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Antioxidant potency of phenolic phytochemicals from the root extract of Acacia confusa
Industrial Crops and Products 49 (2013) 871–878
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Antioxidant potency of phenolic phytochemicals from the root extract of Acacia confusa H.Y. Lin, S.T. Chang ∗ School of Forestry and Resource Conservation, National Taiwan University, Taipei 106, Taiwan
a r t i c l e
i n f o
Article history: Received 15 April 2013 Received in revised form 23 June 2013 Accepted 2 July 2013 Keywords: Acacia confusa Antioxidant activity Flavonoids Melacacidin Okanin Phenolics
a b s t r a c t In the present study, the antioxidant potencies of ethanolic extract and its derived phytochemicals from the root of Acacia confusa, the indigenous Taiwanese species, were investigated and reported for the first time. Among all the soluble fractions of ethanolic extract, the EtOAc soluble fraction exhibited the best antioxidant activity. The bioassay-guided fractionation was carried out and yielded 9 potent antioxidative phytochemicals from EtOAc fraction. Among them, okanin (9) exhibited the lowest IC50 DPPH radical and superoxide anion radical scavenging activity values and the highest TEAC and reducing power activity. Besides, melacacidin (3) was the most abundant antioxidant compound in A. confusa root extract with an absolute content of 109.1 mg/g of crude extract. The structure–activity relationships of antioxidant flavonoids isolated from A. confusa root extract were also discussed. Therefore, with its excellent antioxidant activities, A. confusa root extract has great potential as a source for natural health products. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Reactive oxygen species (ROS) are the endogenous free radicals of normal cellular metabolism. In living organisms, moderate concentrations of ROS benefit physiological functions such as intracellular signaling, cellular defense against infective agents, and induction of a mitogenic response. However, overproduction of ROS can cause cellular lipid peroxidation, protein degradation, and DNA mutation, resulting in cancer, cardiovascular diseases, diabetes, neurodegenerative diseases, inflammations, and aging (Valko et al., 2007). Recently, numerous studies have focused on the phenolic compounds from plant sources to serve as antioxidants against various diseases induced by free radicals. Among the various medicinal and dietary plants, endemic species are of particular interest because of their ability to produce phytochemicals with significant antioxidant activities and health benefits. Generally, phenolic compounds display their antioxidant properties through the roles of electron donors, hydrogen donors, and free radical scavengers to neutralize or quench the ROS (Yizhong et al., 2004). Nowadays, synthetic antioxidants, such as BHT and BHA, are widely used to improve shelf life and safety for food preservation. However, several studies have shown synthetic antioxidants may be involved in latent carcinogenesis, leading to efforts to replace the synthetic antioxidants with natural antioxidants (Jennings and Akoh, 2009). Besides, although natural antioxidants possess weaker abilities and
∗ Corresponding author. Tel.: +886 2 3366 4626; fax: +886 2 2365 4520. E-mail address: [email protected] (S.T. Chang).
cost more than synthetic ones, they are extensively accepted by consumers and health officials with the labels of potentially positive effects and flavorings of sensory properties. Acacia confusa Merr. (Leguminosae), an indigenous species of Taiwan widely distributed over the hills and lowlands, has traditionally been used as a folk medicine (Wu et al., 2005). This plant is an important source for feedstock, charcoal-making, and construction materials. Moreover, the particularly significant of this plant is the conservation of soil and water as well as remarkable carbon sequestration (Chang et al., 2001; Wang, 2011). However, utilization of this plant has declined gradually as emergence of the novel construction materials. The aqueous extract of A. confusa leaves was used for wound healing and anti-blood-stasis in Taiwan (Kan, 1978). Recent studies have demonstrated the crude extracts of heartwood, bark, leaves, branch, twigs, and flowers of A. confusa exhibit strong antioxidative effects, and contain a wide variety of phenolic compounds (Chang et al., 2001; Tung et al., 2009a,c, 2011; Wu et al., 2005, 2008; Hsieh and Chang, 2010). Moreover, some of these phenolic compounds revealed impressive bioactivities. The bark extract and its active constituent gallic acid exhibit potent hepatoprotection against CCl4 -induced liver damages in rats due to the modulation of antioxidant enzymes activities and inhibition of lipid peroxidation and cytochrome P4502E1 protein expression (Tung et al., 2009b). Okanin and melanoxetin isolated from the heartwood show excellent inhibition of xanthine oxidase in noncompetitive and competitive mode, respectively, and their inhibitory activity was better than that of allopurinol (Tung and Chang, 2010). Further, melanoxetin reduced mice serum uric acid levels against the potassium oxanate-induced hyperuricemic effect (Tung et al., 2010).
0926-6690/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.07.001
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Luteolin isolated from the branch bark also revealed good inhibitory effect on xanthine oxidase (Hsieh and Chang, 2010). These findings let us to be interested in investigating the bioactivity of the phenolic compounds from A. confusa root extract, and to the best of our knowledge its potential health benefits have never been studied. The aims of this study were to evaluate the antioxidant activities of A. confusa root extract and its derived fractions, followed by isolating the major active compounds. Additionally, the structureactivity relationships of the antioxidant flavonoids isolated from A. confusa root extract were also discussed. The results obtained from this study can be utilized as basic information for further applications of A. confusa root extract, such as antioxidant additives for commercial food products, antioxidant dietary supplements, and medicines of illnesses induced by oxidative stress.
linear gradients elution from 10/90 to 40/60 (MeOH/water) during 45 min. The 2,3-trans-3,7,8,3 ,4 -pentahydroxydihydroflavone (5), and 2,3-cis-3,7,8,3 ,4 -pentahydroxydihydroflavone (6) were isolated from EA5 subfraction by the isocratic mobile phase with MeOH/water = 20/80. Melanoxetin (7), transilitin (8), and okanin (9) were isolated from EA16, EA17, and EA18 by the isocratic mobile phase with MeOH/water = 35/65, 40/60, and 45/55, respectively. NMR spectra were recorded by a Bruker Avance 500 MHz FT-NMR spectrometer, and ESI-MS data were collected using a Finnigan MAT-95S mass spectrometer. The structures of the antioxidant compounds 1–9 (Fig. 1) were identified by NMR and ESI-MS, and all spectral data were consistent with those reported in the literatures.
2. Materials and methods
The DPPH radical scavenging activity of test samples was carried out based on the method reported by Chang et al. (2001) with slight modifications. Test samples were made up by dissolving each sample in MeOH to achieve final concentration at 1, 5, 10, 20 and 50 g/ml, respectively. Then, 10 l of test samples were mixed with 90 l of 50 mM Tris–HCl buffer (pH 7.4) and 200 l of 0.1 mM DPPH-ethanol solution. After 30 min of incubation at ambient temperature, the reduction of the DPPH free radical was measured by reading the absorbance at 517 nm using the ELISA reader. Quercetin and (+)-catechin were used as positive controls. Each sample was tested for three individual replicates. The inhibition ratio (%) was calculated according to the following equation % inhibition = [(absorbance of control − absorbance of test sample)/absorbance of control] × 100.
2.1. Chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), nitroblue tetrazolium chloride (NBT), (+)-catechin, disodium ethylenediaminetetraacetate (Na2 EDTA), Folin–Ciocalteu reagent, trichloroacetic acid (TCA), potassium phosphate monobasic (KH2 PO4 ), and xanthine oxidase were purchased from Sigma Chemical Co. (St. Louis, MO). Gallic acid, hypoxanthine, potassium persulfate, potassium hydroxide, quercetin, ferric ammonium sulfate, and sodium carbonate were purchased from Acros Chemicals (Morris Plains, NJ). The other chemicals and solvents were of analytical grade. 2.2. Plant material The 35-year-old A. confusa root was sampled from the Experimental Forest of National Taiwan University in Nan-Tou County. The species was identified by Sheng-You Lu of the Taiwan Forestry Research Institute and a voucher specimen (AC001) was deposited at the School of Forestry and Resource Conservation, National Taiwan University. 2.3. Extraction and isolation The dried samples (8.9 kg) were cut into small pieces and soaked in 95% ethanol (80 l) at ambient temperature 3 times (7 days for one time). The extracts were decanted, filtered under a vacuum, concentrated in a rotary evaporator and then lyophilized to yield crude extract (337 g). The crude extracts (300 g) were suspended in water then extracted successively with n-hexane (n-Hex), ethyl acetate (EtOAc), and n-butanol (n-BuOH) to yield n-Hex soluble fraction (1.93 g, 0.64%), EtOAc soluble fraction (180.59 g, 60.19%), n-BuOH soluble fraction (80.12 g, 26.71%) and a water soluble fraction (27.19 g, 9.06%). Afterward, 20 grams of EtOAc soluble fraction sample was subjected to reversephase column chromatography (50 cm × 5 cm i.d.) eluted with MeOH/water (gradient elution was performed by varying from 5/95 to 100/0) and 20 subfractions (EA1–EA20) were obtained. Nine compounds (as shown in Fig. 1) isolated and purified from EA2, EA3, EA5, EA15, EA16, and EA17 subfractions by using Agilent 1100 HPLC Series instrument (Agilent Technologies, Inc., Santa Clara, CA) with a 250 mm × 10 mm i.d., 5 m Luna RP18 semipreparative column (Phenomenex, Torrance, CA) at flow rate of 4 ml/min. From EA2 subfraction, 3,4-dihydroxybenzoic acid (1) was isolated by the isocratic mobile phase with MeOH/water = 13/87. Isomelacacidin (2), melacacidin (3), and 4O-methyl-melacacidin (4) were isolated from EA3 subfraction by
2.4. 1,1-Diphenyl-2-picryhydrazyl assay (DPPH assay)
2.5. Total antioxidant capacity by trolox equivalent antioxidant capacity assay (TEAC assay) TEAC of various samples were measured following the procedure described by Re et al. (1999) with slight modifications using trolox as a standard. The ABTS radical cation (ABTS•+ ) stock solution (7 mM) was generated by mixing ABTS reagent with 2.45 mM potassium persulfate buffer. This stock solution was kept at ambient temperature for 12–16 h until the reaction was complete and the absorbance was stable. The ABTS•+ solution was prepared by diluting ABTS•+ stock solution with water to give an absorbance value of 0.700 ± 0.020 at 730 nm. The sample solution (15 l) was mixed with 1485 l of the ABTS•+ solution. After 6 min of incubation at ambient temperature, the absorbance value of the mixture was measured at 730 nm in a Jasco V-550 UV–vis spectrophotometer. Quercetin, a well-known antioxidant, was used as a positive control. Three replicates were made for each test sample. A dose–response calibration curve of trolox was made up by the decreased absorbance value between the vehicle and the addition of trolox. Then, the TEAC value of each sample was calculated and expressed as trolox equivalent (TE) in millimole per gram of sample or per millimole of pure compound. 2.6. Determination of total phenolic contents Total phenolic contents were determined according to the Folin–Ciocalteu method (Kujala et al., 2000) using gallic acid as the standard. The test samples were dissolved in MeOH. 500 l of sample solution was mixed with 500 l of 1 N Folin–Ciocalteu reagent. The mixture was allowed to stand for 5 min, followed by the addition of 1 ml of 20% Na2 CO3 . After 8 min of incubation at ambient temperature, the mixture was centrifuged for 10 min (150 g), and the absorbance of the supernatant was measured at 730 nm. The total phenolic contents were expressed as gallic acid equivalents (GAE) in milligrams per gram of sample (mg of GAE/g).
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Fig. 1. Phytochemicals isolated from the root extract of A. confusa. (1) 3,4-Dihydroxybenzoic acid; (2) isomelacacidin; (3) melacacidin; (4) 4-O-methyl-melacacidin; (5) 2,3-trans-3,7,8,3 ,4 -pentahydroxydihydroflavone; (6) 2,3-cis-3,7,8,3 ,4 -pentahydroxydihydroflavone; (7) melanoxetin; (8) transilitin; (9) okanin.
2.7. Reducing power assay This assay was determined according to the method reported by Oyaizu (1986), with slight modifications. Briefly, 1 ml of reaction mixture, containing 500 l of the test samples and 500 l phosphate buffer (0.2 M, pH 6.6), was incubated with 500 l of potassium ferricyanide (1%, w/v) at 50 ◦ C for 20 min. Following incubation, the mixture was cooled by iced water followed by adding trichloroacetic acid (10%, w/v) to terminate the reaction and then the mixture was centrifuged at 3000 rpm for 10 min. We added 500 l of supernatant solution to distilled water (500 l) and 100 l of ferric chloride (0.1%, w/v) solution, and then measured the optical density (OD) at 700 nm. Three replicates were made for each test sample. Increased absorbance of the reaction mixture indicated increased reducing power. Further, the reducing power of each sample was expressed as trolox equivalent (TE) in millimole per gram of sample or per millimole of pure compound. 2.8. Total flavonoid contents Total flavonoid contents were determined by the AlCl3 method (Quettier-Deleu et al., 2000), using rutin as a standard. The test samples were dissolved in MeOH. The sample solution (150 l) was mixed with 150 l of 2% aqueous AlCl3 . After 10 min of incubation at ambient temperature, the absorbance of the supernatant was measured at 435 nm. Three replicates were made for each test sample. The total flavonoid contents were expressed as rutin equivalents (RE) in milligrams per gram of sample. 2.9. Superoxide anion radical scavenging assay (NBT assay) Measurement of superoxide anion radical scavenging activity was carried out according to the method reported by Chang et al. (2001). First, 20 l of 15 mM Na2 EDTA in buffer (50 mM
KH2 PO4 /KOH, pH 7.4), 50 l of 0.6 mM nitroblue tetrazolium chloride (NBT) in buffer, 30 l of 3 mM hypoxanthine in 50 mM KOH, 5 l of the test samples in MeOH, and 145 l of buffer were mixed in 96-well microplates. The reaction was started by adding 50 l of xanthine oxidase in buffer (1 unit in 10 ml of buffer) to the mixture. Then, the reaction mixture was incubated at ambient temperature, and the absorbance at 570 nm was determined every 30 s up to 5 min using the ELISA reader. Quercetin was used as a positive control. Three replicates were made for each test sample. The percent inhibition ratio (%) was calculated according to the following equation: % inhibition = [(rate of control reaction − rate of sample reaction)/rate of control reaction] × 100. 2.10. HPLC analysis The quantification of antioxidant phytochemicals from the EtOAc soluble fraction of ethanolic extract of root of A. confusa was carried out using the Agilent 1200 Series instrument HPLC-PDA (Agilent Technologies, Inc., Santa Clara, CA) with a 250 mm × 4 mm i.d., 5 m LiChrospher 100 RP-18e analytical column (Merck, Darmstadt, Germany). The column temperature was set at 40 ◦ C. The mobile phase was composed of 1% (v/v) formic acid in methanol (A) and H2 O (B). The flow rate was constant of 1 ml/min. Elution conditions were 0–30 min of 5–35% A (linear gradient); 30–35 min of 35–42.5% A (linear gradient); 35–50 min of 42.5–50% A (linear gradient). For preparation of the calibration curve, standard stock solutions of compounds were prepared in methanol, filtered through 0.45 m filters (Millipore), and appropriately diluted to obtain the desired concentrations in the quantification range. Quercetin was use as the internal standard. The calibration curves were calculated by linear regression of the peak area ratios which were obtained from the peak area of compounds over the peak area of quercetin versus concentrations of samples. For each sample, 5 l was injected into HPLC in three replications.
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previous reports and our results herein, the extracts of root, heartwood, and bark showed similar activities (IC50 < 5 g/ml), followed by branch bark (7.4 g/ml), leaves (∼20 g/ml), twig (24 g/ml), branch wood (24.8 g/ml) and flowers (13.1–90.9 g/ml) (Chang et al., 2001; Tung et al., 2009a, 2009c, 2011; Wu et al., 2005, 2008; Hsieh and Chang, 2010). The aforesaid results indicate there are plentiful amount of antioxidant phytochemicals present in the root extract of A. confusa, especially in the EtOAc soluble fraction. 3.2. Trolox equivalent antioxidant capacity assay (TEAC assay) of A. confusa root extract
Fig. 2. DPPH radical scavenging activity of ethanolic extract from A. confusa root. () Crude extract; () n-Hex fraction; () EtOAc fraction; (×) n-BuOH fraction; (䊉) water fraction; and (+) (+)-catechin. Results are mean ± SD (n = 3).
2.11. Statistical analysis All results were expressed as mean ± SD (n = 3). The significance of difference was calculated by Scheffe’s test, and values p < 0.05 were considered significant. 3. Results and discussion
TEAC assay is widely utilized to determine the antioxidant capacity of foods and nutraceuticals (Re et al., 1999). The antioxidant activity of crude extract and its fractions of A. confusa determined by the TEAC method are shown in Table 1. Trolox equivalence (TE) values were expressed as mmol of trolox per gram of test sample. The highest ABTS•+ scavenging activity was found for the EtOAc fraction, while the lowest was found for the n-Hex fraction. The TE value decreased in the order of the EtOAc fraction (8.1 mmol of TE/g) > crude extract (7.2 mmol of TE/g) > n-BuOH fraction (2.9 mmol of TE/g) > water fraction (2.1 mmol of TE/g) n-Hex fraction (0.2 mmol of TE/g), which was similar to the DPPH radical scavenging activity results. Surprisingly, the trolox equivalence value of EtOAc fraction was 8.1 mmol of TE/g, which was 2.0-fold better than trolox (4.0 mmol of TE/g). This result reveals the EtOAc fraction of A. confusa root extract is worth further investigation for its phytochemical composition and other antioxidant activities.
3.1. DPPH radical scavenging activity of A. confusa root extract The DPPH radical scavenging activity of ethanolic extract from A. confusa root and its derived soluble fractions, including n-Hex, EtOAc, n-BuOH and water was shown to occur in a dose-dependent manner (Fig. 2). Among them, the EtOAc soluble fraction exhibited the strongest activity, followed by, in sequence, crude extract, n-BuOH soluble fraction, water-soluble fraction and n-Hex fraction. Meanwhile, except for the n-Hex fraction, all extracts showed good inhibitory activity against the DPPH radical. The concentration required to inhibit the 50% radical scavenging effect (IC50 ) was determined from the results of a series of tested concentrations. A lower IC50 value corresponds to a stronger scavenging activity. The IC50 values of the crude extract, n-Hex fraction, EtOAc fraction, n-BuOH fraction and water fraction were 3.8, 81.5, 2.8, 5.2 and 5.8 g/ml, respectively. By comparing the DPPH radical scavenging activity, the crude extract of A. confusa root exhibited a lower IC50 value than that of green tea (IC50 = 5.0 g/ml), a wellknown antioxidant beverage (Yu et al., 2007). Additionally, the EtOAc fraction exhibited a similar IC50 value to the positive control (+)-catechin (IC50 = 3.0 g/ml), which is one of the major components of green tea extract. To evaluate the antioxidant potential, it is imperative compare the DPPH scavenging activity between crude extracts from different plant parts of A. confusa. From the
3.3. Superoxide anion radical scavenging activity of A. confusa root extract Superoxide anion radical was generated by the hypoxanthinexanthine oxidase and NBT systems in this assay. Fig. 3 shows the superoxide anion radical scavenging activity of crude extract and its derived fractions compared to that of (+)-catechin. At the 5 g/ml test concentration, the superoxide anion radical inhibition of A. confusa root extract and its derived fractions decreased in the following order: EtOAc fraction (80.4%) > crude extract (66.3%) > n-BuOH fraction (52.4%) > water fraction (33.1%) n-Hex fraction (0.9%). The IC50 values of (+)-catechin, crude extract, n-Hex fraction, EtOAc fraction, n-BuOH fraction, and water fraction were 3.2, 3.3, >100, 2.1, 4.7 and 7.9 g/ml, respectively. Superoxide anion radical, the most important radical in vivo, produced in mitochondria during electron chain transfer, regularly leaks out of mitochondria. Although superoxide anion radical is a weak oxidant, it can take part in further reactions leading to the formation of more reactive species, such as hydrogen peroxide (H2 O2 ) and hydroxyl radical (OH• ). These reactive species are implicated in many diseases including atherosclerosis, respiratory tract disorders, neurodegenerative diseases, inflammatory bowel disease, cancer and also in aging (Meyer and Isaksen, 1995). The above-mentioned results
Table 1 DPPH radical scavenging activity, superoxide radical scavenging activity, trolox equivalent antioxidant capacity, reducing power, total phenolic content and total flavonoid content of ethanolic extract from A. confusa root and its soluble fractions.a Specimens
IC50 (g/ml) DPPH radical
Crude n-Hex EtOAc n-BuOH Water (+)-Catechinb a b
3.8 81.5 2.8 5.2 5.8 3.0
± ± ± ± ± ±
0.1 c 0.6 a 0.3 d 0.4 b 0.2 b 0.2 d
TEAC (mmol of TE/g)
Reducing power (mmol of TE/g)
TPC (mg of GAE/g)
TFC (mg of RE/g)
Superoxide radical 3.3 ± 0.1 d >100 a 2.1 ± 0.2 e 4.7 ± 0.2 c 7.9 ± 0.4 b 3.2 ± 0.1 d
7.2 0.3 8.1 2.9 2.1 7.8
± ± ± ± ± ±
0.1 b 0.1 e 0.2 a 0.2 c 0.1 d 0.1 a
8.0 0.2 11.2 4.2 3.5 10.3
± ± ± ± ± ±
0.2 c 0f 0.3 a 0.1 d 0.2 e 0.2 b
652.2 ± 25.6 ± 777.0 ± 368.1 ± 320.3 ± –
35.4 b 0.4 e 13.0 a 9.9 c 5.8 d
Each value expressed as the mean ± SD (n = 3). Each value with different letters in the same column was considered significantly different (p < 0.05). Positive control.
69.7 ± 0.7 b Trace e 118.0 ± 1.0 a 7.6 ± 0.5 c 4.9 ± 0.1 d –
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et al. (2008), where the ethanolic extract of twigs of Cinnamomum osmophloeum with abundant phenolic components is well correlated with their antioxidant activity. Therefore, sequential studies must be carried out to isolate and identify the antioxidant phenolic compounds from the EtOAc fraction of A. confusa root extract. 3.6. Determination of total flavonoid contents of A. confusa root extract
Fig. 3. Superoxide anion radical scavenging activity of ethanolic extract from A. confusa root. () Crude extract; () n-Hex fraction; () EtOAc fraction; (×) n-BuOH fraction; (䊉) water fraction; and (+) (+)-catechin. Results are mean ± SD (n = 3).
demonstrate crude extract and EtOAc fraction of A. confusa root show better superoxide anion radical scavenging activity than the famous antioxidant-(+)-catechin. Among them, EtOAc fraction exhibited the strongest radical scavenging activity and its IC50 value was 1.4-fold lower than that of (+)-catechin. Therefore, the EtOAc fraction of A. confusa root extract would be an excellent source of natural antioxidant. 3.4. Reducing power of A. confusa root extract Previous reports have demonstrated the reducing power is often used as an indicator of electron-donating ability, which is an important mechanism of phenolic antioxidant activity (Dorman et al., 2003). Ferric (Fe3+ ) can reduce to ferrous (Fe2+ ) by antioxidants because of their reductive capabilities. Table 1 shows the trolox equivalence (TE) of reducing power of crude extract and its derived fractions, with their ranking order being as follows: EtOAc fraction (11.2 mmol of TE/g) > crude extract (8.0 mmol of TE/g) > n-BuOH fraction (4.2 mmol of TE/g) > water fraction (3.5 mmol of TE/g) nHex fraction (0.2 mmol of TE/g). Again, EtOAc fraction exhibited the strongest activity and its TE was higher than (+)-catechin (10.3 mmol of TE/g), which was used as a positive control. Previously, Tung et al. (2009a) reported hot water extract from leaves of A. confusa possessed good antioxidant activity. However, the reducing power of hot water extract of A. confusa leaves and its derived fractions were lower than that of (+)-catechin. Accordingly, the reducing power of EtOAc fraction of A. confusa root extract is better than the hot water extract of A. confusa leaves. 3.5. Determination of total phenolic contents of A. confusa root extract Phenolic compounds from plant extract often display antioxidant activity. This activity is believed to be mainly due to their hydrogen donor and electron donor properties, which play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides (Adeolu et al., 2009). Table 1 shows the gallic acid equivalence (GAE) of the total phenolic contents of crude extract and its derived fractions, with their ranking order being as follows: EtOAc fraction (777.0 mg of GAE/g) > crude extract (652.2 mg of GAE/g) > n-BuOH fraction (368.1 mg of GAE/g) > water fraction (320.2 mg of GAE/g) n-Hex fraction (25.6 mg of GAE/g). The ranking order of phenolic content of A. confusa root extract and its derived fractions is consistent with their antioxidant results mentioned above, indicating the antioxidant activities of these samples should be connected to their phenolic contents. A similar finding has been demonstrated by Chua
Flavonoids, a group of phenolic compounds widely distributed in the plant kingdom, exhibit many biochemical and pharmacological effects, including antioxidation, antiinflammation and antiallergy (Havsteen, 1983). Herein, a total flavonoid contents assay was carried out to estimate their correlation with antioxidant activity. Table 1 expresses the rutin equivalence of the total flavonoid contents of A. confusa root extract and its derived fractions, and its decreasing order ranks as EtOAc fraction (118.0 mg of Rutin/g) > crude extract (69.7 mg of Rutin/g) n-BuOH fraction (7.6 mg of Rutin/g) > water fraction (4.9 mg of Rutin/g) > n-Hex fraction (trace). This result indicated most flavonoid components of A. confusa root extract are present in its EtOAc fraction. It is notable total flavonoid contents are much lower than total phenolic contents, suggesting A. confusa root extract and its derived fractions contain not only flavonoid components, but also many other types of phenolic components with antioxidant activity. 3.7. Quantitative analysis and antioxidant activities of phytochemicals from EtOAc fraction of A. confusa root extract According to the above-mentioned results, the EtOAc fraction exhibited the best DPPH radical scavenging, superoxide anion radical scavenging, TEAC values, and reducing power. Therefore, a phytochemicals study of the EtOAc fraction was carried out to separate and purify active compounds by column chromatography and HPLC. Then, nine compounds were isolated and their chemical structures were identified by comparing their NMR and MS data with the literatures. Fig. 1 shows the chemical structures of these nine compounds, including 3,4-dihydroxybenzoic acid (1), isomelacacidin (2), melacacidin (3), 4-O-methyl-melacacidin (4), 2,3-trans-3,7,8,3 ,4 -pentahydroxydihydroflavone (5), 2,3-cis3,7,8,3 ,4 -pentahydroxydihydroflavone (6), melanoxetin (7), transilitin (8), and okanin (9) (Wu et al., 2005; Foo and Wong, 1986; Foo, 1986). Among them, compounds 2 and 6 were identified from this species for the first time. To determine the antioxidant activities of nine phytochemicals, DPPH, TEAC, NBT, and reducing power assays were carried out, and these results are expressed in Table 2. Quercetin was used as a positive control. Concerning DPPH radical scavenging activity, compounds 7–9 exhibited better activity than quercetin with an IC50 value of 3.1 M. In addition, compounds 2–6 exhibited parallel activity to quercetin. Then, the superoxide anion radical scavenging activity of these nine phytochemicals can be ranked in a decreasing order of 9 = 7 > 8 > 4 3 2 > 5 > 6 1. Moreover, compounds 7 and 9 exhibited stronger superoxide anion radical scavenging activity than quercetin, and their IC50 values were 2.5 and 2.2 M, respectively. Xanthine oxidase is an enzyme that catalyzes the oxidation of hypoxanthine to xanthine and that of xanthine to uric acid, along with the generation of hydrogen peroxide and superoxide anion (Rasmussen et al., 2000). Over expression of xanthine oxidase may cause overproduction of hydrogen peroxide and superoxide anion, leading to oxidative stress and tissue injury. Previously, Tung and Chang (2010) reported melanoxetin (7) and okanin (9) are excellent xanthine oxidase inhibitors with competitive and noncompetitive mode, respectively. Therefore, it can be suggested compounds 7 and 9 revealed better NBT assay activity than other compounds through not only the superoxide anion radical scavenging effect,
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Table 2 DPPH radical scavenging activity, superoxide radical scavenging activity, trolox equivalent antioxidant capacity, reducing power and content of major phytochemicals from A. confusa root.a Phytochemicals
IC50 (M) DPPH radical
3,4-Dihydroxybenzoic acid (1) Isomelacacidin (2) Melacacidin (3) 4-O-Methyl-melacacidin (4) 2,3-Trans-3,7,8,3 ,4 -pentahydroxydihydroflavone (5) 2,3-Cis-3,7,8,3 ,4 -pentahydroxydihydroflavone (6) Melanoxetin (7) Transilitin (8) Okanin (9) Quercetinb a b
5.9 4.5 3.9 4.3 4.6 3.9 3.1 3.1 3.1 3.9
± ± ± ± ± ± ± ± ± ±
0.4 a 0.5 b 0.6 bc 0.7 b 0.3 b 0.2 b 0.2 c 0.1 c 0.1 c 0.2 b
TEAC (mmol of TE/mmol)
Reducing power (mmol of TE/mmol)
Contents (mg/g of crude extract)
Superoxide radical >50 a 5.0 ± 0.1 d 4.7 ± 0.1 de 4.4 ± 0.1 e 11.9 ± 0.9 b 10.2 ± 0.3 c 2.5 ± 0.1 g 3.4 ± 0.1 f 2.2 ± 0.1 g 3.7 ± 0.2 f
2.5 4.2 4.4 4.4 4.3 4.6 4.4 2.9 5.4 3.9
± ± ± ± ± ± ± ± ± ±
0.2 c 0.1 b 0.4 b 0.3 b 0.2 b 0.3 ab 0.2 b 0.2 c 0.3 a 0.2 b
2.7 5.2 5.0 5.1 4.5 4.7 5.0 4.5 5.3 4.2
± ± ± ± ± ± ± ± ± ±
0.2 c 0.3 a 0.2 a 0.1 a 0.2 ab 0.1 ab 0.1 a 0.1 ab 0.3 a 0.1 b
7.8 ± 41.5 ± 109.1 ± 23.6 ± 19.7 ± 34.6 ± 65.7 ± 28.3 ± 6.9 ± –
0.3 h 1.0 c 2.2 a 0.4 f 0.4 g 0.6 d 1.5 b 0.4 e 0.4 h
Each value expressed as the mean ± SD (n = 3). Each value with different letters in the same column was considered significantly different (p < 0.05). Positive control.
but also the xanthine oxidase inhibitory effect. Moreover, due to the xanthine oxidase inhibitory ability, melanoxetin and okanin may possess cellular antioxidant potential. As for TEAC and reducing power assays, the antioxidant abilities were present in trolox equivalence (TE/mmol of sample). Among all nine phytochemicals, okanin (9) exhibited the best TEAC activities with TE value of 5.4, which is 1.4-fold higher than quercetin. Otherwise, TEAC activities of compounds 2–7 were also comparable to quercetin. On the other hand, compounds 2–4, 7 and 9 revealed obviously higher reducing power ability than that of quercetin, whereas compounds 5, 6 and 8 had similar activities to quercetin. Based on the above-mentioned results, okanin (9) is the best antioxidant compound among these nine phytochemicals. Fig. 4 gives the HPLC profile of the EtOAc fraction of the ethanolic extract of A. confusa root. By following this gradient condition, the quantitative analysis of compounds 1–9 were determined to be 7.8, 41.5, 109.1, 23.6, 19.7, 34.6, 65.7, 28.3 and 6.9 mg/g of crude extract, respectively (Table 2). Accordingly, melacacidin (3) is the most abundant phenolic compound in the ethanolic extract
of A. confusa root, and it also reveals remarkable antioxidant activities. 3.8. Structure–activity relationships of flavonoids from A. confusa root extract This study also investigated the structure–activity relationships of flavonoids in term of their antioxidant activities, including DPPH, NBT, TEAC and reducing power assays. Previously, Heim et al. (2002) reported conjugated flavonoids exhibit better antioxidant activities than the corresponding non-conjugated ones because they permit electron delocalization and then increase the stability of the corresponding flavonoid radicals generated by the ROS. Thus, flavonoids from A. confusa can be divided into conjugated flavonoids (7–9) and non-conjugated flavonoids (2–6). For conjugated flavonoids, okanin (9) exhibited the best antioxidant activities, probably because it is free of both catechol and pyrogallol structures as well as the connection between 2 aromatic groups by an ␣-,-unsaturated carbonyl group. Melanoxetin
Fig. 4. HPLC profile of the EtOAc soluble fraction of root extract from A. confusa.
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(7) showed less antioxidant activities than okanin (9) because of a closed C-ring result in (i) etherization of pyrogallol of the Aring, and (ii) distortion of planarity of the conjugating system. Etherization of the phenol group by the alkyl groups or sugars decreases the antioxidant activities of flavonoids. Distortion of the planarity of the conjugated system lowers the delocalizing effect, diminishes the stability of flavonoid radicals, and reduces the antioxidant activities (Heim et al., 2002). Although the DPPH scavenging activity of transilitin (8) and melanoxetin (7) were comparable, melanoxetin revealed better NBT, TEAC and reducing power activities than transilitin. Van Acker et al. (1996) provided an explanation for this result. They found flavon-3-ol shows better antioxidant activities than its 3-O-methyl derivative because of the intramolecular hydrogen bonding between the 3-hydroxy and 4-oxo groups, resulting in the enhancement of planarity of the conjugated system. Tung et al. (2009a) also reported myricetin showed better DPPH, NBT and reducing power activities than myricetin-3-O-methyl ether, which is consistent with our result. For non-conjugated flavonoids, the DPPH and NBT activities of compounds 2–4 are slightly lower than compounds 7–9, agreeing with the report of Heim et al. (2002). However, it is interesting their TEAC and reducing power activities are similar to melanoxetin (7). Obviously, the 7,8-dihydroxyl configuration of the A-ring is the most significant structural feature of flavonoids isolated from A. confusa root extract. It is well known phenolic compounds with ortho- and para-dihydroxyl groups exhibit better antioxidant activities than that with meta-dihydroxyl groups (Heim et al., 2002). Therefore, it can be speculated the contribution of antioxidant abilities from these flavonoids is through not only the catechol structure in the B-ring, but also the pyrogallol derivative structure in the A-ring, leading to strong enhancement of their activities. Accordingly, these 7,8-dihydroxyl flavonoids should exhibit better antioxidant activities than the corresponding 5,7-dihydroxyl flavonoids. For example, compounds 2–4 showed lower IC50 values of DPPH and NBT than (+)-catechin (10.3 and 11.0 M) and higher TEAC and reducing power values than (+)-catechin (2.3 and 3.0 mmol of TE/mmol). Melanoxetin (7) also showed better activities than quercetin for four assays (Table 2). Compounds 5 and 6 revealed similar DPPH, TEAC and reducing power activities with compounds 2–4, but their NBT assay activity were apparently lower than compounds 2–4. This may be due to the electron-withdrawing effect caused by the 4-oxo group of compounds 5 and 6, leading to a decrease in activities (Heim et al., 2002).
4. Conclusions In this study, the antioxidant activities of the ethanolic extract of A. confusa root and its active compounds were reported for the first time. The ethyl acetate soluble fraction showed the best antioxidant ability and the highest yield. Afterwards, nine major phenolic compounds were isolated from this fraction, and their chemical structures were successfully elucidated by comparing the spectra with literature data. Flavonoids are the major antioxidant phytochemicals from A. confusa root and their strong antioxidant capacities are derived from 7,8-dihydroxyl groups of the A-ring as well as 3 ,4 -dihydroxyl groups of the B-ring. Therefore, these results demonstrated A. confusa root extract has great potential in preventing diseases caused by overproduction of ROS. In addition, it could serve as a potent source of natural antioxidant in the nutraceutical, beneficial additives, and food industries. Further studies should focus on the in vivo pharmacological research of these strong antioxidant flavonoids.
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Acknowledgements The authors thank the National Science Council for the financial support (100-2313-B-002-032-MY3) and the Experimental Forest of National Taiwan University for providing A. confusa materials. References Adeolu, A.A., Florence, O.J., Srinivas, V., Patrick, J.M., Anthony, J.A., 2009. Assessment of the medicinal potentials of the methanol extracts of the leaves and stems of Buddleja saligna. BMC Complem. Altern. Med. 9, 21, http://dx.doi.org/10.1186/1472-6882-9-21. Chang, S.-T., Wu, J.-H., Wang, S.-Y., Kang, P.-L., Yang, N.-S., Shyur, L.-F., 2001. Antioxidant activity of extracts from Acacia confusa bark and heartwood. J. Agric. Food Chem. 49, 3420–3424. Chua, M.-T., Tung, Y.-T., Chang, S.-T., 2008. Antioxidant activities of ethanolic extracts from the twigs of Cinnamomum osmophloeum. Bioresour. Technol. 99, 1918–1925. Dorman, H.J.D., Peltoketo, A., Hiltunen, R., Tikkanen, M.J., 2003. 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Antioxidant potency of phenolic phytochemicals from the root extract of Acacia confusa H.Y. Lin, S.T. Chang ∗ School of Forestry and Resource Conservation, National Taiwan University, Taipei 106, Taiwan
a r t i c l e
i n f o
Article history: Received 15 April 2013 Received in revised form 23 June 2013 Accepted 2 July 2013 Keywords: Acacia confusa Antioxidant activity Flavonoids Melacacidin Okanin Phenolics
a b s t r a c t In the present study, the antioxidant potencies of ethanolic extract and its derived phytochemicals from the root of Acacia confusa, the indigenous Taiwanese species, were investigated and reported for the first time. Among all the soluble fractions of ethanolic extract, the EtOAc soluble fraction exhibited the best antioxidant activity. The bioassay-guided fractionation was carried out and yielded 9 potent antioxidative phytochemicals from EtOAc fraction. Among them, okanin (9) exhibited the lowest IC50 DPPH radical and superoxide anion radical scavenging activity values and the highest TEAC and reducing power activity. Besides, melacacidin (3) was the most abundant antioxidant compound in A. confusa root extract with an absolute content of 109.1 mg/g of crude extract. The structure–activity relationships of antioxidant flavonoids isolated from A. confusa root extract were also discussed. Therefore, with its excellent antioxidant activities, A. confusa root extract has great potential as a source for natural health products. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Reactive oxygen species (ROS) are the endogenous free radicals of normal cellular metabolism. In living organisms, moderate concentrations of ROS benefit physiological functions such as intracellular signaling, cellular defense against infective agents, and induction of a mitogenic response. However, overproduction of ROS can cause cellular lipid peroxidation, protein degradation, and DNA mutation, resulting in cancer, cardiovascular diseases, diabetes, neurodegenerative diseases, inflammations, and aging (Valko et al., 2007). Recently, numerous studies have focused on the phenolic compounds from plant sources to serve as antioxidants against various diseases induced by free radicals. Among the various medicinal and dietary plants, endemic species are of particular interest because of their ability to produce phytochemicals with significant antioxidant activities and health benefits. Generally, phenolic compounds display their antioxidant properties through the roles of electron donors, hydrogen donors, and free radical scavengers to neutralize or quench the ROS (Yizhong et al., 2004). Nowadays, synthetic antioxidants, such as BHT and BHA, are widely used to improve shelf life and safety for food preservation. However, several studies have shown synthetic antioxidants may be involved in latent carcinogenesis, leading to efforts to replace the synthetic antioxidants with natural antioxidants (Jennings and Akoh, 2009). Besides, although natural antioxidants possess weaker abilities and
∗ Corresponding author. Tel.: +886 2 3366 4626; fax: +886 2 2365 4520. E-mail address: [email protected] (S.T. Chang).
cost more than synthetic ones, they are extensively accepted by consumers and health officials with the labels of potentially positive effects and flavorings of sensory properties. Acacia confusa Merr. (Leguminosae), an indigenous species of Taiwan widely distributed over the hills and lowlands, has traditionally been used as a folk medicine (Wu et al., 2005). This plant is an important source for feedstock, charcoal-making, and construction materials. Moreover, the particularly significant of this plant is the conservation of soil and water as well as remarkable carbon sequestration (Chang et al., 2001; Wang, 2011). However, utilization of this plant has declined gradually as emergence of the novel construction materials. The aqueous extract of A. confusa leaves was used for wound healing and anti-blood-stasis in Taiwan (Kan, 1978). Recent studies have demonstrated the crude extracts of heartwood, bark, leaves, branch, twigs, and flowers of A. confusa exhibit strong antioxidative effects, and contain a wide variety of phenolic compounds (Chang et al., 2001; Tung et al., 2009a,c, 2011; Wu et al., 2005, 2008; Hsieh and Chang, 2010). Moreover, some of these phenolic compounds revealed impressive bioactivities. The bark extract and its active constituent gallic acid exhibit potent hepatoprotection against CCl4 -induced liver damages in rats due to the modulation of antioxidant enzymes activities and inhibition of lipid peroxidation and cytochrome P4502E1 protein expression (Tung et al., 2009b). Okanin and melanoxetin isolated from the heartwood show excellent inhibition of xanthine oxidase in noncompetitive and competitive mode, respectively, and their inhibitory activity was better than that of allopurinol (Tung and Chang, 2010). Further, melanoxetin reduced mice serum uric acid levels against the potassium oxanate-induced hyperuricemic effect (Tung et al., 2010).
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Luteolin isolated from the branch bark also revealed good inhibitory effect on xanthine oxidase (Hsieh and Chang, 2010). These findings let us to be interested in investigating the bioactivity of the phenolic compounds from A. confusa root extract, and to the best of our knowledge its potential health benefits have never been studied. The aims of this study were to evaluate the antioxidant activities of A. confusa root extract and its derived fractions, followed by isolating the major active compounds. Additionally, the structureactivity relationships of the antioxidant flavonoids isolated from A. confusa root extract were also discussed. The results obtained from this study can be utilized as basic information for further applications of A. confusa root extract, such as antioxidant additives for commercial food products, antioxidant dietary supplements, and medicines of illnesses induced by oxidative stress.
linear gradients elution from 10/90 to 40/60 (MeOH/water) during 45 min. The 2,3-trans-3,7,8,3 ,4 -pentahydroxydihydroflavone (5), and 2,3-cis-3,7,8,3 ,4 -pentahydroxydihydroflavone (6) were isolated from EA5 subfraction by the isocratic mobile phase with MeOH/water = 20/80. Melanoxetin (7), transilitin (8), and okanin (9) were isolated from EA16, EA17, and EA18 by the isocratic mobile phase with MeOH/water = 35/65, 40/60, and 45/55, respectively. NMR spectra were recorded by a Bruker Avance 500 MHz FT-NMR spectrometer, and ESI-MS data were collected using a Finnigan MAT-95S mass spectrometer. The structures of the antioxidant compounds 1–9 (Fig. 1) were identified by NMR and ESI-MS, and all spectral data were consistent with those reported in the literatures.
2. Materials and methods
The DPPH radical scavenging activity of test samples was carried out based on the method reported by Chang et al. (2001) with slight modifications. Test samples were made up by dissolving each sample in MeOH to achieve final concentration at 1, 5, 10, 20 and 50 g/ml, respectively. Then, 10 l of test samples were mixed with 90 l of 50 mM Tris–HCl buffer (pH 7.4) and 200 l of 0.1 mM DPPH-ethanol solution. After 30 min of incubation at ambient temperature, the reduction of the DPPH free radical was measured by reading the absorbance at 517 nm using the ELISA reader. Quercetin and (+)-catechin were used as positive controls. Each sample was tested for three individual replicates. The inhibition ratio (%) was calculated according to the following equation % inhibition = [(absorbance of control − absorbance of test sample)/absorbance of control] × 100.
2.1. Chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), nitroblue tetrazolium chloride (NBT), (+)-catechin, disodium ethylenediaminetetraacetate (Na2 EDTA), Folin–Ciocalteu reagent, trichloroacetic acid (TCA), potassium phosphate monobasic (KH2 PO4 ), and xanthine oxidase were purchased from Sigma Chemical Co. (St. Louis, MO). Gallic acid, hypoxanthine, potassium persulfate, potassium hydroxide, quercetin, ferric ammonium sulfate, and sodium carbonate were purchased from Acros Chemicals (Morris Plains, NJ). The other chemicals and solvents were of analytical grade. 2.2. Plant material The 35-year-old A. confusa root was sampled from the Experimental Forest of National Taiwan University in Nan-Tou County. The species was identified by Sheng-You Lu of the Taiwan Forestry Research Institute and a voucher specimen (AC001) was deposited at the School of Forestry and Resource Conservation, National Taiwan University. 2.3. Extraction and isolation The dried samples (8.9 kg) were cut into small pieces and soaked in 95% ethanol (80 l) at ambient temperature 3 times (7 days for one time). The extracts were decanted, filtered under a vacuum, concentrated in a rotary evaporator and then lyophilized to yield crude extract (337 g). The crude extracts (300 g) were suspended in water then extracted successively with n-hexane (n-Hex), ethyl acetate (EtOAc), and n-butanol (n-BuOH) to yield n-Hex soluble fraction (1.93 g, 0.64%), EtOAc soluble fraction (180.59 g, 60.19%), n-BuOH soluble fraction (80.12 g, 26.71%) and a water soluble fraction (27.19 g, 9.06%). Afterward, 20 grams of EtOAc soluble fraction sample was subjected to reversephase column chromatography (50 cm × 5 cm i.d.) eluted with MeOH/water (gradient elution was performed by varying from 5/95 to 100/0) and 20 subfractions (EA1–EA20) were obtained. Nine compounds (as shown in Fig. 1) isolated and purified from EA2, EA3, EA5, EA15, EA16, and EA17 subfractions by using Agilent 1100 HPLC Series instrument (Agilent Technologies, Inc., Santa Clara, CA) with a 250 mm × 10 mm i.d., 5 m Luna RP18 semipreparative column (Phenomenex, Torrance, CA) at flow rate of 4 ml/min. From EA2 subfraction, 3,4-dihydroxybenzoic acid (1) was isolated by the isocratic mobile phase with MeOH/water = 13/87. Isomelacacidin (2), melacacidin (3), and 4O-methyl-melacacidin (4) were isolated from EA3 subfraction by
2.4. 1,1-Diphenyl-2-picryhydrazyl assay (DPPH assay)
2.5. Total antioxidant capacity by trolox equivalent antioxidant capacity assay (TEAC assay) TEAC of various samples were measured following the procedure described by Re et al. (1999) with slight modifications using trolox as a standard. The ABTS radical cation (ABTS•+ ) stock solution (7 mM) was generated by mixing ABTS reagent with 2.45 mM potassium persulfate buffer. This stock solution was kept at ambient temperature for 12–16 h until the reaction was complete and the absorbance was stable. The ABTS•+ solution was prepared by diluting ABTS•+ stock solution with water to give an absorbance value of 0.700 ± 0.020 at 730 nm. The sample solution (15 l) was mixed with 1485 l of the ABTS•+ solution. After 6 min of incubation at ambient temperature, the absorbance value of the mixture was measured at 730 nm in a Jasco V-550 UV–vis spectrophotometer. Quercetin, a well-known antioxidant, was used as a positive control. Three replicates were made for each test sample. A dose–response calibration curve of trolox was made up by the decreased absorbance value between the vehicle and the addition of trolox. Then, the TEAC value of each sample was calculated and expressed as trolox equivalent (TE) in millimole per gram of sample or per millimole of pure compound. 2.6. Determination of total phenolic contents Total phenolic contents were determined according to the Folin–Ciocalteu method (Kujala et al., 2000) using gallic acid as the standard. The test samples were dissolved in MeOH. 500 l of sample solution was mixed with 500 l of 1 N Folin–Ciocalteu reagent. The mixture was allowed to stand for 5 min, followed by the addition of 1 ml of 20% Na2 CO3 . After 8 min of incubation at ambient temperature, the mixture was centrifuged for 10 min (150 g), and the absorbance of the supernatant was measured at 730 nm. The total phenolic contents were expressed as gallic acid equivalents (GAE) in milligrams per gram of sample (mg of GAE/g).
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Fig. 1. Phytochemicals isolated from the root extract of A. confusa. (1) 3,4-Dihydroxybenzoic acid; (2) isomelacacidin; (3) melacacidin; (4) 4-O-methyl-melacacidin; (5) 2,3-trans-3,7,8,3 ,4 -pentahydroxydihydroflavone; (6) 2,3-cis-3,7,8,3 ,4 -pentahydroxydihydroflavone; (7) melanoxetin; (8) transilitin; (9) okanin.
2.7. Reducing power assay This assay was determined according to the method reported by Oyaizu (1986), with slight modifications. Briefly, 1 ml of reaction mixture, containing 500 l of the test samples and 500 l phosphate buffer (0.2 M, pH 6.6), was incubated with 500 l of potassium ferricyanide (1%, w/v) at 50 ◦ C for 20 min. Following incubation, the mixture was cooled by iced water followed by adding trichloroacetic acid (10%, w/v) to terminate the reaction and then the mixture was centrifuged at 3000 rpm for 10 min. We added 500 l of supernatant solution to distilled water (500 l) and 100 l of ferric chloride (0.1%, w/v) solution, and then measured the optical density (OD) at 700 nm. Three replicates were made for each test sample. Increased absorbance of the reaction mixture indicated increased reducing power. Further, the reducing power of each sample was expressed as trolox equivalent (TE) in millimole per gram of sample or per millimole of pure compound. 2.8. Total flavonoid contents Total flavonoid contents were determined by the AlCl3 method (Quettier-Deleu et al., 2000), using rutin as a standard. The test samples were dissolved in MeOH. The sample solution (150 l) was mixed with 150 l of 2% aqueous AlCl3 . After 10 min of incubation at ambient temperature, the absorbance of the supernatant was measured at 435 nm. Three replicates were made for each test sample. The total flavonoid contents were expressed as rutin equivalents (RE) in milligrams per gram of sample. 2.9. Superoxide anion radical scavenging assay (NBT assay) Measurement of superoxide anion radical scavenging activity was carried out according to the method reported by Chang et al. (2001). First, 20 l of 15 mM Na2 EDTA in buffer (50 mM
KH2 PO4 /KOH, pH 7.4), 50 l of 0.6 mM nitroblue tetrazolium chloride (NBT) in buffer, 30 l of 3 mM hypoxanthine in 50 mM KOH, 5 l of the test samples in MeOH, and 145 l of buffer were mixed in 96-well microplates. The reaction was started by adding 50 l of xanthine oxidase in buffer (1 unit in 10 ml of buffer) to the mixture. Then, the reaction mixture was incubated at ambient temperature, and the absorbance at 570 nm was determined every 30 s up to 5 min using the ELISA reader. Quercetin was used as a positive control. Three replicates were made for each test sample. The percent inhibition ratio (%) was calculated according to the following equation: % inhibition = [(rate of control reaction − rate of sample reaction)/rate of control reaction] × 100. 2.10. HPLC analysis The quantification of antioxidant phytochemicals from the EtOAc soluble fraction of ethanolic extract of root of A. confusa was carried out using the Agilent 1200 Series instrument HPLC-PDA (Agilent Technologies, Inc., Santa Clara, CA) with a 250 mm × 4 mm i.d., 5 m LiChrospher 100 RP-18e analytical column (Merck, Darmstadt, Germany). The column temperature was set at 40 ◦ C. The mobile phase was composed of 1% (v/v) formic acid in methanol (A) and H2 O (B). The flow rate was constant of 1 ml/min. Elution conditions were 0–30 min of 5–35% A (linear gradient); 30–35 min of 35–42.5% A (linear gradient); 35–50 min of 42.5–50% A (linear gradient). For preparation of the calibration curve, standard stock solutions of compounds were prepared in methanol, filtered through 0.45 m filters (Millipore), and appropriately diluted to obtain the desired concentrations in the quantification range. Quercetin was use as the internal standard. The calibration curves were calculated by linear regression of the peak area ratios which were obtained from the peak area of compounds over the peak area of quercetin versus concentrations of samples. For each sample, 5 l was injected into HPLC in three replications.
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previous reports and our results herein, the extracts of root, heartwood, and bark showed similar activities (IC50 < 5 g/ml), followed by branch bark (7.4 g/ml), leaves (∼20 g/ml), twig (24 g/ml), branch wood (24.8 g/ml) and flowers (13.1–90.9 g/ml) (Chang et al., 2001; Tung et al., 2009a, 2009c, 2011; Wu et al., 2005, 2008; Hsieh and Chang, 2010). The aforesaid results indicate there are plentiful amount of antioxidant phytochemicals present in the root extract of A. confusa, especially in the EtOAc soluble fraction. 3.2. Trolox equivalent antioxidant capacity assay (TEAC assay) of A. confusa root extract
Fig. 2. DPPH radical scavenging activity of ethanolic extract from A. confusa root. () Crude extract; () n-Hex fraction; () EtOAc fraction; (×) n-BuOH fraction; (䊉) water fraction; and (+) (+)-catechin. Results are mean ± SD (n = 3).
2.11. Statistical analysis All results were expressed as mean ± SD (n = 3). The significance of difference was calculated by Scheffe’s test, and values p < 0.05 were considered significant. 3. Results and discussion
TEAC assay is widely utilized to determine the antioxidant capacity of foods and nutraceuticals (Re et al., 1999). The antioxidant activity of crude extract and its fractions of A. confusa determined by the TEAC method are shown in Table 1. Trolox equivalence (TE) values were expressed as mmol of trolox per gram of test sample. The highest ABTS•+ scavenging activity was found for the EtOAc fraction, while the lowest was found for the n-Hex fraction. The TE value decreased in the order of the EtOAc fraction (8.1 mmol of TE/g) > crude extract (7.2 mmol of TE/g) > n-BuOH fraction (2.9 mmol of TE/g) > water fraction (2.1 mmol of TE/g) n-Hex fraction (0.2 mmol of TE/g), which was similar to the DPPH radical scavenging activity results. Surprisingly, the trolox equivalence value of EtOAc fraction was 8.1 mmol of TE/g, which was 2.0-fold better than trolox (4.0 mmol of TE/g). This result reveals the EtOAc fraction of A. confusa root extract is worth further investigation for its phytochemical composition and other antioxidant activities.
3.1. DPPH radical scavenging activity of A. confusa root extract The DPPH radical scavenging activity of ethanolic extract from A. confusa root and its derived soluble fractions, including n-Hex, EtOAc, n-BuOH and water was shown to occur in a dose-dependent manner (Fig. 2). Among them, the EtOAc soluble fraction exhibited the strongest activity, followed by, in sequence, crude extract, n-BuOH soluble fraction, water-soluble fraction and n-Hex fraction. Meanwhile, except for the n-Hex fraction, all extracts showed good inhibitory activity against the DPPH radical. The concentration required to inhibit the 50% radical scavenging effect (IC50 ) was determined from the results of a series of tested concentrations. A lower IC50 value corresponds to a stronger scavenging activity. The IC50 values of the crude extract, n-Hex fraction, EtOAc fraction, n-BuOH fraction and water fraction were 3.8, 81.5, 2.8, 5.2 and 5.8 g/ml, respectively. By comparing the DPPH radical scavenging activity, the crude extract of A. confusa root exhibited a lower IC50 value than that of green tea (IC50 = 5.0 g/ml), a wellknown antioxidant beverage (Yu et al., 2007). Additionally, the EtOAc fraction exhibited a similar IC50 value to the positive control (+)-catechin (IC50 = 3.0 g/ml), which is one of the major components of green tea extract. To evaluate the antioxidant potential, it is imperative compare the DPPH scavenging activity between crude extracts from different plant parts of A. confusa. From the
3.3. Superoxide anion radical scavenging activity of A. confusa root extract Superoxide anion radical was generated by the hypoxanthinexanthine oxidase and NBT systems in this assay. Fig. 3 shows the superoxide anion radical scavenging activity of crude extract and its derived fractions compared to that of (+)-catechin. At the 5 g/ml test concentration, the superoxide anion radical inhibition of A. confusa root extract and its derived fractions decreased in the following order: EtOAc fraction (80.4%) > crude extract (66.3%) > n-BuOH fraction (52.4%) > water fraction (33.1%) n-Hex fraction (0.9%). The IC50 values of (+)-catechin, crude extract, n-Hex fraction, EtOAc fraction, n-BuOH fraction, and water fraction were 3.2, 3.3, >100, 2.1, 4.7 and 7.9 g/ml, respectively. Superoxide anion radical, the most important radical in vivo, produced in mitochondria during electron chain transfer, regularly leaks out of mitochondria. Although superoxide anion radical is a weak oxidant, it can take part in further reactions leading to the formation of more reactive species, such as hydrogen peroxide (H2 O2 ) and hydroxyl radical (OH• ). These reactive species are implicated in many diseases including atherosclerosis, respiratory tract disorders, neurodegenerative diseases, inflammatory bowel disease, cancer and also in aging (Meyer and Isaksen, 1995). The above-mentioned results
Table 1 DPPH radical scavenging activity, superoxide radical scavenging activity, trolox equivalent antioxidant capacity, reducing power, total phenolic content and total flavonoid content of ethanolic extract from A. confusa root and its soluble fractions.a Specimens
IC50 (g/ml) DPPH radical
Crude n-Hex EtOAc n-BuOH Water (+)-Catechinb a b
3.8 81.5 2.8 5.2 5.8 3.0
± ± ± ± ± ±
0.1 c 0.6 a 0.3 d 0.4 b 0.2 b 0.2 d
TEAC (mmol of TE/g)
Reducing power (mmol of TE/g)
TPC (mg of GAE/g)
TFC (mg of RE/g)
Superoxide radical 3.3 ± 0.1 d >100 a 2.1 ± 0.2 e 4.7 ± 0.2 c 7.9 ± 0.4 b 3.2 ± 0.1 d
7.2 0.3 8.1 2.9 2.1 7.8
± ± ± ± ± ±
0.1 b 0.1 e 0.2 a 0.2 c 0.1 d 0.1 a
8.0 0.2 11.2 4.2 3.5 10.3
± ± ± ± ± ±
0.2 c 0f 0.3 a 0.1 d 0.2 e 0.2 b
652.2 ± 25.6 ± 777.0 ± 368.1 ± 320.3 ± –
35.4 b 0.4 e 13.0 a 9.9 c 5.8 d
Each value expressed as the mean ± SD (n = 3). Each value with different letters in the same column was considered significantly different (p < 0.05). Positive control.
69.7 ± 0.7 b Trace e 118.0 ± 1.0 a 7.6 ± 0.5 c 4.9 ± 0.1 d –
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et al. (2008), where the ethanolic extract of twigs of Cinnamomum osmophloeum with abundant phenolic components is well correlated with their antioxidant activity. Therefore, sequential studies must be carried out to isolate and identify the antioxidant phenolic compounds from the EtOAc fraction of A. confusa root extract. 3.6. Determination of total flavonoid contents of A. confusa root extract
Fig. 3. Superoxide anion radical scavenging activity of ethanolic extract from A. confusa root. () Crude extract; () n-Hex fraction; () EtOAc fraction; (×) n-BuOH fraction; (䊉) water fraction; and (+) (+)-catechin. Results are mean ± SD (n = 3).
demonstrate crude extract and EtOAc fraction of A. confusa root show better superoxide anion radical scavenging activity than the famous antioxidant-(+)-catechin. Among them, EtOAc fraction exhibited the strongest radical scavenging activity and its IC50 value was 1.4-fold lower than that of (+)-catechin. Therefore, the EtOAc fraction of A. confusa root extract would be an excellent source of natural antioxidant. 3.4. Reducing power of A. confusa root extract Previous reports have demonstrated the reducing power is often used as an indicator of electron-donating ability, which is an important mechanism of phenolic antioxidant activity (Dorman et al., 2003). Ferric (Fe3+ ) can reduce to ferrous (Fe2+ ) by antioxidants because of their reductive capabilities. Table 1 shows the trolox equivalence (TE) of reducing power of crude extract and its derived fractions, with their ranking order being as follows: EtOAc fraction (11.2 mmol of TE/g) > crude extract (8.0 mmol of TE/g) > n-BuOH fraction (4.2 mmol of TE/g) > water fraction (3.5 mmol of TE/g) nHex fraction (0.2 mmol of TE/g). Again, EtOAc fraction exhibited the strongest activity and its TE was higher than (+)-catechin (10.3 mmol of TE/g), which was used as a positive control. Previously, Tung et al. (2009a) reported hot water extract from leaves of A. confusa possessed good antioxidant activity. However, the reducing power of hot water extract of A. confusa leaves and its derived fractions were lower than that of (+)-catechin. Accordingly, the reducing power of EtOAc fraction of A. confusa root extract is better than the hot water extract of A. confusa leaves. 3.5. Determination of total phenolic contents of A. confusa root extract Phenolic compounds from plant extract often display antioxidant activity. This activity is believed to be mainly due to their hydrogen donor and electron donor properties, which play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides (Adeolu et al., 2009). Table 1 shows the gallic acid equivalence (GAE) of the total phenolic contents of crude extract and its derived fractions, with their ranking order being as follows: EtOAc fraction (777.0 mg of GAE/g) > crude extract (652.2 mg of GAE/g) > n-BuOH fraction (368.1 mg of GAE/g) > water fraction (320.2 mg of GAE/g) n-Hex fraction (25.6 mg of GAE/g). The ranking order of phenolic content of A. confusa root extract and its derived fractions is consistent with their antioxidant results mentioned above, indicating the antioxidant activities of these samples should be connected to their phenolic contents. A similar finding has been demonstrated by Chua
Flavonoids, a group of phenolic compounds widely distributed in the plant kingdom, exhibit many biochemical and pharmacological effects, including antioxidation, antiinflammation and antiallergy (Havsteen, 1983). Herein, a total flavonoid contents assay was carried out to estimate their correlation with antioxidant activity. Table 1 expresses the rutin equivalence of the total flavonoid contents of A. confusa root extract and its derived fractions, and its decreasing order ranks as EtOAc fraction (118.0 mg of Rutin/g) > crude extract (69.7 mg of Rutin/g) n-BuOH fraction (7.6 mg of Rutin/g) > water fraction (4.9 mg of Rutin/g) > n-Hex fraction (trace). This result indicated most flavonoid components of A. confusa root extract are present in its EtOAc fraction. It is notable total flavonoid contents are much lower than total phenolic contents, suggesting A. confusa root extract and its derived fractions contain not only flavonoid components, but also many other types of phenolic components with antioxidant activity. 3.7. Quantitative analysis and antioxidant activities of phytochemicals from EtOAc fraction of A. confusa root extract According to the above-mentioned results, the EtOAc fraction exhibited the best DPPH radical scavenging, superoxide anion radical scavenging, TEAC values, and reducing power. Therefore, a phytochemicals study of the EtOAc fraction was carried out to separate and purify active compounds by column chromatography and HPLC. Then, nine compounds were isolated and their chemical structures were identified by comparing their NMR and MS data with the literatures. Fig. 1 shows the chemical structures of these nine compounds, including 3,4-dihydroxybenzoic acid (1), isomelacacidin (2), melacacidin (3), 4-O-methyl-melacacidin (4), 2,3-trans-3,7,8,3 ,4 -pentahydroxydihydroflavone (5), 2,3-cis3,7,8,3 ,4 -pentahydroxydihydroflavone (6), melanoxetin (7), transilitin (8), and okanin (9) (Wu et al., 2005; Foo and Wong, 1986; Foo, 1986). Among them, compounds 2 and 6 were identified from this species for the first time. To determine the antioxidant activities of nine phytochemicals, DPPH, TEAC, NBT, and reducing power assays were carried out, and these results are expressed in Table 2. Quercetin was used as a positive control. Concerning DPPH radical scavenging activity, compounds 7–9 exhibited better activity than quercetin with an IC50 value of 3.1 M. In addition, compounds 2–6 exhibited parallel activity to quercetin. Then, the superoxide anion radical scavenging activity of these nine phytochemicals can be ranked in a decreasing order of 9 = 7 > 8 > 4 3 2 > 5 > 6 1. Moreover, compounds 7 and 9 exhibited stronger superoxide anion radical scavenging activity than quercetin, and their IC50 values were 2.5 and 2.2 M, respectively. Xanthine oxidase is an enzyme that catalyzes the oxidation of hypoxanthine to xanthine and that of xanthine to uric acid, along with the generation of hydrogen peroxide and superoxide anion (Rasmussen et al., 2000). Over expression of xanthine oxidase may cause overproduction of hydrogen peroxide and superoxide anion, leading to oxidative stress and tissue injury. Previously, Tung and Chang (2010) reported melanoxetin (7) and okanin (9) are excellent xanthine oxidase inhibitors with competitive and noncompetitive mode, respectively. Therefore, it can be suggested compounds 7 and 9 revealed better NBT assay activity than other compounds through not only the superoxide anion radical scavenging effect,
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Table 2 DPPH radical scavenging activity, superoxide radical scavenging activity, trolox equivalent antioxidant capacity, reducing power and content of major phytochemicals from A. confusa root.a Phytochemicals
IC50 (M) DPPH radical
3,4-Dihydroxybenzoic acid (1) Isomelacacidin (2) Melacacidin (3) 4-O-Methyl-melacacidin (4) 2,3-Trans-3,7,8,3 ,4 -pentahydroxydihydroflavone (5) 2,3-Cis-3,7,8,3 ,4 -pentahydroxydihydroflavone (6) Melanoxetin (7) Transilitin (8) Okanin (9) Quercetinb a b
5.9 4.5 3.9 4.3 4.6 3.9 3.1 3.1 3.1 3.9
± ± ± ± ± ± ± ± ± ±
0.4 a 0.5 b 0.6 bc 0.7 b 0.3 b 0.2 b 0.2 c 0.1 c 0.1 c 0.2 b
TEAC (mmol of TE/mmol)
Reducing power (mmol of TE/mmol)
Contents (mg/g of crude extract)
Superoxide radical >50 a 5.0 ± 0.1 d 4.7 ± 0.1 de 4.4 ± 0.1 e 11.9 ± 0.9 b 10.2 ± 0.3 c 2.5 ± 0.1 g 3.4 ± 0.1 f 2.2 ± 0.1 g 3.7 ± 0.2 f
2.5 4.2 4.4 4.4 4.3 4.6 4.4 2.9 5.4 3.9
± ± ± ± ± ± ± ± ± ±
0.2 c 0.1 b 0.4 b 0.3 b 0.2 b 0.3 ab 0.2 b 0.2 c 0.3 a 0.2 b
2.7 5.2 5.0 5.1 4.5 4.7 5.0 4.5 5.3 4.2
± ± ± ± ± ± ± ± ± ±
0.2 c 0.3 a 0.2 a 0.1 a 0.2 ab 0.1 ab 0.1 a 0.1 ab 0.3 a 0.1 b
7.8 ± 41.5 ± 109.1 ± 23.6 ± 19.7 ± 34.6 ± 65.7 ± 28.3 ± 6.9 ± –
0.3 h 1.0 c 2.2 a 0.4 f 0.4 g 0.6 d 1.5 b 0.4 e 0.4 h
Each value expressed as the mean ± SD (n = 3). Each value with different letters in the same column was considered significantly different (p < 0.05). Positive control.
but also the xanthine oxidase inhibitory effect. Moreover, due to the xanthine oxidase inhibitory ability, melanoxetin and okanin may possess cellular antioxidant potential. As for TEAC and reducing power assays, the antioxidant abilities were present in trolox equivalence (TE/mmol of sample). Among all nine phytochemicals, okanin (9) exhibited the best TEAC activities with TE value of 5.4, which is 1.4-fold higher than quercetin. Otherwise, TEAC activities of compounds 2–7 were also comparable to quercetin. On the other hand, compounds 2–4, 7 and 9 revealed obviously higher reducing power ability than that of quercetin, whereas compounds 5, 6 and 8 had similar activities to quercetin. Based on the above-mentioned results, okanin (9) is the best antioxidant compound among these nine phytochemicals. Fig. 4 gives the HPLC profile of the EtOAc fraction of the ethanolic extract of A. confusa root. By following this gradient condition, the quantitative analysis of compounds 1–9 were determined to be 7.8, 41.5, 109.1, 23.6, 19.7, 34.6, 65.7, 28.3 and 6.9 mg/g of crude extract, respectively (Table 2). Accordingly, melacacidin (3) is the most abundant phenolic compound in the ethanolic extract
of A. confusa root, and it also reveals remarkable antioxidant activities. 3.8. Structure–activity relationships of flavonoids from A. confusa root extract This study also investigated the structure–activity relationships of flavonoids in term of their antioxidant activities, including DPPH, NBT, TEAC and reducing power assays. Previously, Heim et al. (2002) reported conjugated flavonoids exhibit better antioxidant activities than the corresponding non-conjugated ones because they permit electron delocalization and then increase the stability of the corresponding flavonoid radicals generated by the ROS. Thus, flavonoids from A. confusa can be divided into conjugated flavonoids (7–9) and non-conjugated flavonoids (2–6). For conjugated flavonoids, okanin (9) exhibited the best antioxidant activities, probably because it is free of both catechol and pyrogallol structures as well as the connection between 2 aromatic groups by an ␣-,-unsaturated carbonyl group. Melanoxetin
Fig. 4. HPLC profile of the EtOAc soluble fraction of root extract from A. confusa.
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(7) showed less antioxidant activities than okanin (9) because of a closed C-ring result in (i) etherization of pyrogallol of the Aring, and (ii) distortion of planarity of the conjugating system. Etherization of the phenol group by the alkyl groups or sugars decreases the antioxidant activities of flavonoids. Distortion of the planarity of the conjugated system lowers the delocalizing effect, diminishes the stability of flavonoid radicals, and reduces the antioxidant activities (Heim et al., 2002). Although the DPPH scavenging activity of transilitin (8) and melanoxetin (7) were comparable, melanoxetin revealed better NBT, TEAC and reducing power activities than transilitin. Van Acker et al. (1996) provided an explanation for this result. They found flavon-3-ol shows better antioxidant activities than its 3-O-methyl derivative because of the intramolecular hydrogen bonding between the 3-hydroxy and 4-oxo groups, resulting in the enhancement of planarity of the conjugated system. Tung et al. (2009a) also reported myricetin showed better DPPH, NBT and reducing power activities than myricetin-3-O-methyl ether, which is consistent with our result. For non-conjugated flavonoids, the DPPH and NBT activities of compounds 2–4 are slightly lower than compounds 7–9, agreeing with the report of Heim et al. (2002). However, it is interesting their TEAC and reducing power activities are similar to melanoxetin (7). Obviously, the 7,8-dihydroxyl configuration of the A-ring is the most significant structural feature of flavonoids isolated from A. confusa root extract. It is well known phenolic compounds with ortho- and para-dihydroxyl groups exhibit better antioxidant activities than that with meta-dihydroxyl groups (Heim et al., 2002). Therefore, it can be speculated the contribution of antioxidant abilities from these flavonoids is through not only the catechol structure in the B-ring, but also the pyrogallol derivative structure in the A-ring, leading to strong enhancement of their activities. Accordingly, these 7,8-dihydroxyl flavonoids should exhibit better antioxidant activities than the corresponding 5,7-dihydroxyl flavonoids. For example, compounds 2–4 showed lower IC50 values of DPPH and NBT than (+)-catechin (10.3 and 11.0 M) and higher TEAC and reducing power values than (+)-catechin (2.3 and 3.0 mmol of TE/mmol). Melanoxetin (7) also showed better activities than quercetin for four assays (Table 2). Compounds 5 and 6 revealed similar DPPH, TEAC and reducing power activities with compounds 2–4, but their NBT assay activity were apparently lower than compounds 2–4. This may be due to the electron-withdrawing effect caused by the 4-oxo group of compounds 5 and 6, leading to a decrease in activities (Heim et al., 2002).
4. Conclusions In this study, the antioxidant activities of the ethanolic extract of A. confusa root and its active compounds were reported for the first time. The ethyl acetate soluble fraction showed the best antioxidant ability and the highest yield. Afterwards, nine major phenolic compounds were isolated from this fraction, and their chemical structures were successfully elucidated by comparing the spectra with literature data. Flavonoids are the major antioxidant phytochemicals from A. confusa root and their strong antioxidant capacities are derived from 7,8-dihydroxyl groups of the A-ring as well as 3 ,4 -dihydroxyl groups of the B-ring. Therefore, these results demonstrated A. confusa root extract has great potential in preventing diseases caused by overproduction of ROS. In addition, it could serve as a potent source of natural antioxidant in the nutraceutical, beneficial additives, and food industries. Further studies should focus on the in vivo pharmacological research of these strong antioxidant flavonoids.
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