Scheffe’s test, and values of p < 0.05 were considered significant.

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RESULTS AND DISCUSSION In Vitro Antioxidant Activities of a Hot Water Extract from C. obtusa var. formosana Leaves. Strong activity in scavenging DPPH and superoxide radical of hot water extracts is shown in Table 1. The DPPH free radical inhibitory (IC50)

Table 1. DPPH Radical and Superoxide Radical Scavenging Activity and Total Phenolic Contents of a Hot Water Extract from C. obtusa var. formosana Leaves and Its Soluble Fractionsa DPPH

NBT

TPC

specimen

IC50 (μg/mL)

IC50 (μg/mL)

(mg GAE/g)

HWE EAF BF WF

8.5 ± 0.2 b 6.3 ± 0.2 c 7.2 ± 0.1 c 30.2 ± 3.1 a

2.6 ± 0.2 b 1.6 ± 0.0 c 3.1 ± 0.1 b 8.4 ± 0.1 a

282.4 ± 7.3 c 649.8 ± 9.6 a 368.2 ± 12.1 b 147.7 ± 5.2 d

a HWE: hot water extract; EAF: ethyl acetate-soluble fraction; BF: nbutanol-soluble fraction; WF: water-soluble fraction. DPPH: DPPH free radical scavenging effect; NBT: superoxide radical scavenging assay; TPC: total phenolic content. Different letters (a−d) are significantly different at p < 0.05 according to Scheffe’s test (mean ± SE, n = 3). The IC50 value of catechin is 2.9 μg/mL (DPPH) and 2.7 μg/mL (NBT), respectively.

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acetate-soluble fraction was selected to evaluate the ability of lifespan extension using the C. elegans model. Adult wild-type worms grown under our standard laboratory conditions at 20 °C (feeding with UV-killed bacteria) have a mean lifespan of 20.0 ± 0.5 days with a maximum lifespan of 35.0 ± 0.9 days (the age at death of the oldest animals) (Figure 4). Ethyl

Figure 2. Survival of C. elegans (wild-type) treated with 2 μg/mL hot water extract of C. obtusa var. formosana leaves and its soluble fractions under oxidative stress induced by juglone. HWE: hot water extract; EAF: ethyl acetate-soluble fraction; BF: n-butanol-soluble fraction; WF: water-soluble fraction. Different letters (a, b) are significantly different at α = 0.05 according to Scheffe’s test (mean ± SE, n = 3).

elegans. After pretreatment with extracts, the intracellular ROS levels in C. elegans were then examined. Nonfluorescent DCF-DA is a freely cell-permeable dye, which is readily converted to the fluorescent 2′,7′-dichlorofluorescein (DCF) due to an interaction with intracellular H2O2. As shown in Figure 3, the fluorescence intensities of the control and hot

Figure 4. Effects of the ethyl acetate-soluble fraction of a hot water extract from C. obtusa var. formosana leaves on C. elegans (wild-type) lifespan. Survival curve’s p value (compared with the control by logrank test) of EAF 2 and 20 μg/mL are 0.0035 and 0.0008, respectively. EAF: ethyl acetate-soluble fraction.

acetate soluble fraction-treated worms had a statistically significant increased lifespan compared with untreated ones (log-rank test p = 0.0035 and 0.0008 for 2 and 20 μg/mL, respectively, untreated vs treated) (Figure 4, Table 2). This result suggests that the ethyl acetate-soluble fraction of a hot water extract from C. obtusa var. formosana has a beneficial effect against aging for organisms. Table 2. Effects of Ethyl Acetate Soluble Fraction of a Hot Water Extract from C. obtusa var. formosana Leaves on C. elegans (Wild-Type) Lifespan Valuesa Figure 3. Effects of 2 μg/mL hot water extract from C. obtusa var. formosana leaves and its soluble fractions on ROS accumulation. Results are expressed as relative fluorescence units (RFU), fluorescence relative to total proteins in worm lysates. HWE: hot water extract; EAF: ethyl acetate-soluble fraction; BF: n-butanolsoluble fraction; WF: water-soluble fraction. Different letters (a−c) are significantly different at α = 0.05 according to Scheffe’s test (mean ± SE, n = 3).

conc (μg/mL) lifespan

control

2

20

mean maximum log-rank test

20.0 ± 0.5 35.0 ± 0.9

21.5 ± 0.4 38.0 ± 0.6 p = 0.0035**

24.0 ± 0.6 41.0 ± 1.1 p = 0.0008***

a Results are expressed as relative fluorescence units (RFU), fluorescence relative to total proteins in worm lysates.

water crude extract were 36.9 ± 1.1 and 29.7 ± 2.3 units, respectively, revealing that the hot water crude extract significantly decreased the intracellular ROS in wild-type L1 larvae pretreated at 2 μg/mL. Among three subfractions of crude extract, the ethyl acetate-soluble fraction showed a significant decrease in the fluorescence intensity (26.2 ± 1.5 units) compared with the control and hot water crude extract. These results demonstrated that the crude extract and its ethyl acetate-soluble fraction may act against oxidative stress due to its ROS-scavenging ability. Effects of Ethyl Acetate-Soluble Fraction of a Hot Water Extract from C. obtusa var. formosana Leaves on Lifespan Extension of C. elegans. The ethyl acetate-soluble fraction of a hot water extract from C. obtusa var. formosana leaves not only had the highest phenolic contents but also exhibited the best antioxidant activity in both in vitro and in vivo antioxidant tests (Table 1, Figure 2). Accordingly, the ethyl

Effects of Ethyl Acetate-Soluble Fraction of a Hot Water Extract from C. obtusa var. formosana Leaves on Lipofuscin Levels in C. elegans. Lipofuscin (aging pigments) levels in the intestines of the C. elegans were examined, as lipofuscin is an endogenous intestinal autofluorescent material that accumulates in aged C. elegans. The lipofuscin levels in the intestines of control and ethyl acetate soluble fraction-treated worms on the fourth and eighth days of adulthood were measured. On the fourth day of adulthood, the intestinal lipofuscin levels of ethyl acetate soluble fraction-treated worms were similar to those of the control (Figure 5). On the eighth day of adulthood, there was a significant decrease in intestinal lipofuscin level between the control (12.4 ± 0.6 unit) and ethyl acetate soluble fraction-treated worms (2 and 20 μg/mL: 9.7 ± 0.2 units and 9.1 ± 0.6 units). The results suggest that the ethyl acetate-soluble fraction of the hot water extract from C. obtusa 4162



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Table 3. Contents and Antioxidant Activities of Six Phytochemicals from the Ethyl Acetate-Soluble Fraction of Hot Water Extracts from C. obtusa var. formosana Leavesa phytochemical catechin (1) quercetin (2) quercetin-3-O-αrhamnopyranoside (3) myricetin-3-O-αrhamnopyranoside (4) vanillic acid (5) 4-hydroxybenzoic acid (6) control (0.1% DMSO)

Figure 5. Effects of the ethyl acetate-soluble fraction of a hot water extract from C. obtusa var. formosana leaves on the lipofuscin accumulation of C. elegans (wild-type). Results are expressed as relative fluorescence units (RFU), fluorescence relative to total proteins in worm lysates. Different letters (a, A, B) are significantly different at α = 0.05 according to Scheffe’s test (mean ± SE, n = 3). EAF: ethyl acetate-soluble fraction.

contents (mg/ g of crude)

IC50 (μM) DPPH radical

C. elegansb survival (%)

5.89 ± 0.1 4.17 ± 0.1 17.70 ± 0.6

10.6 ± 0.2 a 6.6 ± 0.4 d 8.5 ± 0.1 b

61.7 ± 1.0 d 77.3 ± 0.8 b 90.5 ± 0.5 a

6.79 ± 0.2

7.2 ± 0.2 c

70.1 ± 0.7 c

2.34 ± 0.3 1.21 ± 0.0

>50 >50

53.4 ± 0.7 e 54.3 ± 0.6 e 48.3 ± 0.5 f

Different letters (a−f) are significantly different at α = 0.05 according to Scheffe’s test (mean ± SE, n = 3). bConcentration of sample is 10 μM and observed after 2.5 h of 250 μM juglone exposure. a

of C. obtusa var. formosana leaves might play a key role in the observed oxidative stress resistance. Additionally, quercetin (2) is a well-known antioxidant in both the in vitro assay and the C. elegans model.35,36 It is noted that compound 3 is quercetin with a rhamnopyranoside group at the 3-position. This study found that the antioxidant activity in the C. elegans model was enhanced when quercetin contains a sugar. The antioxidant effect may be attributed to the moiety promoted by the rhamnopyranoside that can facilitate flavonoid absorption as described in the animal model.37 In conclusion, results obtained from this study revealed that hot water extracts from C. obtusa var. formosana leaves had excellent in vitro antioxidant activities via free radical and superoxide radical scavenging and total phenolic content assays. Furthermore, the hot water extracts promoted the resistance of oxidative stress in C. elegans. Among all soluble fractions obtained from hot water extracts, the ethyl acetate-soluble fraction exhibited the best antioxidant activities in both in vitro and C. elegans models. Moreover, it delayed the aging phenomenon (reduction of lipofuscin deposition and lifespan extension). Moreover, six potent antioxidant constituents, namely, catechin, quercetin, quercetin-3-O-α-rhamnopyranoside, myricetin-3-O-α-rhamnopyranoside, vanillic acid, and 4hydroxybenzoic acid, were identified from the ethyl acetatesoluble fraction by bioactivity-guided fractionation. Among them, quercetin-3-O-α-rhamnopyranoside exhibited the best in vivo antioxidant activity and was of the highest content in hot water extracts, suggesting that it might play the key role in the observed antioxidant activity of extracts. The results in the present study indicated that hot water extracts from C. obtusa var. formosana leaves have the potential to be used as a source for an antioxidant or delayed aging health food and warrants further investigations.

var. formosana leaves could delay aging in C. elegans by inhibiting lipofuscin accumulation. Constituents of Ethyl Acetate-Soluble Fraction of a Hot Water Extract from C. obtusa var. formosana Leaves and Their Antioxidant Activities. Constituents from the ethyl acetate-soluble fraction that might contribute to the observed oxidative stress resistance were then determined. By following the bioactivity-guided fractionation procedure, phytochemicals in the ethyl acetate-soluble fraction of a hot water extract from C. obtusa var. formosana leaves were isolated using semipreparative high-performance liquid chromatography (HPLC). NMR spectra and mass spectroscopy were employed to identify six constituents (Figure 1): catechin (1), quercetin (2), quercetin-3-O-α-rhamnopyranoside (3), myricetin-3-O-αrhamnopyranoside (4), vanillic acid (5), and 4-hydroxybenzoic acid (6). The highest content of phytochemicals in C. obtusa var. formosana leaves was 3 (17.70 ± 0.6 mg/g of crude extracts), followed by 4 (6.79 ± 0.2 mg/g of crude extracts) > 1 (5.89 ± 0.1 mg/g of crude extracts) > 2 (4.17 ± 0.1 mg/g of crude extracts) > 5 (2.34 ± 0.3 mg/g of crude extracts) > 6 (1.21 ± 0.0 mg/g of crude extracts). In vitro and in vivo antioxidant activities of each chemical were investigated. In the DPPH free radical scavenging assay, 2 had the lowest IC50 value (6.6 ± 0.4 μM), followed by 4 < 3 < 1, with IC50 values of 7.2 ± 0.2, 8.5 ± 0.1, and 10.6 ± 0.2 μM, respectively. Compounds 5 and 6 did not have significant inhibitory effects on scavenging DPPH free radicals, and their IC50 values were higher than 50 μM (Table 3). Furthermore, in the C. elegans oxidative stress resistance assay, the antioxidant activities of phytochemicals can be ranked as 3 > 2 > 4 > 1 > 5 = 6 (Table 3). All compounds exhibited higher survival than that of the control (48.3 ± 0.5%). Previous studies had reported that 1 and 2 were excellent antioxidant agents in C. elegans.33,34 It is interesting to note that 5 and 6 did not show significant in vitro antioxidant activity, whereas they showed a beneficial antioxidant effect in the C. elegans model. Among the six compounds derived from C. obtusa var. formosana leaves, quercetin-3-O-α-rhamnopyranoside (3) has the highest content and was the most effective antioxidant in the C. elegans model. This finding suggests that quercetin-3-Oα-rhamnopyranoside (3) from the ethyl acetate-soluble fraction

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(V. H.-C. Liao) Phone: +886-2-33665239. Fax: +886-233663462. E-mail: [email protected]. 4163



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*(S.-T. Chang) Phone: +886-2-33664626. Fax: +886-223654520. E-mail: [email protected].

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Prof. B. T. Guan and Prof. S. T. Lin for providing samples and all members of the CHEMTREE laboratory for technical assistance. We also thank Shou-Ling Huang (Department of Chemistry, National Taiwan University) for NMR spectral analyses and Dr. T. F. Yeh for his constructive and valuable comments.

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