Food Chemistry 139 (2013) 1015–1020

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Food Chemistry

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Inhibitory effects of water extract of Flos Inulae on mutation and tyrosinase

Ming-Hsing Huang a, Huo-Mu Tai a, Bor-Sen Wang b,⇑, Lee-Wen Chang c,⇑a Department of Cosmetic Science, Chia-Nan University of Pharmacy and Science, No. 60, Sec.1, Erren Rd., Rende Dist., Tainan City 717, Taiwan, ROCb Department of Applied Life Science & Health, Chia-Nan University of Pharmacy and Science, No. 60, Sec.1, Erren Rd., Rende Dist., Tainan City 717, Taiwan, ROCc Department of Food Science & Technology, Chia-Nan University of Pharmacy and Science, No. 60, Sec.1, Erren Rd., Rende Dist., Tainan City 717, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history:Received 31 October 2012Received in revised form 12 January 2013Accepted 23 January 2013Available online 7 February 2013

Keywords:Flos InulaeMutationTyrosinaseOxidative damage

0308-8146/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2013.01.066

⇑ Corresponding authors. Fax: +886 6 2667321 (L.(B.-S. Wang).

E-mail addresses: wangbs@mail.chna.edu.tw (B.-Sedu.tw (L.-W. Chang).

In this study, the effects of a water extract of Flos Inulae (WFI) on antioxidant, antimutation and antityr-osinase were investigated. The results showed that WFI inhibited the mutagenicity of 2-aminoanthracene(2-AA), an indirect mutagen; and 4-nitroquinoline-N-oxide (4-NQO), a direct mutagen toward Salmonellatyphimurium TA 98 and TA 100. In addition, WFI, in the range of 0.2–0.6 mg/ml, showed radical scaveng-ing, reducing activities and chelating activity as well as decreased lipid oxidative damage. Meanwhile,WFI also inhibited tyrosinase activity and NO generation in lipopolysaccharide (LPS) stimulated macro-phages. High performance liquid chromatography analysis suggests that the major phenolic constituentsin WFI are chlorogenic acid, rutin, quercetin, luteolin and kaempferol. These bioactive components maycontribute to the protective effects of WFI. The obtained data suggests that Flos Inulae can be applied toantimutation, antityrosinase and anti-inflammation.

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1. Introduction

The lipid components in food are susceptible to oxidation,which results in some annoying effects in the course of food stor-age. Lipid oxidation not only plays a destructive role, decreasingthe quality of the food, but also contaminates the food with harm-ful substances. In addition, the browning reaction in food is an-other unfavourable effect which decreases its appearance, offflavour and nutritional value during storage. Reports have indi-cated that tyrosinase has a crucial role in the enzymatic browningreaction in food processing by catalysing the oxidation of phenolsand regulating the initial step of melanin production. Tyrosinasenot only catalyses undesirable reactions to change colour and fla-vour, deteriorate nutritional value of foods but also increases oxi-dative risk in different physiological systems (Sanchez-Ferrer,Rodriguez-Lopez, Garcia-Canovas, & Garcia-Carmona, 1995).Therefore, many studies have focused on natural additives in orderto avoid the oxidation of lipids and decrease tyrosinase activity infood production (Germanas, Wang, Miner, Hao, & Ready, 2007).

Various mutagens of contaminated foods induce oxidativestress and mutations production through different mechanism incells (Felton & Knize, 1991). For instance, 2-aminoanthracene (2-AA), a typical mutagen-carcinogen polycyclic aromatic amine,can induce tumours primarily in the liver (Baker et al., 2001).

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-W. Chang), +886 6 2667097

. Wang), leewen@mail.chna.

However, 2-AA must submit to metabolism activation to displayits carcinogenicity. In addition to 4-nitroquinoline-N-oxide (4-NQO), a direct strong mutagen, is a quinoline derivative carcinogenand also induce potent intracellular production of reactive oxygenspecies (ROS) (Arima et al., 2006). The mutagenic compounds canbe inserted between the base pairs of double strand DNA and in-crease oxidative stress in cells, which also induces cell damageand causes mutations. In addition, under inflammatory stimula-tion, macrophages may produce a substantial amount of NO andsuperoxide, which promotes the production of other toxic reactivenitrogen species (RNS), such as peroxynitrite, nitrogen dioxide anddinitrogen trioxide (Mocellin, Bronte, & Nitti, 2007). The inhibitionof nitrosative stress in cells may play an important step in prevent-ing mutation and aging diseases. Therefore, taking anti-inflamma-tory agents as dietary supplements may reduce the incidence andmortality rate for a variety of chronic disorders including cancer.Thus, studies investigating antimutagenic and anti-inflammatoryagents have received increasing attention in recent years.

Inula japonica is widely distributed in China, Japan and Korea.Flos Inulae, the dried flower of I. japonica and an edible flower, isa commonly used Chinese herb drug for treatment of digestive dis-orders, bronchitis, and inflammation, etc. (Hsu et al., 1985). It hasmedicinal properties as well as nutritional value and is now beingpromoted as a healthy food. Recently, an herbal or function teaprepared from Flos Inulae is available in teabags on the shelvesof markets in Taiwan and China. Extracts of Flos Inulae are re-ported to have a wide variety of activity, such as antioxidant, anti-bacterial, antitumour, and anti-inflammatory (Park, Kim, & Kim,2000). Flos Inulae contains high amounts of bioactive compounds,

1016 M.-H. Huang et al. / Food Chemistry 139 (2013) 1015–1020

such as phenolic acids, flavonoids, and sesquiterpene lactones (Baiet al., 2005; Zhou, Bai, Lin, & Cordell, 1994). A number of com-pounds in the herb have been isolated and identified, such as chlor-ogenic acid, rutin, quercetin, luteolin, kaempferol, arachidic acidand apigenin, among others. To establish the fingerprint chromato-gram for the quality control of Flos Inulae, chlorogenic acid, rutin,quercetin, luteolin and kaempferol were used as markers. An opti-mised HPLC-DAD technique was employed. Although the flower ofI. japonica has shown some physiological effects, there are fewstudies focusing on their effects on the mutation and tyrosinaseactivity so far as we know. Thus, the objective of the present studywas to investigate the modulation capacity of Flos Inulae on tyros-inase activity, environmental mutation and the protection pro-vided against oxidative damage.

2. Materials and methods

2.1. Materials

The compounds 4-nitroquinoline-N-oxide (4-NQO), 2-aminoan-thracene (2-AA), lipopolysaccharide (LPS, Escherichia coli O127:B8),N-(1-naphthyl) ethylenediamine dihydrochloride, sulphanilamide,thiobarbituric acid (TBA), 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphe-nyltetrazolium bromide (MTT), and mushroom tyrosinase werepurchased from Sigma–Aldrich (St. Louis, MO, USA). The com-pounds L-3,4-dihydroxyphenylalanine (L-DOPA) was obtained fromAcros Organic (Geel, Belgium). Culture medium was prepared bydissolving 0.8 g of nutrient broth in 100 ml of water. The minimalagar plates were made up by 2% glucose, 1.67 mM NaHNH4PO4-

�4H2O, 57.41 mM K2HPO4, 9.51 mM citric acid monohydrate,0.81 mM MgSO4�7H2O and 15 g agar in 1000 ml of sterile water.Top agar was prepared by adding 5 g NaCl and 6 g agar in1000 ml of sterile water. Dried samples of Flos Inulae were ob-tained from Sun Ten Pharmaceutical Co., Ltd., in Taipei, Taiwan.

2.2. Sample preparation

A 100 g sample of pulverized Flos Inulae was extracted withwater (1000 ml) at 100 �C for 60 min and then centrifuged at10,000g for 20 min. The extract was filtered and the residue wasre-extracted under the same conditions. The combined filtratewas then freeze-dried. The yield obtained was 13.5% (w/w). The fi-nal sample was named as WFI (water extract of Flos Inulae).

2.3. Total polyphenolics assay

Total polyphenolics were determined as gallic acid equivalents.The different concentrations of WFI were added to a 10 ml volu-metric flask, to which 2 ml sodium carbonate (20% (w/v)) wasadded. After 5 min, 0.1 ml Folin–Ciocalteu reagent (50% (v/v))was added and the volume was made up to 10 ml with H2O. After1 h incubation at 30 �C, the absorbance was measured at 750 nmand compared to a gallic acid calibration curve (Shahidi, Liyana-Pathirana, & Wall, 2006).

2.4. Total flavonoid content assay

WFI (1.0 ml) was incubated with 0.1 ml (2-aminoethyl) diphe-nyl borate (0.2% in ethanol). After 20 min of incubation, the absor-bance was measured at 405 nm. The absorbance of rutin solutionswas detected under the same conditions. The amount of flavonoidsin WFI (in rutin equivalents) was calculated (Oomah & Mazza,1996).

2.5. Determination of effect on DPPH radical

The effect of samples on the DPPH radical was estimated as de-scribed previously (Hatano, Kagawa, Yasuhara, & Okuda, 1988).The samples (0.2–1.0 mg/ml, 1 ml) were added to a methanolicsolution (1 ml) of DPPH radical (final concentration of DPPH was0.2 mmol/l). The mixture was shaken vigorously and allowed tostand at room temperature for 30 min; the absorbance of theresulting solution was then measured spectrophotometrically at517 nm.

2.6. Determination of ABTS radical inhibition

The ABTS�+ scavenging activity was measured as previously de-scribed (Arnao, Cano, & Acosta, 2001). The ABTS�+ was generated byreacting 1 mM ABTS with 0.5 mM hydrogen peroxide and 10 units/ml horseradish peroxidase in the dark at 30 �C for 2 h. After 1 mlABTS�+ was added to samples, the absorbance at 734 nm was re-corded after 10 min. A lower level of absorbance indicated a stron-ger protective activity of samples.

2.7. Determination of reducing activity

The reducing power of sample was determined as previouslydescribed (Oyaizu, 1986). The samples were added to potassiumferricyanide (2.5 ml, 10 mg/ml), and the mixture was incubatedat 50 �C for 20 min. TCA (2.5 ml, 100 mg/ml) was added to the mix-ture, which was then centrifuged at 650g for 10 min. The superna-tant (2.5 ml) was mixed with distiled water (2.5 ml) and ferricchloride (0.5 ml, 1.0 mg/ml), and then the absorbance was readat 700 nm.

2.8. Determination of chelating activity

The chelating activity of samples on Fe2+ was measured as pre-viously described (Carter, 1971). The samples thus prepared(0.6 ml) were reacted with FeCl2 (2 mM, 0.2 ml) and ferrozine(5 mM, 0.2 ml) for 10 min, and the absorbance at 562 nm wasdetermined. A lower level of absorbance indicated a higher chelat-ing activity of samples.

2.9. Determination of liposome oxidation

A solution containing the lecithin (580 mg) and phosphate buf-fer (58 ml, 10 mM, pH 7.4) was sonicated by an ultrasonic cleaner(Branson 8210, Branson ultrasonic Corporation, Danbury, CT, USA)in an ice-cold water bath for 2 h. The sonicated solution, FeCl3,ascorbic acid and samples (0.2 ml) were mixed to produce a finalmixture with a concentration of 3.12 lM FeCl3 and 125 lM ascor-bic acid. The mixture was incubated at 37 �C for 1 h. The levels ofliposome oxidation were determined by the thiobarbituric acid(TBA) method (Tamura & Shibamoto, 1991). The absorbance ofthe sample group was read at 532 nm against a blank, which con-tained all reagents except lecithin. A lower level of absorbanceindicated a stronger protective activity of samples.

2.10. Determination of nitric oxide scavenging

Nitric oxide (NO) scavenging activity was determined by usingthe DAF-2 fluorescence probe as previously described (Wang,Duh, Wu, & Huang, 2011). Sodium nitroprusside (SNP, 5 mM) andvarious concentrations of WFI in PBS were incubated at 25 �C for30 min in the presence of DAF-2 (5 lM). After incubation, NOproduced from SNP was determined using a Bio-Tek FLx800 micro-plate fluorescence reader with excitation and emission wave-lengths of 485 and 535 nm, respectively.

1

2

3

5

4

Retention Time (min)

Abs

orba

nce

(AU

)

Fig. 1. HPLC chromatogram of the water extract of Flos Inulae (WFI). The peaksindicate the following: 1. chlorogenic acid; 2. rutin; 3. quercetin; 4. luteolin; 5.kaempferol; IS, butyl p-hydroxybenzoate.

M.-H. Huang et al. / Food Chemistry 139 (2013) 1015–1020 1017

2.11. Mushroom tyrosinase activity determination

The mushroom tyrosinase was used for the bioassay. The tyros-inase inhibitory activity was determined with the degree of inhibi-tion on tyrosinase-catalysed oxidation of L-DOPA as previouslydescribed (Kubo et al., 2000). Diphenolase inhibitory activity wasdetermined by measuring the dopachrome accumulation at475 nm using spectrophotometer. All the experiments were per-formed in sodium phosphate buffer (pH 6.8). The reaction mixtureconsisting of 0.1 ml of samples, 0.1 ml of mushroom tyrosinase(1000 unit/ml) and L-DOPA (3.8 mM) was added in this order toread the absorbance at 475 nm for 5 min. The reaction wasperformed at 25 �C. The value in the absence of samples wasrepresented as the control. The inhibition of tyrosinase activitywas calculated with the following formula: Inhibition (%) =(1 � (OD475 in sample/OD475 in control)) � 100%.

2.12. Mutagenicity assay

The mutagenicity of WFI was tested according to the Ames testwith a 20 min first incubation at 37 �C (Maron & Ames, 1983). Thehistidine-requiring strains of Salmonella typhimurium TA 98 and TA100 were obtained from Taiwan Agricultural Chemicals and ToxicSubstances Research Institute (Taichung, Taiwan). The externalmetabolic activation system, S9 mix (Organ Teknika Co., Switzer-land) was prepared from Sprague–Dawley male rats treated withAroclor 1254. Samples (0.1 ml, 50–100 mg/ml corresponding to5–10 mg/plate, respectively) were added to the overnight culturedS. typhimurium TA98 or TA 100 (0.1 ml) and S9 mix (0.5 ml) or0.1 M phosphate buffer (0.5 ml, pH 7.4) in place of the S9 mix.The entire mixture was incubated at 37 �C for 20 min before mol-ten top agar (2.0 ml) was added and then spread out in a Petri dishcontaining 20 ml of minimum agar. The mixture was counted afterincubating at 37 �C for 48 h. The toxic effects of WFI on S. typhimu-rium TA 98 and TA 100 was determined as previously described(Wang et al., 2011).

2.13. Antimutagenic activity assay

The antimutagenic activitity of WFI was assayed according tothe Ames method except for the addition of mutagen before incu-bation (Maron & Ames, 1983). The concentrations of mutagenswere tested as in a previous study (Mazzei et al., 2007). The muta-gens used were 4-NQO (0.5 lg/plate), a direct mutagen; 2-AA(2.5 lg/plate), which required S9 mix for metabolic activation.Mutagen (0.1 ml) was added to the mixture of a strain (TA 98 orTA 100), and samples were added with the S9 mix for 2-AA or withphosphate buffer (0.1 M, pH 7.4) for 4-NQO. The mutagenicity ofeach mutagen in the absence of samples is defined as 100%. Thenumber of spontaneous revertants in the absence of mutagensand samples was used as reference. The inhibition (%) of mutage-nicity of the sample was calculated as following:Inhibition(%) = {1 � [(No. of his+ revertants with mutagen and sample � No.of spontaneous revertant)/(No. of his+ revertants with muta-gen � No. of spontaneous revertant)]} � 100

2.14. High performance liquid chromatography (HPLC) assay

HPLC was performed with a Hitachi Liquid Chromatograph (Hit-achi Ltd., Tokyo, Japan), consisting of two model L-7100 pumps,and one model L-7455 photodiode array detector. Sample(10 mg/ml) was filtered through a 0.45 lm filter and injected intothe HPLC column. The injection volume was 20 ll and the flow ratewas 0.8 ml/min. The separation temperature was 25 �C. The col-umn was a Mightysil RP-18 GP (5 lm, 250 � 4.6 mm I.D.; KantoCorporation, Portland, OR, USA). The method involved the use of

a binary gradient with mobile phases containing: (A) phosphoricacid in water (0.1%, v/v) and (B) H2O/CH3CN (2:8, v/v). The solventgradient elution program was as follows: 0–10 min, 100–85% A,0–15% B; 10–15 min, 85% A, 15% B; 15–30 min, 85–75% A,15–25% B; 30–40 min, 70–65% A, 30–35% B; 40–50 min, 65–55%A, 35–45% B; 50–65 min, 55–0% A, 45–100% B; and finally65–70 min, 0% A, 100% B.

2.15. Statistical analysis

All data were presented as means ± SD. Statistical analysis in-volved use of the Statistical Analysis System software package.Analysis of variance was performed by ANOVA procedures. Signif-icant differences between means were determined by Duncan’smultiple range tests at a level of p < 0.05.

3. Results

The HPLC chromatographic analysis showed that there were fivebioactive phenolic components present in WFI. These phenoliccomponents have been identified as chlorogenic acid, rutin, querce-tin, luteolin and kaempferol by their retention time and UV absor-bance of purified standards. In the HPLC system, by making theplot of the peak-area (y) vs. concentration (x, lg/ml), the regressionequations of the five constituents and their correlation coefficients(r) were as follows: chlorogenic acid, y = 0.0155x + 0.0532(r2 = 0.993); rutin, y = 0.0332x + 0.0487 (r2 = 0.999); quercetin,y = 0.0913x � 0.0695 (r2 = 0.997); luteolin, y = 0.0653x + 0.2309(r2 = 0.999); and kaempferol, y = 0.0691x � 0.0507 (r2 = 0.999).Fig. 1 shows the concentrations of the five phenolic compounds inWFI. The relative amounts of these compounds found in WFI werein the order of quercetin (10.1 mg/g extract) > rutin (9.4 mg/gextract) > kaempferol (5.5 mg/g extract) > chlorogenic acid(3.8 mg/g extract) > luteolin (1.8 mg/g extract). Meanwhile, the lev-els of total polyphenols and total flavonoid in WFI were determinedas gallic acid and rutin equivalents, were 210.7 and 125.2 mg/g ex-tract, respectively (data not shown).

The effects of WFI and five maker constituents on radical scav-enging and reducing activities are summarised in Table 1. Reducingactivity of natural products is regarded as a hydrogen donatingcapacity, which can terminate the radicals’ chain reaction. Thereducing ability of WFI and its marker constituents were deter-mined. In the range of 0.05–0.2 mg/ml, WFI exhibits reducing ef-fect that increased with increasing extract concentrations, thereducing capacity of WFI at 0.1 mg/ml showed 72.1%. Further,the reducing activities of the five markers in WFI, at the concentra-tion of 0.01 mg/ml, was in the order of quercetin > chlorogenicacid > luteolin > kaempferol > rutin. DPPH and ABTS inhibition is

Table 1Effects of WFI and five marker compounds on radical scavenging and reducingactivity.

Sample (mg/ml) DPPH inhibition(%)

ABTS inhibition(%)

Reducing activity(%)

WFI 0.05 56.7 ± 0.8 74.7 ± 1.8 51.8 ± 1.40.1 91.2 ± 4.0 96.1 ± 2.8 72.1 ± 1.10.2 95.0 ± 2.5 98.6 ± 4.1 82.5 ± 1.5

Chlorogenicacid

0.01 42.7 ± 1.2 97.3 ± 0.6 30.6 ± 1.3

Rutin 0.01 22.2 ± 2.3 87.4 ± 0.2 17.8 ± 0.9Quercetin 0.01 84.0 ± 2.1 101.0 ± 0.3 33.9 ± 1.1Luteolin 0.01 93.9 ± 3.2 101.4 ± 0.2 30.1 ± 0.5Kaempferol 0.01 31.6 ± 0.7 100.6 ± 0.8 27.0 ± 2.1

Table 3Effects of WFI and five marker compounds on nitric oxide (NO) production andmushroom tyrosinase activity.

Sample (mg/ml) Inhibition of NO production (%)

WFI 0.05 27.3 ± 1.50.1 46.0 ± 1.60.2 90.8 ± 0.7

Inhibition of tyrosinase activity (%)

WFI 0.5 34.0 ± 3.21.0 59.3 ± 2.21.5 90.3 ± 2.8

Chlorogenic acid 0.01 66.2 ± 1.2Rutin 0.01 50. 8 ± 0.9Quercetin 0.01 79. 4 ± 1.0Luteolin 0.01 38.1 ± 1.4Kaempferol 0.01 62. 9 ± 2.1

Results are displayed with mean ± SD (n = 3).

Table 4The mutagenicity of WFI toward S. typhimurium TA98 and TA100 with and without S9

A

1018 M.-H. Huang et al. / Food Chemistry 139 (2013) 1015–1020

generally recognised as popular methods to determine antioxidantcapacities of natural products. At 0.05–0.2 mg/ml, the radicalsscavenging activities on DPPH and ABTS of WFI were 56.7–95.0%and 74.7–98.6%, respectively, indicating that WFI is a potential freeradical scavenger as well as a strong radical terminator. Table 1also shows, with the concentration of 0.01 mg/ml, the inhibitoryeffects of chlorogenic acid, rutin, quercetin, luteolin and kaempfer-ol on DPPH and ABTS radical as 42.7%, 22.2%, 84.0%, 93.9% and31.6% and 97.3%, 87.4%, 101.0%, 101.4% and 100.6%, respectively.These data indicate that these phenolic constituents of WFI mightcontribute to the radical scavenging activity as well as the reducingactivity of WFI.

The chelating activity of natural resources could serve as a sig-nificant inhibitory factor in oxidation processes; thus the metalions chelating capacity of WFI was determined. As shown in Ta-ble 2, WFI in the range of 0.2–0.6 mg/ml exhibited 27.5–81.8% che-lating activity on ferrous ions. Lipid peroxidation is a harmfulprocess, producing toxic aldehydes and promoting cellular patho-logical metabolism. WFI in the range of 0.2–0.6 mg/ml exhibiteda concentration-dependent inhibitory effect, 51.3–100.2%, on theliposome oxidation induced by the Fe3+/H2O2 system. These dataindicated WFI could be an effective protector in preventing lipidperoxidation in vitro. In addition, Table 3 shows the protection pro-vided by WFI from nitric oxide (NO) production in LPS inducedmacrophages. The NO produced by activated macrophages wasmeasured as nitrite in a cell culture medium, using the Griess reac-tion. WFI at levels of 0.05–0.2 mg/ml inhibited NO production by27.3–90.8%, compared to that observed for the LPS group. No celltoxicity was noted in the presence of WFI, as measured by theMTT cell viability test. These data suggest that WFI reduces theRNS stress in LPS stimulated macrophages. Further, as shown in Ta-ble 3, WFI also showed a potent inhibitory action on mushroomtyrosinase activity with an IC50 value of 0.8 mg/ml. In the rangeof 0.5–1.5 mg/ml, WFI exhibit 34.0–90.3% inhibitory action on

Table 2Effects of WFI and five marker compounds on chelating activity and liposomeperoxidation.

Sample (mg/ml) Chelatingactivity (%)

Liposomeprotection (%)

WFI 0.2 27.5 ± 2.6 51.3 ± 8.50.4 47.5 ± 0.8 89.8 ± 5.10.6 81.8 ± 1.2 100.2 ± 1.6

Chlorogenic acid 0. 01 28.6 ± 1.1 25.8 ± 2.0Rutin 0.01 24.7 ± 1.6 29.6 ± 1.8Quercetin 0.01 31.5 ± 1.3 89.7 ± 2.1Luteolin 0.01 29.1 ± 1.0 85.6 ± 1.3Kaempferol 0.01 26.6 ± 1.5 86.7 ± 0.7

Results are displayed with mean ± SD (n = 3).

tyrosinase. In this test, the tyrosinase inhibitory action of WFI in-creased with increasing sample concentration. Table 3 also shows,with the concentration of 0.01 mg/ml, the inhibitory effects ofchlorogenic acid, rutin, quercetin, luteolin and kaempferol ontyrosinase were 66.2%, 50.8%, 79.4%, 38.1% and 62.9%, respectively.Quercetin showed greatest liposome protection and antityrosinaseactivity among the maker constituents.

The Ames assay is a common method to determine the mutage-nicity of natural products. In the mutation study, if a lethal toxicityoccurred in a test treated with samples, the results of the mutage-nicity could be affected and confuse the numbers of revertants ofTA 98 and TA 100. In this study, WFI in the range of 2–10 mg/platedid not show any toxicity against TA 98 and TA 100, respectively,indicating that the WFI did not display toxicity in TA 98 and TA100 (data not shown). Further, the mutagenicity of WFI, wasmeasured by comparing the ratio of induced revertants and spon-taneous revertants in plates. According to Table 4, in the range of2–10 mg/plate, WFI did not significantly (p > 0.05) increase thenumbers of colonies in S. typhimurium TA 98 and TA 100 with orwithout S9 activation. Therefore, the dose below 10 mg/plate wasselected for the antimutagenic assay. As reported in Table 5, WFIdisplayed dose-dependent protective effects against 4-NQO in-duced mutagenicity in S. typhimurium TA 98 and TA 100 withoutS9 activation. WFI at levels of 2–10 mg/plate showed 12–31%inhibitory effects against 4-NQO induced mutagenicity in TA 98;meanwhile, they showed 27–80% inhibitory effects in TA 100.Table 5 also reports the antimutagenicity of WFI on 2-AA inducedmutation in S. typhimurium TA 98 and TA 100 with S9 activation.

mix.

Sample (mg/plate) His+ revertants/plate (% of spontaneous)B

TA98 TA100

Spontaneous group 19 ± 1 (100)a 137 ± 2 (100)a

2 17 ± 3 (89)a 137 ± 4 (100)a

5 19 ± 2 (100)a 137 ± 6 (100)a

10 16 ± 3 (84)a 135 ± 6 (99)a

TA98 + S9 TA100 + S9

57 ± 1 (100)b 203 ± 6 (100)a

2 56 ± 2 (98)b 195 ± 1 (96)a

5 41 ± 1(72)a 192 ± 2 (95)a

10 40 ± 1 (71)a 200 ± 26 (99)a

A Data are means ± SD of three plates. Values with different superscripts in acolumn are significantly different (p < 0.05).

B % of spontaneous = [(No. of his + revertants in the presence of sample)/(No. ofspontaneous revertants)] � 100. The number of spontaneous revertants wasdetermined without samples and mutagens.

Table 5The antimutagenicity of WFI toward S. typhimurium TA98 and TA100A.

Sample (mg/plate) His + revertants/plate (% of inhibition)B

TA98 + 4-NQO TA100 + 4-NQO

Spontaneous group 191 ± 7 (0)c 1204 ± 10 (0)d

2 168 ± 5 (12)b 881 ± 11 (27)c

5 164 ± 4 (14)b 757 ± 16 (51)b

10 132 ± 4 (31)a 595 ± 16 (80)a

TA98 + S9 + 2-AA TA100 + S9 + 2-AA

3457 ± 46 (0)d 3796 ± 6 (0)d

2 620 ± 23 (82)c 517 ± 22 (86)c

5 171 ± 2 (95)b 274 ± 1 (93)b

10 84 ± 4 (98)a 265 ± 4 (93)a

A Data are means ± SD of three plates. 4-NQO, 4-nitroquinoline-N-oxide. 2-AA, 2-anthramine.Values with different superscripts in a column are significantly differ-ent (p < 0.05).

B % of inhibition = {1 � [(No. of revertants with mutagen and sample � No. ofspontaneous revertants)/(No. of revertants with mutagen � No. of spontaneousrevertants)]} � 100. The number of spontaneous revertants was determined with-out samples and mutagen.

M.-H. Huang et al. / Food Chemistry 139 (2013) 1015–1020 1019

The inhibitory effects of WFI on 2-AA induced mutation in S.typhimurium TA 98 and TA 100 were dose dependent. WFI at levelsof 2–10 mg/plate showed 82–98%, inhibitory effects against 2-AAinduced mutagenicity in TA 98; meanwhile, they showed 86–93%, protective effects in TA 100.

4. Discussion

Phytochemicals rich natural sources with a complex array ofantioxidant exhibit different protective effects against harmfulstress in various models (Ness & Powles, 1997). Much evidencehas shown that the antioxidantive effects of phenolic compoundscould play a critical role, not only in free radical inhibition, but alsoin the prevention of cell damage. In this study, the results showedthat these significant protective effects of WFI could be attributedmainly to the high levels of total polyphenols and total flavonoid.Additionally, the HPLC chromatogram of WFI demonstrates fivephenolic components identified as chlorogenic acid, rutin, querce-tin, luteolin and kaempferol. These compounds have been reportedto possess strong antioxidant activities, such as inhibiting ROS gen-eration, inflammation and tumour activities, etc. (Nirmala & Rama-nathan, 2011; Shan et al., 2009; Shen et al., 2002; Xie et al., 2012).As shown in Table 1, chlorogenic acid, rutin, quercetin, luteolin andkaempferol found in WFI have shown significant antioxidant ef-fects by various antioxidation assays. They also exhibited potentreducing ability. In particular, quercetin and luteolin showedgreater reducing effects than the other marker constituents. Quer-cetin has a catechol structure in ring B, as well as a 2,3-doublebond in conjunction with a 4-carbonyl group in ring C, allowingfor delocalisation of the phenoxyl radical electron to the flavonoidnucleus. The combined presence of a 3-hydroxy group with a 2,3-double bond additionally increases the resonance stabilisation forelectron delocalisation; hence it has a higher antioxidant value.Luteolin has also identical numbers of hydroxyl groups, in ringsB and A. Thus, the five markers, especially quercetin found inWFI in high contents, exhibited the high efficiency, which mightcorrespond to reducing effect of this plant and could benefit thepharmaceutical and food industry.

Occurring in cell membranes, lipid peroxidation releases arachi-donic acid, which is responsible for long term damage to cells. Asshown in Table 2, in the liposome model system, WFI showedprotective activity against the damage caused by lipid oxidation.Besides, three flavonoids, including quercetin, luteolin and

kaempferol, showed the greatest liposome protective actionamong the reference phenolic compounds. Obviously, rutin, alsoone of the plant flavonoids, showed about one third liposomeprotective activity of the three. In general, the antioxidant potencyof a given compound is thought to be closely linked to its structuralfeatures, such as, the ortho-dihydroxy structure in the B-ring, the2,3-double bond in conjugation with a 4-oxo function and thepresence of the 3- and 5-OH functions, or influenced by the pres-ence of glycosidic moieties and position of hydroxyl and methoxygroups.

The synthesis of melanin will cause glutathione depletion andthe formation of hydrogen peroxide. WFI clearly decreased tyrosi-nase activity in vitro (Table 3). This finding shows that WFI can de-crease the danger posed by a reduction in glutathione and thequinone induced cell damage. Recent studies have supported therole of metal ion chelation in the processes of tyrosinase inhibition(Kubo et al., 2000). Moreover, the tested standard, rutin, is strongertyrosinase inhibitor than luteolin, but lower with its metal ion che-lating activity. The result is not consistent with Kubo’s report.These findings suggest that WFI exhibited the inhibition of tyrosi-nase could not be associated with their metal ion chelating activi-ties. Other unknown active components present in WFI could alsoplay critical roles in their biological effects. In addition, some stud-ies have indicated that NO is easily diffusible and fairly reactive.Excess NO, produced under pathological conditions, interacts withsuperoxide to produce toxic peroxynitrite, which can destroy cellu-lar nucleic acids, which induces formation of 8-nitroguanine andaccelerates the progression of cancers (Goetz & Luch, 2008; Pinlaoret al. 2005). The results also show that WFI suppresses NO gener-ation in LPS stimulated macrophages. In recent years, the sesqui-terpene have been under intensive investigation due to theirpossible role in preventing some diseases, such as inflammationand cancer (Bai et al. 2006; Jin et al. 2006). Consequently, it is pos-sible that the polyphenolic constituents in the WFI were able tocontribute to their physiological activities.

In fact, neither toxicity nor mutagenicity was found in WFI in S.typhimurium TA 98 and TA 100 with or without S9 activation.These data suggested that none of WFI and their constituentsshowed frame shift, base pair mutagenicity or cytotoxicity. Further,WFI also showed inhibitory effects on the mutagenicity of 4-NQOand 2-AA in the S. typhimurium bacterial model (Tables 4 and 5).These data implied that WFI could play an antimutagenic role byscavenging the active metabolic electrophile of 4-NQO and 2-AA.On the other hand, the conjugated reaction between WFI and thetoxic electrophile could be an important detoxification pathway.Aside from these, the antimutagenic effects of the WFI could beattributed to the decreased metabolic activation and the levels oftoxic reactive intermediates, which would further prevent cellularoxidative stress, and thereby decrease the DNA damage by modu-lation of DNA replication and repair of DNA. From the data of pres-ent study, WFI showed a significant capacity in radical scavenging,lipid peroxidation, tyrosinase, and mutation inhibition. It sug-gested that the many different activities of the tested extractscould be also closely associated with their phenolic constituents.In conclusion, these obtained data suggested that WFI can exhibittheir biological activities in antimutation, antityrosinase, antioxi-dant and anti-inflammation in vitro. However, this paper is a pre-liminary screen test for various activities of WFI. Furtherinvestigations of the nutritional and physiological effects of WFIare still intensively required.

Acknowledgements

This work was supported by Chia Nan University of Pharmacy &Science and National Science Council of Taiwan, ROC (NSC 99-2321-B-041-001-MY2).

1020 M.-H. Huang et al. / Food Chemistry 139 (2013) 1015–1020

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