Journal of the Science of Food and Agriculture J Sci Food Agric 88:1400–1405 (2008)

Antioxidant activity of the ethanolicextract from the bark of Chamaecyparisobtusa var. formosanaPalanisamy Marimuthu, Chi-Lin Wu, Hui-Ting Chang and Shang-Tzen Chang∗School of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan

Abstract

BACKGROUND: Chamaecyparis obtusa var. formosana (Taiwan hinoki) is an endemic conifer in Taiwan andthe purpose of this study is to evaluate the antioxidant activity of various fractions obtained from the bark ofthis plant material. The ethanolic extract of the bark was sequentially separated into three fractions, includingn-hexane, ethyl acetate and ethanol soluble fractions, by liquid–liquid partition. Then the antioxidant activities ofcrude extract and three fractions along with 13 subfractions obtained from the ethyl acetate (EA) soluble fractionwere tested for several antioxidant assays.

RESULTS: The total phenolic content of the samples varied from 27.71 to 102.86 mg GAE g−1 dry weight forfractions, and from 49.94 to 206.46 mg GAE g−1 for subfractions (where GAE is milligrams of gallic acid pergram of extract). The Trolox equivalent antioxidant capacity (TEAC) ranged from 0.15 to 0.26 mmol L−1 Troloxequivalents. The EA soluble fraction was found to be the best antioxidant-rich fraction in terms of DPPH andreducing power assays. With further data analysis it was found that there was a positive correlation between thetotal phenolic content of extracts and TEAC is R2 = 0.61.

CONCLUSION: Results from various antioxidant assays showed that the EA fraction possessed strong antioxidantactivity. This would provide additional information about the antioxidant activity of bark extract of this plantspecies. 2008 Society of Chemical Industry

Keywords: antioxidant activity; bark extract; β-carotene bleaching assay; Chamaecyparis obtusa var. formosana;total phenolic content; Trolox equivalent antioxidant capacity

INTRODUCTIONPlants have been used in many domains includingmedicine, nutrition, flavourings, beverages, dyeing,repellents, fragrances, cosmetics and other industrialpurposes. Since the prehistoric era, plants havebeen the basis for nearly all medicinal therapyuntil synthetic drugs were developed in the 19thcentury.1,2 The preservative effect of many plantextracts suggests the presence of antioxidative andantimicrobial constituents in their tissues.3,4 Recently,interest has increased considerably in finding naturallyoccurring antioxidants for use in foods or medicinalmaterials to replace synthetic antioxidants, which arebeing restricted due to their carcinogenicity.5

Many medicinal plants contain large amounts ofantioxidants such as polyphenols, which can playan important role in adsorbing and neutralising freeradicals, quenching singlet and triplet oxygen, ordecomposing peroxides. Many of these phytochem-icals possess significant antioxidant capacities that areassociated with lower occurrence and lower mortalityrates of several human diseases.6 It has been reported

that there is an inverse relationship between the antiox-idative status occurrences of human diseases.7 In addi-tion, antioxidant compounds which are responsible forsuch antioxidant activity could be isolated and thenused as antioxidant for the prevention and treatmentof free radical-related disorders.8 Therefore, researchto identify antioxidative compounds is an importantissue. Although it remains unclear which of the com-pounds from medical plants are the active ones,polyphenols have recently received increasing atten-tion because of some interesting new findings regard-ing their biological activities. From pharmacologicaland therapeutic points of view, the antioxidant proper-ties of polyphenols, such as free-radical scavenging andinhibition of lipid peroxidation, are the most crucial.

There are seven species of the genus Chamaecyparis(Cupressaceae), but only two endemic species, C. for-mosensis and C. obtusa var. formosana, are found in thecentral mountains of Taiwan. Five new cadinane-typesesquiterpenes were isolated from the heartwoodextract of C. obtusa var. formosana9 and also a recentreport explains the dominancy of this plant partly due

∗ Correspondence to: Shang-Tzen Chang, School of Forestry and Resource Conservation, National Taiwan University, Taipei 106, TaiwanE-mail: peter@ntu.edu.tw(Received 29 November 2007; revised version received 11 January 2008; accepted 17 January 2008)Published online 17 April 2008; DOI: 10.1002/jsfa.3231

2008 Society of Chemical Industry. J Sci Food Agric 0022–5142/2008/$30.00

Antioxidant activity of C. obtusa

to the allelopathic potential, when it was planted alongwith other plant species.10 Both of these species areimportant building materials, and the latter species isbelieved to be more resistant to wood-decaying fungi.

The choice of our investigated plant is based on twocriteria: firstly, in this domain there is no antioxidativestudy that deals with this plant; and secondly, thisplant is believed to be more resistant to wood-decayingfungi.11 Although there are several earlier studieson the chemical constituents12,13 and antitermite14

studies on C. obtusa var. formosana, there are no studiesreported to antioxidant activity of bark of this plantspecies. This evaluation is related to the total phenoliccontent and antioxidant activity to determine newpotential sources of natural antioxidants from the barkof C. obtusa var. formosana.

MATERIALS AND METHODSPlant materialThe bark was collected from the 600-year-oldChamaecyparis obtusa var. formosana tree locatedin the central Taiwan. A voucher specimen wasdeposited in the laboratory of wood chemistry, Schoolof Forestry and Resource Conservation, NationalTaiwan University.

Chemicals2,2′-Diphenyl-1-picrylhydrazyl radical (DPPH),potassium dihydrogen phosphate, trichloracetic acid,3-(-2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid), β-carotene, lipoxidase (type I) from Glycine max Mer-rill (soybean), Folin–Ciocalteu reagent, quercetin,β-carotene and (+)-catechin were purchased fromSigma Chemical Co., St Louis, MO. Linoleic acidwas from Acros, Morris Plains, NJ, USA. All othersolvents and reagents were purchased from Sigma.

Extraction and isolationFifty kilograms of bark from the plant material wasdried at room temperature and milled. The milledmaterial was percolated in 95% ethanol (2 × 60 L) atroom temperature for 6 days. Then, the extract wasfiltered and solvent was evaporated under reducedpressure gave an extract (472.4 g), which was subjectedto liquid–liquid partition successively with n-hexanethen ethyl acetate (EA) to give the n-hexane (180.5 g)and EA soluble fractions (166 g), respectively. Theremaining fraction was considered as the ethanolsoluble fraction (22.6 g).

Based on the results obtained from antioxidantassays (DPPH and reducing power), the EA solublefraction was considered to be the antioxidant-richfraction. One hundred grams of the EA soluble fractionwas applied to a silica gel open column and elutedwith a stepped gradient consisting of n-hexane, EA,acetone, ethanol and water. The samples collectedwere screened by thin-layer chromatography (TLC)profile and fractions having similar TLC patterns werecombined: 13 fractions (SF1–SF13) were obtained.

Based on the DPPH screening assay, fraction 11was found to be the antioxidant-rich fraction. It wasapplied (20 g) to a RP-18 open column and eluted witha gradient of MeOH–H2O and further fractionatedinto 13 subfractions. Then the fractions were studiedfor antioxidant activity by using the DPPH assay anddetermining the reducing power, and also for theirtotal phenolic content.

Antioxidant assaysDPPH assayThe DPPH assay was carried out as reportedpreviously.15 Fifty microliters of sample solution (100,50, 25, 12.5 µg mL−1 as per final concentration)were added to 450 µL of Tris-HCl buffer and1 mL of 0.1 mmol L−1 methanol solution of DPPH.After a 30 min incubation at room temperature, theabsorbance was read against a blank at 515 nm in aJasco V-550 UV–visible spectrophotometer (Tokyo,Japan). The assay was carried out in triplicate andresults were averaged. (+)-Catechin was used as apositive reference.

Reducing powerThe reducing power was determined as describedpreviously.16 Various amounts (final concentration of50, 25, 12.5, 6.25 µg mL−1) of fractions or subfractions(dissolved in methanol) were mixed with 0.5 mLof 0.2 mol L−1 phosphate buffer (pH = 6.6) and0.5 mL of 1% potassium ferricyanide, and the mixturewas incubated at 50 ◦C for 20 min. After adding0.5 mL of 10% trichloroacetic acid, the mixture wascentrifuged at 976 × g for 10 min in a Hettich Micro22R model centrifuge (Tuttlingen, Germany). Thesupernatant (0.5 mL) was mixed with 0.55 mL ofdistilled water and 0.1 mL of 0.1% ferric chlorideand the absorbance read at 700 nm in a Jasco V-500UV–visible spectrophotometer. Quercetin was used asa positive control.

β-Carotene bleaching assayThe β-carotene antioxidant assay was carried outas given by Chaillou and Nazareno17 and Kulisicaet al.18 with slight modifications. All the reagents andsolutions were prepared according to the procedurereported in the literature. Initially, 2.5 mL of β-carotene solution was thoroughly mixed with 200 µLof linoleic acid. Then, 200 µL of lipoxidase were addedfollowed by 100 µL of sample (final concentrations of100, 50, 25, 12.5, 6.25 µg mL−1). The absorbanceof the control sample was measured immediately(t = 0) and t = 10 min. Reading of samples containingantioxidants were measured at 10 min at 460 nm ina Jasco V-550 UV–visible spectrophotometer. Alldeterminations were performed in triplicate. Thepercentage of β-carotene inhibition was calculated as

% inhibition = (1 − ((AS(0) − AS(10))/

(AC(0) − AC(10)))) × 100

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where AS(0) is the absorbance of the sample att = 0 min; AS(10) is the absorbance of the sample att = 10 min; AC(10) is the absorbance of the control att = 10 min; and AC(0) is the absorbance of the controlat t = 0 min.

Quantification of total antioxidant activityThe total antioxidant activity values were estimated bythe Trolox equivalent antioxidant capacity (TEAC)assay.19 In this test, we measured the relative capac-ity of antioxidants to scavenge the 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammoniumsalt (ABTS) radical compared to the antioxidantpotency of Trolox is used as a standard. The ABTSradical generated by mixing a solution of ABTS(7 mmol L−1) with K2S2O8 (2.45 mmol L−1). Beforeuse, the ABTS solution was diluted with water toobtain an absorbance of 0.700 ± 0.020 at 734 nm.Upon adding 1485 µL of the diluted ABTS solutionto 15 µL of antioxidant sample or Trolox standard,the absorbance at 734 nm was recorded by a JascoV-550 UV–visible spectrophotometer 6 min after ini-tial mixing. Appropriate solvent blanks were run ineach assay, and all measurements are done at leastthree times. Decreases in absorbance were noted andthen calculated and plotted with respect to absorbanceand concentration of the standard and samples. Thefinal TEAC value of the antioxidant compound wascalculated by comparing ABTS decolorisation withTrolox, which gives a useful indication of the antioxi-dant potential of the specimen.

Determination of total phenolics according to theFolin–Ciocalteu methodThe amount of total phenolics was measured by theFolin–Ciocalteu method15 using gallic acid as stan-dard, for which a calibration curve was obtained withsolutions of 0.08, 0.04, 0.02, 0.01, and 0.005 mgmL−1 of this compound (y = 37.907x − 0.093, R2 =0.9991). A 0.4 mL aliquot of diluted extract (allfractions were diluted with methanol to adjust theabsorbance within the calibration limits), 0.4 mL of1 mol L−1 Folin–Ciocalteu reagent, and 0.8 mL ofNa2CO3 (20%, w/v) were mixed. After 8 min, the mix-ture was centrifuged at 15 616 × g for 10 min. Thenthe absorbance of the supernatant solution was mea-sured at 730 nm by using a Jasco V-550 UV–visiblespectrophotometer and against a blank prepared sim-ilarly but containing distilled water instead of extract.The concentration of phenolics thus obtained wasmultiplied by the dilution factor and the results wereexpressed as the equivalent to milligrams of gallic acidper gram of extract (mg GAE g−1).

Statistical analysisFor all the extracts three samples were prepared forassays of every antioxidant attribute. The data werepresented as mean ± standard deviation of threedeterminations. The significance of difference was

analysed using SAS Scheffe’s statistics software anda value of P < 0.05 was considered significant.

RESULTS AND DISCUSSIONAntioxidant activities of bark extract and itsfractions from C. obtusa var. formosanaDPPH and reducing power assayThe scavenging effect of crude (71.72–12.06%),n-hexane (38.68–6.81%), EA (83.06–32.12%) andethanol (80.56–24.31%) soluble fractions on DPPHradical increased linearly with increasing concen-tration (Fig. 1) at 100, 50, 25 and 12.5 µg mL−1.The IC50 values of EA, ethanol, crude and (+)-catechin were found to be 21.88, 31.07 56.65 and2.18 µg mL−1, respectively. Wang et al.20 reported thatthe IC50 of an ethanolic extract of Calocedrus for-mosana bark, which belongs to the same family, was23 µg mL−1, which is higher in comparison with ourplant. The reducing power of various soluble frac-tions increased with increasing concentration (Fig. 2).Based on optical density values of the fractions, theantioxidant activity can be ranked in the followingdescending order: EA fraction > ethanol fraction >

crude extract > n-hexane fraction.

Total phenolic content and TEAC assayThe amount of total phenolics varied in differentfractions and ranged from 27.71 to 102.86 mgGAE g−1 of dry material (Table 1) while for thesubfractions it was ranged from 49.94–206.46 mgGAE g−1 (Table 2). EA soluble fraction showed higherphenolic content (102.86 mg GAE g−1) followedby ethanol, crude and n-hexane extract (27.71 mgGAE g−1). TEAC values are expressed in mmol L−1

Trolox equivalent. The ethanol fraction has a higherTEAC value of 0.26 mmol L−1 Trolox equivalentfollowed by the EA, crude and n-hexane fractions(Table 1). Wang et al.20 also demonstrated that thetotal phenolic content of the ethanol extract fromCalocedrus formosana heartwood (159.5 ± 1.9 mg GAE

Figure 1. Antioxidant activity of extract and various soluble fractionsof C. obtusa var. formosana bark in terms of the DPPH radicalscavenging assay.

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Figure 2. Antioxidant activity of extract and various soluble fractionsof C. obtusa var. formosana bark in terms of the reducing powerassay.

g−1) was higher than that of the bark extract (115.3 mgGAE g−1), which is relatively lower (90.72 ± 0.18)in Chamaecyparis obtusa var. formosana. The report21

on antioxidant activity of n-hexane, EA, n-butanoland water extracts from the bark of Chamecyparislawosoniana revealed that the EA fraction exhibiteda higher total phenolic content (337 mg GAE g−1)and a lower IC50 value (6.53 µg mL−1) in the DPPHassay. The EA fraction from our bark extract belongingto the same family showed lower total phenoliccontent (102.86 mg GAE g−1) and higher IC50 value(21.88 µg mL−1) in the DPPH assay. At the sametime, our crude bark extract showed relatively highertotal phenolic content in comparison with Juniperusoxycedrus22 from the same family.

β-Carotene bleaching assayThis method is based on the loss of the yellow colourof β-carotene due to its reaction with radicals thatare formed by oxidation of linoleic acid, inducedby lipoxidase in the emulsion. It was reported thatlinoleic acid is the preferred substrate for lipoxidaseas it particularly attacks the fatty acid containinga 1-cis, 4-cis-pentadiene system.23 The rate of β-carotene bleaching can be reduced in the presenceof antioxidants. This fact is used in the determination

Table 1. Total phenolic content and Trolox equivalent antioxidant

capacity values for extract and various fractions of C. obtusa var.

formosana bark

SpecimenTotal phenolic content

(mg GAE g−1)TEAC (mmol L−1

trolox equivalent)

Crude extract 50.86 ± 1.61c 0.19 ± 0.01b

n-Hexane fraction 27.71 ± 0.18d 0.15 ± 0.00b

EA fraction 102.86 ± 0.78a 0.21 ± 0.05b

Ethanol fraction 90.72 ± 0.18b 0.26 ± 0.01b

Quercetin – 3.55 ± 0.11a

Numbers followed by different letters (a–d) are statistically different atthe probability level of P < 0.05 according to Scheffe’s analysis.Each value is mean ± SD of three measurements.

Table 2. Total phenolic content, DPPH (IC50 value) and reducing

power of subfractions fractionated on a reverse-phase open column

Specimen

Total phenoliccontent

(mg GAE g−1)DPPH IC50

(µg mL−1)Reducingpower∗

SF1 49.94 ± 1.15i 82.87 ± 1.73a 0.75 ± 0.01h

SF2 84.86 ± 0.75h 49.93 ± 0.22b 1.29 ± 0.10f

SF3 134.77 ± 0.50e 37.52 ± 0.07c 1.27 ± 0.03f

SF4 177.52 ± 0.50b 26.43 ± 0.72d 1.77 ± 0.04e,d

SF5 206.46 ± 1.34a 14.82 ± 0.47g 2.22 ± 0.01b

SF6 141.02 ± 0.57d 16.82 ± 0.17g,f 2.10 ± 0.05c,b

SF7 138.52 ± 0.78e,d 17.00 ± 0.24g,f 1.94 ± 0.01c,d

SF8 172.58 ± 1.78c 15.05 ± 0.21g 2.06 ± 0.03c,b

SF9 139.75 ± 0.32d 17.30 ± 0.29g,f 1.77 ± 0.07e,d

SF10 135.04 ± 0.98e 23.89 ± 1.41e 1.39 ± 0.15g,f

SF11 113.02 ± 0.76f 26.79 ± 0.52d 1.66 ± 0.05e,f

SF12 91.60 ± 0.84g 22.63 ± 0.18f 2.19 ± 0.05c,b

SF13 109.21 ± 1.37f 23.14 ± 0.25e 1.76 ± 0.09e,d

Catechin – 2.18 ± 0.03h –Querectin – – 2.49 ± 0.01a

Numbers followed by different letters (a–d) are statistically differentat the probability level of P < 0.05 according to Scheffe’s analysis.SF, subfraction. ∗ Absorbance value at 700 nm, subfractions at finalconcentration of 50 µg mL−1. Each value is mean ± SD of threemeasurements.

of antioxidant activity of fractions obtained from C.obtusa var. formosana. Figure 3 shows the antioxidantactivity of various fractions, among which the EAfraction showed strong antioxidant activity with75.82% inhibition of β-carotene at a concentrationof 100 µg mL−1. IC50 values of the ethanol fraction,EA fraction, n-hexane fraction, crude extract andquercetin were found to be 43.16, 43.90, 92.45, 65.76and 4.27 µg mL−1, respectively. In terms of percentinhibition of β-carotene bleaching at 100 µg mL−1,antioxidant activity of various fractions can be rankedas EA fraction = ethanol fraction > crude extract >

n-hexane fraction.In the modified β-carotene assay, the percent

inhibition of various fractions at higher concentration(100 µg mL−1) were significantly different, but at lower

Figure 3. Antioxidant activity of extract and various soluble fractionsof C. obtusa var. formosana bark in terms of the modified β-carotenebleaching assay.

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concentration, almost all the specimens (except the n-hexane fraction) showed similar percent inhibition.At the lower concentration (6.25 µg mL−1), the n-hexane fraction gave a negative antioxidant value(−0.81%); this represents a pro-oxidant effect of then-hexane fraction in this system. This result is inaccord with the pro-oxidant effect of cinnamic acidshown by Chaillou and Nazareno.17 The presence ofdifferent antioxidant components in the plant tissuesmakes it relatively difficult to quantify each antioxidantcomponent separately. Therefore, in many studies,several intermediate extractions are used to ensure amaximum extraction of the available antioxidants.24

The antioxidant activity of phenolics is mainly dueto their redox properties which make them act asreducing agents, hydrogen donors, and singlet oxygenquenchers. They may also have a metallic chelatingpotential.25

Antioxidant activities of the subfractionsfrom the ethyl acetate soluble fractionDPPH and reducing power assaySubfractions obtained from the RP-18 open columnhave IC50 values in the range 14.82–82.87 µg mL−1

(Table 2). Subfractions SF5, SF6, SF7, SF8 andSF9 exhibited strong radical scavenging effects andthese subfractions have IC50 values of 14.82–17.30 µg mL−1. As for the reducing power, subfrac-tions SF5, SF6, SF8 and SF12 showed higher reducingpower (2.10–2.22) in comparison with other samples(Table 2).

Total phenolic contentRP open column considerably increased total phenoliccontent in the subfractions SF5, SF4 and SF8.Subfraction SF5 exhibited higher total phenoliccontent followed by subfractions SF4 and SF8 incomparison with other fractions (Table 2).

The correlation coefficient, R2, between TEAC andtotal phenolic content of various solvent fractions is0.61 (Fig. 4). The antioxidant activity of fractionsmay not only be due to the presence of phenoliccompounds but also related to the presence of someindividual active components in the extracts. Theunclear relationship between the antioxidant activityand total phenolic content may be explained by the factthat the total phenolic content does not incorporate allthe antioxidants. In addition, the synergism betweenthe antioxidants in the mixture makes the antioxidantactivity not only dependent on the concentration butalso on the structure and interaction between theantioxidants. This is why the EA and ethanol fractions,which have similar total phenolic contents, varied inantioxidant performance in the TEAC assay.

Phenolic groups play an important role in antiox-idant activity.26,27 It has been reported that mostnatural antioxidative compounds often work synergis-tically with each other to produce a broad spectrum ofantioxidative activities that create an effective defencesystem against free-radical attack.28 The composition

Figure 4. Linear correlation of Trolox equivalent antioxidant capacity(TEAC) with respect to the total phenolic content of various fractionsobtained from C. obtusa var. formosana.

of the extract is very complex; it consists of variousclasses of organic compound which may exert oppo-site effects on the process of lipid oxidation. Basedon the results obtained, it is highly possible that someconstituents of different polarity may contribute to theantioxidative activity of the extract.

CONCLUSIONSIt has also been noted in this study that the EAfraction of C. obtusa var. formosana bark extract showedstrong radical scavenging and can be considered agood source of natural antioxidants for medicinaland commercial use. However, due to the diversityand complexity of the natural mixtures of phenoliccompounds in this plant extract, it is not easyto characterise every compound and assess theantioxidant activity of each one. Each plant generallycontains different phenolic compounds with differentamounts of antioxidant activity. As a result of thisstudy, we believe that in vivo studies are needed tofurther confirm the advantageous quality of thesenatural products. In order to confirm the antioxidativeeffect of these promising plant extracts, a furthersurvey, which uses other types of antioxidant assay,is now under way. This survey also includes thecharacterisation of active phenolic antioxidants.

ACKNOWLEDGEMENTWe thank the National Science Council, Taiwanfor generous financial support (NSC95-2313-B-002-012).

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