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Antioxidant activity of herbaceous plant extracts protect against hydrogenperoxide-induced DNA damage in human lymphocytes



Herbaceous plants containing antioxidants can protect against DNA damage. Thepurpose of this study was to evaluate the antioxidant substances,antioxidant activity, and protection of DNA from oxidative damage in humanlymphocytes induced by hydrogen peroxide (H2O2). Ourmethods used acidic methanol and water extractions from six herbaceousplants, including Bidens alba (BA), Lycium chinense (LC),Mentha arvensis (MA), Plantago asiatica (PA),Houttuynia cordata (HC), and Centella asiatica(CA).


Antioxidant compounds such as flavonol and polyphenol were analyzed.Antioxidant activity was determined by the inhibition percentage ofconjugated diene formation in a linoleic acid emulsion system and bytrolox-equivalent antioxidant capacity (TEAC) assay. Their antioxidativecapacities for protecting human lymphocyte DNA fromH2O2-induced strand breaks was evaluated by cometassay.


The studied plants were found to be rich in flavonols, especially myricetinin BA, morin in MA, quercetin in HC, and kaemperol in CA. In addition,polyphenol abounded in BA and CA. The best conjugated diene formationinhibition percentage was found in the acidic methanolic extract of PA.Regarding TEAC, the best antioxidant activity was generated from the acidicmethanolic extract of HC. Water and acidic methanolic extracts of MA and HCboth had better inhibition percentages of tail DNA% and tail moment ascompared to the rest of the tested extracts, and significantly suppressedoxidative damage to lymphocyte DNA.


Quercetin and morin are important for preventing peroxidation and oxidativedamage to DNA, and the leaves of MA and HC extracts may have excellentpotential as functional ingredients representing potential sources ofnatural antioxidants.


Herbaceous plants have a long history of use as medicine, food, and a variety ofdaily needs. Many epidemiological studies suggest that an increased consumption ofseveral medicinal plants containing antioxidants can protect against DNA damage andcarcinogenesis, and often exhibit a wide range of pharmacological activities such asantiflammatory, anti-bacterial, and anti-fungal properties [1]. Flavonoids have strong antioxidant efficiencies and are common in leafyvegetables. Trolox, for example, is a water-soluble derivative of vitamin E thatblocks DNA fragmentation in irradiated MOLT-4 cells, a human lymphocytic leukemialine [2]. Hence, a number of phytochemicals commonly used in research haveantioxidant activity that can protect cells from reactive oxygen species(ROS)-mediated DNA damage that results in mutation and subsequent carcinogenesis [3, 4]. Cao et al.[5] indicated that increased consumption of vegetables and fruits increasesthe plasma antioxidant capacity in humans. Some common vegetables like purple-leavedsweet potato and the outer layers of purple onions abound in quercetin andmyricetin, which scavenge 2, 2-diphenyl-1-picrylhydrazyl (DPPH), superoxide, andhydroxyl radicals, and inhibit lipid peroxidation [6]. The search for phytochemicals and dietary compounds with potentantioxidant and otherwise preventive properties continues to be of great importancein the search for remedies against free radical-mediated diseases. There is greatinterest in the use of potent dietary antioxidants in preventive strategies forapplications ranging from the prevention of oxidative reactions in foods andpharmaceuticals to the role of ROS in chronic degenerative diseases [7].

In recent years, increasing attention has been paid by consumers to the health andnutritional benefits of herbaceous plants. Some herbs, such as pilosa beggarticks(Bidens alba L. var. minor) (BA), Chinese wolfberry(Lycium chinense Mill.) (LC), wild mint or corn mint (Menthaarvensis L. var. piperascens Malinv.) (MA), Asiatic plantain (Plantagoasiatica L.) (PA), heartleaf (Houttuynia cordata Thunb.) (HC), andAsiatic centella (Centella asiatica L. Urban) (CA) are favored asfunctional herbals. Some of the health effects of herbaceous plants have beenreported to include antioxidation [810], anti-inflammation [11], and blood pressure reduction [12]. In animal experiments, Chinese wolfberry, heartleaf, Asiatic plantain,Asiatic centella, and pilosa beggarticks showed special detoxification andanti-inflammatory effects [8, 9, 11, 13, 14]. Particularly, HC, LC, and CA showed antioxidant activities [8, 9]. Asiatic centella increased the activity of antioxidant enzymes such assuperoxide dismutase, catalase, and glutathione peroxidase, and enhanced theconcentration of vitamin C and vitamin E in new tissues during wound healings [13]. Both HC and BA were reported to have anti-inflammatory functions due totheir quercetin and luteolin content [8, 11]. Furthermore, LC and BA can reduce the injury to liver cells fromCCl4[9, 13]. Pilosa beggarticks also functions as an anti-fungal and anti-bacterialagent, and lowers high blood pressure [12]. Several herbs are consumed to protect against common, serious diseasessuch as cardiovascular and cerebrovascular events, cancer, and other age-relateddegenerative diseases [15]. These protective effects are considered, in large part, to be related tothe various antioxidants contained in them. Evidence that free radicals causeoxidative damage to lipids, proteins, and nucleic acids is overwhelming.Antioxidants, which can inhibit or delay the oxidation of an oxidizer in a chainreaction, would therefore seem to be important in preventing these diseases [16]. Prevention from oxidative stress might be achieved by the uptake ofantioxidants. Polyphenols and flavonols can act as antioxidants in two ways: byscavenging free radicals and chelating redox active metal ions (direct antioxidantactivity), and by inducing cellular antioxidant defense and repair. These benefitshave significantly contributed to their antioxidant activity and have stimulatedresearch into the content, ability, capacity, and function of antioxidant systems inherbaceous plants. Polyphenolic and flavonol substances are the most commoncompounds in herbs having strong antioxidant activity [6]. Previously, we also demonstrated that purple-leaved sweet potatoexhibits free radical scavenging and has high polyphenolic content [17]. Although a variety of medicinal herbs are known to be potent sources ofpolyphenolic and flavonol compounds, studies that isolate polyphenols, evaluatetheir antioxidative effects, and determine their efficacy or ability to preventoxidative damage to DNA are either scarce or little known. The bioactive componentsof these herbal plants might be responsible for anti-cancer effects through growthinhibition and apoptosis in human chronic myeloid leukemia K562 cells [18]. The objective of this study was to isolate, identify, and evaluate theantioxidant components, antioxidant activity, and extent to which methanolic acidhydrolysates and water extracts of six herbaceous plants could protect DNA in humanlymphocytes from oxidative damage induced by H2O2. Our studyexplores the relationships between the composition and content of flavonols andpolyphenol having antioxidant efficiency, and the prevention of DNA oxidative damageafforded by the herbaceous plants.


Chemicals and reagent

Methanol, ethanol, hydrochloric acid, di-sodium hydrogen phosphate, potassiumdihydrogen phosphate, formic acid, sodium chloride (NaCl), potassium chloride(KCl), Tris–HCl, Tris (hydroxymethyl) aminomethane (Tris base), dimethylsulfoxide (DMSO), ethylenediamine tetraacetic acid (EDTA), Trolox, and butylatedhydroxyltoluene were purchased from Merck (Darmstadt, Germany). Linoleic acid,d-glucose, calcium chloride dihydrate, sodium lauryl sarcosinate, gallic acid,2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS), peroxidase,H2O2, sodium carbonate (Na2CO3),tetrazolium/formazan, Folin-Ciocalteau reagent, and ethidium bromide wereprocured from Sigma Chemical (St Louis, MO, USA). Myricetin, morin, quercetin,kaempferol, cynidin, and malvidin were obtained from ROTH (Rheinzabern,Denmark). Ficoll-Paque was acquired from Amersham Biosciences (Uppsala, Sweden).Low-melting gel agrose and Triton X-100 were purchased from BDH (Poole,England). Normal-melting gel agarose was purchased from Pantech Instruments(Darmstadt, Germany). AIM V serum-free lymphocyte medium was purchased fromGibco Invitrogen (Carlsbad, CA, USA).

Herbaceous plants

The tested plants were Bidens alba L. var. minor, Lyciumchinense Mill., Mentha arvensis L. var. piperascens Malinv.,Plantago asiatica L., Houttuyni acordata Thunb., andCentella asiatica L. Urban. These were generously provided by Dr.Kuang-Chuan Liu, Taoyuan District Agricultural Research and Extension StationCouncil of Agriculture, Executive Yuan, Taiwan.

Preparation of plant extracts

The plants were weighed, lyophilized, and ground to powder. Each lyophilizedpowder was extracted by distilled deionized (dd)H2O. The extractionmixture was then heated to 90°C in a steam bath and refluxed for 2 h,allowed to cool in a refrigerator, sonicated for 5 min, and diluted to50 mL with ddH2O to prepare the final extract. These waterextracts were ready for the comet assay. For high-performance liquidchromatography (HPLC), only the edible portions of plants were weighed,lyophilized, and ground into powder. Lyophilized vegetable powders were preparedaccording to Justesen et al. [19] with modifications as follows: 10 ml of 62.5% aqueous methanolcontaining butylated hydroxyltoluene (2 g/L) were added to 1.25 g oflyophilized samples, followed by adding 5 mL of 6 M HCl to bring totalvolume up to 12.5 mL. The final mixture consisted of 1.2 M HCl in 50%aqueous methanol. The extraction mixture was thereafter heated to 90°C in asteam bath and refluxed for 2 h, allowed to cool in a refrigerator,sonicated for 5 min, and diluted to 50 mL with methanol to form thefinal extract. The acid hydrolysates methanolic extract was ready forhigh-performance liquid chromatography (HPLC), inhibition of conjugated dieneformation in the linoleic acid assay, TEAC assay, and comet assay.

Polyphenol assay

Polyphenol content was determined according to the method of Taga et al. [20]. Briefly, standard gallic acid and an aliquot of methanolic extractwere diluted with an ethanol/water (60:40, v/v) solution containing 0.3% HCl.Two mL of 2% Na2CO3 was mixed into each sample of100 μL and allowed to equilibrate for 2 min before adding 50%Folin-Ciocalteau reagent. Absorbance at 750 nm was measured at roomtemperature. The standard curve of gallic acid was used to calculate polyphenollevels.

Flavonols analysis by HPLC

One mL of acid hydrolysates methanolic extract was filtered through a0.45 μm filter prior to 20 μL being injected into the HPLC.Samples were analyzed with a SpectraSYSTEMUV6000LP Photodiode Array DetectionSystem (Thermo Separation Products, San Jose, USA) and an ODS column (250 ×4.6 mm, 5 μm; YMC, Kyoto, Japan). The mobile phase consisted ofmethanol–water (30:70, v/v) with 1% formic acid and 100% methanol. Thegradient was 25 - 74% methanol in 40 min at a flow rate of0.75 mL/min. Spectra were recorded at 365 nm for flavonols [19].

Inhibition of conjugated diene formation in linoleic acid emulsionautoxidation system

The inhibition of conjugated diene formation was determined according to Mitsudaet al. [21]. Briefly, an aliquot of 0.1 mL of diluted plant acidicmethanolic extract or blank was added to 2 mL of 10 mM linoleicacidemulsion (pH 6.6), mixed well, and incubated at 37°C for15 h. A sample of 0.2 mL for 0 and 15 h incubation periods weremixed with 7 mL of 80% methanol, followed by measuring the absorbance at234 nm.

Trolox equivalent antioxidant capacity (TEAC) analysis

The total antioxidant capacity of hydrophilic and lipophilic antioxidants wasdetermined using the horseradish peroxidase catalyzed oxidation of2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) [22]. The reaction mixture contained 0.5 mL of 1000 μM ABTS (inddH2O) and 3.5 mL of 100 μM H2O2(in ddH2O). The reaction was started by adding 0.5 mL of 44 U/mLperoxidase (in 0.1 M PBS). After 1 h, 0.05 mL of plant acidicmethanolic extracts were added to the mixture. After 5 min, absorbance wasmeasured at 730 nm. Trolox (TR) was used as a standard, and the totalantioxidant capacity of plant extracts were measured as mM TR equivalent.

Isolated human peripheral blood lymphocytes

Fasting blood samples were obtained from six donors, including four male and twofemale healthy non-smokers, 24–48 years old. Fresh venous blood(20–30 mL) was collected in lithium heparin tubes (Becton- Dickinson)from volunteers, and lymphocytes were isolated using a separation solution kitsupplemented with Ficoll-Paque Plus lymphocyte isolation sterile solution(Pharmacia Biotech, Sweden) [23]. Cells were harvested within 1 day of taking the blood samplesand cultured with AIM V serum-free lymphocyte medium (Gibco Invitrogen, USA) ina humidified atmosphere of 5% CO2 in air at 37°C for24 h.

Cell viability testing

After culturing, lymphocytes were exposed to each of six different plant acidicmethanolic and water extracts. Each lymphocyte was treated with threeconcentrations of plant acidic methanolic and water extracts (25, 50, and100 μg/mL) for 30 min at 37°C. DNA damage was induced byexposing lymphocytes to H2O2 (10 μM) for5 min on ice to minimize the possibility of cellular DNA repair afterH2O2 injury. Cells were centrifuged(100 g for 10 min), washed, and re-suspended in the samemedium as the comet assay. All experiments were carried out in triplicate. Cellviability was tested using the tetrazolium/formazan (MTT) assay [24] both prior to and after treatment with plant extracts orH2O2.

DNA single strand break damage estimation using the comet assay

The standard comet assay was performed as described in Szeto et al. [3], with acidic methanolic and water extracts from these six herbalplants being used for this study. Cultured lymphocytes (105 cells/mL)were embedded in 75 μL of 1% low-melting-point agarose on a microscopeslide (precoated with agarose) at 37°C. The gel was allowed to set at4°C, and cells were lysed for a period of at least 2 h in lysis bufferat 4°C. Cells were then alkaline-unwound, following which electrophoresiswas carried out using the electrophoresis buffer at 4°C for 15 min at25 V with the current adjusted to 300 mA. All steps were conductedunder dim light to prevent the occurrence of additional DNA damage. Followingelectrophoresis, slides were neutralized with neutralization buffer and stainedwith ethidium bromide. The comet-like images resulting from the extension of DNAwere scored as a reflection of the single strand breaks under a fluorescencemicroscope (Zeiss-Axiovert 100, Zeiss, Germany). Triplicate slides were preparedfor each experimental point sample, and 50 comet-like images selected at randomper slide were evaluated to determine average DNA damage values. A computerizedimage analysis system (VisCOMET 1.6, Impuls GmbH, Germany) was employed todetermine various comet parameters, and used to analyze DNA damage by tail DNA%[(total brightness of tail area / total brightness of total area) × 100%]and tail moment (tail length × tail DNA%). Inhibition percentage of tailDNA% and tail moment were calculated relative to the 10 μMH2O2 treated group.

Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA), and the significancebetween means by the least significant difference (LSD) test. Pearson’slinear correlation was also determined. Means of three replicates werereported.


Antioxidant composition and antioxidant activity

Table 1 documents the content of polyphenol in theleaves of tested plants. Polyphenols were significantly abundant in both BA(32.90 mg gallic acid/g DW) and CA (32.03 mg gallic acid/g) comparedto other plants. Table 2 presents varied amounts offlavonols ranging from 53.33 to 3200 μg/g DW in the acidic methanolicextract of the studied plants. BA and CA were also rich in myricetin, at levelsof 1133.33 and 960.00 μg/g DW, respectively. Morin was present only inMA, CA, and BA plants at a level of 2000.00, 600.00, and 573.33 μg/gDW. Quercetin was abundant in HC (3200.00 μg/g DW), while CA followedat a level of 533.33 μg/g DW. Kaempferol was abundant in CA at a levelof 853.33 μg/g DW, but LC and PA did not contain any kaempferol atall. Thus, these species displayed variations in their polyphenol and flavonollevels.

Table 1 The content of polyphenol in tested herbaceous plants
Table 2 The content of flavonols in acidic methanolic extracts of testedherbaceous plants

The inhibition of linoleic acid peroxidation was observed to be significantlyhigher in PA and BA at both 25 and 50 μg/mL of plant extracts(Table 3). Furthermore, significantly higherpercentages of conjugated diene inhibition were detected in PA (79.31) and CA(77.61) compared to MA (70.31) at 100 μg/mL of the extract. Hence,each species showed significant differences in inhibition percentages ofconjugated dienes at various extract concentrations.

Table 3 Inhibition percentage of conjugated diene formation in the linoleicacid emulsion autoxidation system treated with variousconcentrations of methanolic acid hydrolysates of herbaceousplants

Plant extracts from the six species showed antioxidant activities, proving theircapacity to scavenge the ABTS radical-cation. The antioxidant activity inmethanolic acid hydrolysate extracts of leaf tissues of studied species wereexpressed in Trolox Equivalent Antioxidant Capacity (TEAC) (Table 4). HC showed a significantly higher TEAC value(231.16 mM) than other species.

Table 4 The TEAC values of acidic methanolic extracts in the investigatedherbaceous plants

Effects of acidic methanolic and water extracts from herbaceous plants onH2O2-induced DNA damage to lymphocytes

Lymphocytes were exposed to each of three different herbal extracts at threeconcentrations (25, 50, and 100 μg/mL) for 30 min at 37°C.DNA damage was induced by exposing lymphocytes to H2O2(10 μM) for 5 min on ice. At two lower levels, no extracts werecytotoxic at the concentrations used, with > 98% of cellsremaining viable [25]. Therefore, concentrations only at 25 and 50 μg/mL werechosen for the comet assay. The comet assay was performed to determine the DNAdamaging activity of the plants as it is a sensitive method for monitoringsingle strand DNA breaks at the single cell level. Any DNA damage is representedas tail DNA% and tail moment. The effects of pretreatment of the six testedextracts on 10 μM H2O2-induced DNA oxidativedamage in human lymphocytes are presented in Figure 1. Tail DNA% demonstrated that MA had a significantly greater level ofprotection against H2O2 exposure than lymphocytes thatwere exposed to other tested compounds at two doses (25 and 50 μg/mL)(Figure 1A). The maximum protective effect oflymphocyte pretreatment was observed with pretreatment by 25 μg/mL MA,exhibiting 12.43% of tail DNA% compared to the rest of treated samples.Furthermore, at lower concentrations, all tested samples had lower tail DNA%,indicating better inhibition efficacies. The MA extract at the50 μg/mL was significantly lower than the rest of treated samples,except for HC extract. Tested plants showed at least 707.53 and 1040.63 of tailmoment in HC extract at 25 and 50 μg/mL levels compared to the rest ofthe acidic methanolic extract samples (Figure 1B).

Figure 1
figure 1

Effects of various acidic methanolic extracts from six herbaceousplants onH 2 O 2 -induced DNAdamage to lymphocytes. Tail DNA% (A) and tail moment(B) were measured after exposure to tested compounds at 25and 50 μg/mL of extract. ,BA;, LC; , MA;, PA; , HC; ■,CA. Values with different letters differ significantly with regard tooxidative damage when comparing different plant extracts;*p < 0.05 refers to differences in oxidativedamage as compared with 10 μM H2O2-alone(■) treatment.

HC had the lowest % tail DNA at 11.14% in 25 μg/mL of water extract(Figure 2A). Both HC (18.36%) and MA (18.25%)extracts at 50 μg/mL had lowest % tail DNA compared to the rest of thewater extract of samples. Moreover, HC also had a significantly lower tailmoment (1255.40 ~ 1826.10) than the rest of the water extracts at the same doses(Figure 2B). Hence, the DNA damage induced byH2O2 was significantly high as compared to the treatedextracts, which had 87.26 in tail DNA% and 8328.84 in tail moment.

Figure 2
figure 2

Effects of various water extracts from six herbaceous plants onH 2 O 2 -inducedDNA damage to lymphocytes. Tail DNA% (A) and tail moment(B) were measured after exposure to tested compounds at 25and 50 μg/mL of extract. , BA;, LC; , PA;, HC; ■, CA. Values with different lettersdiffer significantly with regard to oxidative damage when comparingdifferent plant extracts; *p < 0.05 refers todifferences in oxidative damage as compared with 10 μMH2O2-alone (■) treatment.


Antioxidant composition and antioxidant activity

Plant leaves are rich in flavonols and other pigments. BA and CA plants containhigher polyphenol levels than the other plants tested (Table 1). Antioxidant activities are known to increase proportionally tothe polyphenol content, mainly due to their redox properties [1]. Among the diverse roles of polyphenols, they protect cellconstituents against destructive oxidative damage, thus limiting the risk ofvarious degenerative diseases associated with oxidative stress and tending to bepotent free radical scavengers. Their ability to act as antioxidants depends ontheir chemical structure and ability to donate/accept electrons, thusdelocalizing the unpaired electron within the aromatic structure [26]. Phenolic compounds are known as radical scavengers or radical-chainbreakers, and they strongly eliminate oxidative free radicals. Quercetin andmorin are the principal flavonol constituents in HC and MA plants, respectively(Table 2). These antioxidant compounds mayaccount for the high antioxidant power of the plants in the present study.Quercetin, kaempferol, morin, and myricetin are the most common flavonols, andare the most widely distributed flavonoids in plant leaves. Quercetin, the mostabundant flavonoid in the human diet, is an excellent free radical scavengingantioxidant [27]. Polyphenol and flavonol contents found in the extracted plants(Tables 1, 2) were muchlower than those in our previous study where purple-leaved sweet potato appearedto have higher contents [28]. A possible reason is the usage of different extraction methods. Infact, different results were obtained from the water and acidic methanolicextracts, and especially from the water extracts. The antioxidant compositionand activities of herbal plants cannot be evaluated by a single method due tothe complex nature of plants, in which pigments and phytochemicals have specificfunctions. Therefore, several methods should be employed to evaluate the totalantioxidant effects of any plant. Antioxidant compounds presented in plantextracts are therefore multi-functional and their activities and mechanisms ofaction would largely depend on the composition and conditions of the testsystem.

Compared to the inhibition percentage of conjugated diene formation in thelinoleic acid emulsion autoxidation system of tested samples, PA exhibitedrelatively higher effectiveness than the others at all extract concentrations(Table 3). The tested vegetables showed >70%inhibition of linoleic acid peroxidation in 100 μg/mL extracts, and PAin particular exhibited the highest inhibition of linoleic acid peroxidation, upto 79.31%. Therefore, all tested plants were effective inhibitors and exhibitedbetter inhibition efficacy at higher concentrations. Previously, we demonstratedthat water and methanolic extracts from PA both had higher antioxidant activity,and that the antioxidant activity of PA was equivalent to 10-4 Mof Trolox in preventing conjugated diene formation during linolic acidperoxidation at 62.5 μg/mL of methanolic extract [29]. The polyphenol content of methanolic extracts was significantlycorrelated with the delay of the lag phase of low-density lipoprotein (LDL)treated with methanolic extracts. Moreover, the polyphenol content of themethanolic extract of herbaceous plants was significantly correlated withscavenging DPPH radical activity and ferric reducing power [29].

We measured the direct antioxidant activity of acidic methanolic extracts by TEACassay, reflecting the major mechanisms of antioxidant action for evaluatingtheir relevance to cell protection (Table 3).Jastrzebski et al.[30] reported that prolipid, a mixture of herbs used as a plasma lipidlowering medicine, had strong antioxidant activity. The correlation coefficientsbetween the polyphenols, flavonoids, and TEAC of prolipid water extracts were0.97 and 0.90, respectively. They concluded that the content of polyphenol inprolipid was the main contributors to the overall antioxidant activity ofprolipids. The antioxidant activity of leaf extracts from CA was found to have adirect linear relationship between total phenolic content and total antioxidantactivity, indicating that phenolic compounds might be the major contributors tothe antioxidant activities of CA extracts [31]. Chung et al.[29] reported that PA, BA, CA, Curled Spearmint, MA, and Mesona had highertotal phenolic contents compared to LC and Taiwan lily, and that CA and PA hadhigher antioxidant activity. In this study, we found that HC and CA containedabundant quercetin while MA and CA were rich in morin and kaempferol,respectively. Additionally, BA and CA had significantly higher levels ofmyricetin than other tested samples (Table 2). Thesedifferent pigments may exhibit effective antioxidant activity alone orsynergistically, and are a likely cause of cultivar differences. Wang etal.[32] demonstrated that the H donation potential wasquercetin > myricetin > morin > kaempferol,indicating that the presence of a 3′,4′-catechol moiety in the Bring correlated with high activity. Moreover, the structural peculiarity ofdi-OH in the B ring obviously rendered quercetin and morin more potent as ROSinhibitors than myricetin and kaempferol, which have tri- and mono-OH in the Bring, respectively. The unclear relationship between antioxidant activity andflavonol extracts indicates that the structure prerequisite to reinforce freeradical scavenging activity may vary with the type of free radical. Thesynergisms among antioxidants make antioxidant activity dependent not only onthe concentration, but also might be due to their structures and interactionsamong antioxidants [33]. The accumulation of flavonoid metabolites in the appropriate targetsite is probably required to exert their antioxidant activity. Thepolyphenol-rich plant extracts exhibited distinct cell-free antioxidant activity(TEAC) according to their levels of polyphenol and flavonols, with distinctantioxidant activity strongly accounting for the antioxidant activity of theextracts. HC plants containing 3200 μg/g DW quercetin(Table 2) exhibited the highest TEAC value(231.16 mM) within the tested extracts (Table 4).

Estimation of DNA single strand break damage from exposure to acidicmethanolic and water extracts

Quercetin was found to protect against H2O2-induced DNAdamage in human lymphocytes at 10 μM [34] and at 3.1 to 25 μM [35]. However, it was found to induce DNA damage in human lymphocytes athigher concentrations, such as 100 μM or above [34]. Similarly, myricetin was also found to decrease oxidant-induced DNAdamage at 100 μM, although α–tocopherol andβ–carotene did not behave similarly. This might be due to thedihydroxy structure of quercetin and myricetin being essential for protectingDNA against hydrogen peroxide [34]. No such hydroxyl groups are present in the tocopherol molecule. Thismay reflect structure/activity relationships or the localization of theantioxidant relative to free radical generation within cells. Noroozi etal. [36] demonstrated that, in addition to quercetin, kaempferol could alsoinhibit H2O2-induced DNA strand breaks in humanlymphocytes. Zhu and Loft [37] reported that aqueous extracts of cooked and autolysed Brusselssprouts decreased DNA strand breaks in human lymphocytes, with the maximuminhibition being 38 and 39% at cooked and autolysed extract levels of10 μg/mL and 5 μg/mL, respectively, with the inhibitioneffect decreasing at increasing concentrations up to 100 μg/mL.Quercetin-rich onions showed increased resistance of lymphocytic DNA to exvivo- induced oxidation [15]. In addition, several types of natural antioxidants, includingflavonols and polyphenolic compounds, inhibit adhesion molecule expression andthe adhesion of monocytes to endothelial cells, and also suppress cellinflammation, transformation, proliferation, survival, invasion, andangiogenesis [3840]. Free radicals induce cellular damage and are involved in severalhuman diseases such as cancer, atherosclerosis, and inflammatory disorders, andpolyphenols tend to reduce mutagenic activity and oxygen-free radicals [41]. Since the initiation and progress of carcinogenesis involvesmutations of DNA, the chemical alteration of DNA bases is believed to be acrucial factor. As a consequence of increased oxidative stress, DNA oxidationdamage can occur with ROS, leading to mispairing of DNA bases or DNA strandbreaks. ROS are generated endogenously from cellular metabolism and inflammatoryresponses or by exposure to exogenous agents such as ionizing radiation andxenobiotics [42].

In our study, the inhibition percentages of tested plants ranged from 74.51% (BA)to 91.45% (MA) with acidic methanolic extract concentrations at25 μg/mL (Figure 1A). MA plants had a valueof 985.73 (91.95% inhibition percentage) for tail moment at 25 μg/mLof acidic methanolic extracts (Figure 1B). Theresults in inhibition percentage of tail DNA% were not similar to the results ininhibition percentage of tail moment among treated samples. The MA plant extractwas most effective against DNA single strand breaks in tail DNA%, while HC plantextract was most effective against DNA single strand breaks in tail moment(Figure 1A and 1B). Inaddition, HC plant water extracts exhibited 11.14% tail DNA% (Figure 2A) and 1255.40 (92.19% inhibition percentage) tail momentat the 25 μg/mL dose (Figure 2B). Theinhibition percentage of tail DNA% results was similar to the results of theinhibition percentage of tail moment among treated samples. HC plant extractsnot only had the highest Trolox equivalent (Table 4),but were also the most effective against DNA single strand breaks induced byH2O2 in human lymphocytes (Figure 1), indicating that it contains polyphenol (19.82 mg gallicacid/g DW), myricetin (146.67 μg/g DW), quercetin(3200.00 μg/g DW), and kaempferol (53.33 μg/g DW)(Tables 1 and 2). To someextent, the observed efficacy of the extracts against DNA damage can beattributed to specific flavonol constituents. The high levels of quercetin andmorin are believed to account for the high DNA protective potential of HC and MAsince quercetin has also been identified as an efficient reducer of DNA damagein Caco-2 cells [43]. Morin from Psidium guajava was effective in increasing cellviability, decreasing ROS levels, and preventing DNA fragmentation upon exposureto high glucose levels in primary rat hepatocyte cultures [44]. The antioxidant activity of polyphenolic compounds in differentspecies showed higher polyphenolic content and antioxidant activity in allspecies, demonstrating that the tested species are a potent source of novelbioactive compounds with a wide range of medicinal properties. In particular,they have significant free radical scavenging activity. Our present studydemonstrates that, among the six investigated species, the higher content ofpolyphenols, flavonols, and antioxidant properties in HC and MA plants may bethe reason for their wide medicinal use. Both species can be used as potentmedicinal herbs for novel bioactive compounds with high free radical scavengingactivity, and extracts of these plants may been attractive alternative formanaging oxidative stress-induced liver injury and drug-induced gastric ulcer [45, 46]. Recently, Gargouri et al. [47] demonstrated that quercetin could protect against dimethoate-inducedoxidative stress by decreasing lipid peroxidation and protein oxidation, andincreasing superoxide dismutase and catalase activities in human lymphocytes.The herbaceous plant extracts in our study may increase antioxidant enzymeactivities to protect against H2O2-induced DNA damage inhuman lymphocytes.


Polyphenol-rich extracts from the tested plants effectively diminish DNA oxidationdamage. This preventive effectiveness is attributable to the induction of cellulardefenses rather than the radical scavenging activity of polyphenol and flavonols,and might well contribute to the reported health benefits of herbals. The contentsof these bioactive compounds in MA and HC extracts can explain their antioxidantactivity, and there exists a relationship between the content of polyphenol andflavonol to antioxidant activity. This is the first report suggestion that MA and HCplants have abundant antioxidants with strong antioxidant activity, and consequentlycan protect DNA in lymphocytes from oxidative damage.







High-performance liquid chromatography


Trolox equivalent antioxidant capacity




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Correspondence to Pi-Yu Chao.

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The authors declare that they have no competing interests.

Authors’ contributions

KHL prepared the extracts and carried out all the experimental process. PYC designedthe current project, supervised the work and wrote the manuscript. YYY workedclosely with KCL and MYH in the laboratory to carry out the experiments. HFL and HSLevaluated the data and edited the manuscript. CMY participated in statisticalanalysis. All the authors read and approved the final manuscript.

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Lin, KH., Yang, YY., Yang, CM. et al. Antioxidant activity of herbaceous plant extracts protect against hydrogenperoxide-induced DNA damage in human lymphocytes. BMC Res Notes 6, 490 (2013).

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