Food Chemistry 125 (2011) 1430–1435

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Food Chemistry

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Analytical Methods

A novel antioxidant activity index (AAU) for natural products using the DPPH assay

Jing Deng, Wangyuan Cheng, Guangzhong Yang ⇑College of Life Science, South-Central University for Nationalities, Wuhan 430074, China

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

Article history:Received 18 March 2010Received in revised form 22 August 2010Accepted 6 October 2010

Keywords:Evaluation of antioxidative abilityAntioxidant activity unitLignansDPPH assay

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.10.031

⇑ Corresponding author. Fax: +86 27 67841196.E-mail addresses: iceberg430@hotmail.com, yangg

A novel antioxidant activity unit (AAU) was proposed as the antioxidation ability of natural productsusing the DPPH assay. Based on the definition of AAU, using a combination of fitting curve equationfor scavenging ratio of DPPH free radicals with the theoretical relation between DPPH concentrationand absorbance, the equation was deduced. In this study, eight – lignans and the standard samples of gal-lic acid, vitamin C were used. It was demonstrated that the concentration of DPPH was affected more bytemperature than by time; AAU showed its consistency and uniqueness between different concentrationsof each lignan with a relative standard deviation (RSD) of less than 8.07%. The antioxidant strength ofplant extracts and pure compounds were compared. Analysis of the antioxidant activity of lignans in rela-tion with their structure factors was made.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Free radical biology is a burgeoning discipline researched morebroadly in life science in recent years. It mainly explores the forma-tion and the scavenging of free radicals, as well as the damagecaused by free radicals in biological systems. It is now well estab-lished that a series of oxygen-centred free radicals and other reac-tive oxygen species (ROS) contribute to the pathology of manydisorders including atherogenesis, neurodegeneration, chronicinflammation, cancer and physiological senescence (Ani, Varadaraj,& Naidu, 2006). Therefore, antioxidants are considered importantnutraceuticals on account of their many health benefits and theyare widely used in the food industry as potential inhibitors of lipidperoxidation (Scherer & Godoy, 2009). However, it has been dem-onstrated that synthetic antioxidants can accumulate in the bodywhich can result in liver damage and carcinogenesis. These prob-lems are not seen when natural antioxidants, extracted from herbsand spices with high antioxidant activity, are used in food applica-tions. These exacts are safe, potentially nutritional and have ther-apeutic effects.

1,1-Diphenyl-2-picrylhydrazyl (DPPH), is a kind of stable organ-ic radical. The DPPH oxidative assay (Peng, Chen, Lin, & Lin, 2000)adopted in the paper is used worldwide in the quantification ofradical-scavenging capacity (RSC). The capacity of biological re-agents to scavenge the DPPH radical, can be expressed as its mag-nitude of antioxidation ability. The DPPH alcohol solution is deeppurple in colour with an absorption peak at 517 nm, which disap-

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pears with the presence of the radical scavenger in the reactivesystem and when the odd electron of the nitrogen in the DPPHare paired. The reactive rate and the ability of the radical scavengerdepend on the rate and the peak value of disappearance of theDPPH (Chi, Zhang, Zheng, & Mei, 2003). Compared with othermethods, the DPPH assay has many advantages, such as good sta-bility, credible sensitivity, simplicity and feasibility (Jin, Li, &Zhang, 2006; Ozcelik, Lee, & Min, 2003).

The results of the DPPH assay have been presented in manyways. The majority of studies express the results as the IC50 value,defined as the amount of antioxidant necessary to decrease the ini-tial DPPH concentration by 50%. This value is calculated by plottinginhibition percentage against extract concentration (Ani et al.,2006; Elzaawely, Xuan, & Tawata, 2007; Sokmen et al., 2004; Tepe,Sokmen, Akpulat, & Sokmen, 2005). However, for plant extracts orpure compounds, the IC50 value changes according to the final con-centration of the DPPH used. The lack of standardization of the re-sults makes it difficult to compare the antioxidant strength ofdifferent plant extracts, as well as of pure compounds (Scherer &Godoy, 2009; Sun & Ho, 2005; van den Berg, Haenen, van den Berg,& Bast, 1999). Hence, Scherer and Godoy developed a calculationalformula in 2009 named ‘‘antioxidant activity index (AAI)’’ (Scherer& Godoy, 2009). Three concentrations of DPPH were used and nosignificant difference in the AAI for each compound tested was ob-served, indicating that the AAI found was appropriate to comparethe antioxidant strength of plant extracts, as well as of pure com-pounds. However, AAI has some limitations. Firstly, it does nothave a precise definition: a viewpoint should be formed on the ba-sis of theories and principles, even if though its correctness hasbeing proved by experiments. Secondly, according to Beer’s law,the range of accuracy for spectrophotometric measurements falls

J. Deng et al. / Food Chemistry 125 (2011) 1430–1435 1431

within an absorbance of 0.221–0.698 which equals a transmittanceof 20–60%. This corresponds to a DPPH concentration of nearly 25–70 lM (Sharma & Bhat, 2009). However, the concentrations ofDPPH used in the study by Scherer and Godoy (2009), producedabsorbance values between 2 and 3. Since these absorbance valuesfell outside the range of accuracy for spectra measures, their datashould be questioned. Recently, de Brito’s group used a kineticmodel (Rufino, Fabiano, Alves, & de Brito, 2009) to study the radi-cal-scavenging capacity of several tropical fruits. The aim of thisstudy was to characterize kinetically the free radical-scavengingcapacity of certain Brazilian fruits by using DPPH. They determinedthe antioxidant activity of the test substances by comparing thesecond-order rate constants proposed but they neglected the effectof the quantity of the test substance.

A novel evaluation coefficient ‘‘antioxidant activity unit (AAU)’’was proposed in this paper by theoretical deduction and verifiedby two standard scavengers (gallic acid and vitamin C) and eightlignans. The definition and calculation equation for AAU are shown.Experiments demonstrated that AAU, the novel index, was appro-priate to compare the antioxidant strength of plant extracts, aswell as of pure compounds. This index allows for the standardiza-tion of the antioxidant capacity of test compounds even when dif-ferent concentrations are used, which makes this method verypractical.

2. Materials and methods

2.1. Materials and instruments

Eight of lignans were extracted from Zanthoxylum Planispinumsieb.et Zucc. The Z. Planispinum sieb.et Zucc (Fang & Liao, 2006)was picked from Enshi district, Hubei province of China. The stan-dard scavengers, gallic acid was purchased from Tianjin KermelChemical Reagent Co. Ltd. (China) and vitamin C was purchasedfrom Shanghai Zhanyun Chemical Co. Ltd. (China). One gram pervial of 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Sigma, USA) wasused. Methanol and all other solvents were analytic grade andwere purchased from Sinopharm Chemical Reagent Co. Ltd.(China). Double distilled water was used in all experiments.

Absorbance measurements were made using a model 722 Spec-trophotometer (Shanghai spectrum instrument Co. Ltd., China).

2.2. Methods

2.2.1. AAU equationFitting curve equation of the free radical scavenging ratio

y = Bx + D was combined with theoretical relation between DPPHconcentration and absorbance (Abs) y = Kx to deduce the index,antioxidant activity unit (AAU), of all-purpose and practicable,which is defined as ‘‘1 mol of DPPH free radical was completelyscavenged to consume amount (mole number) of the scavenger.’’The lower the AAU value, the stronger the antioxidation ability ofa compound is.

AAU ¼ 394:32� RB� C �Mr

ð1Þ

where R is the solution volume ratio of sample to solution volume ofDPPH for each reaction; B is slope of fitting equation of the free rad-ical scavenging ratio; C is initial concentration of DPPH solution ob-served (g mL�1); Mr is the molecular weight of the sample.

2.2.2. Measurement of antioxidant activity

Step 1. Preparation of a gradient solution of samples: sampleswere accurately weighed, and dissolved in methanol to producea solution of known concentration. One millilitre of solution

was added to 1 mL of methanol, thus diluting the concentrationof sample by twofold. Sample were serially diluted 1/4, 1/8, 1/16, 1/32 and 1/64.Step 2. DPPH system: 2.9 mg, 4.4 mg and 5.9 mg of DPPH wereaccurately weighed in 250 mL volumetric flasks. Methanolwas added until a total of 250 mL was reached. Solutions werestored in the dark and in a dry environment.Step 3. Reactive system: to seven ground-glass test tubes 3.9 mLof DPPH solution was added. To one tube 100 lL of methanolwas added. This tube served as the blank. To the other six tubes100 lL of six solutions prepared in step 1 was added. Thesesolutions were mixed and allowed to react for 40 min at25 �C. The solutions were transferred to a 1 cm colorimeter celland the absorbance read at 517.

These experiments, from step 1 to step 3, were repeated threetimes in 2 days.

Equation of free radical scavenging ratio (Guerrero, Guirado,Fuentes, and Pérez, 2006)

I% ¼ Abs0 � Absl

Abs0

� �� 100% ð2Þ

The AAU correlative curves of samples were plotted in light ofsample concentration on the x axis and their absorbance on the yaxis.

3. Results and discussion

3.1. Evaluation of AAU index and accuracy

Lignans, a type of compounds that possess phenolic hydroxylgroups, widely exist in food plants (Zhang, Wang, & Wang, 1996).Phenol is an admirable proton and electron donor just becauseits hydroxyl group is high at activity and has the ability absorbingfree radicals. It can release hydrogen atoms to combine with theradicals produced from auto-oxidation, breaking the chain reac-tion. On the other hand, the resonance and non-localized effectin the phenol produces stable radical intermediates which has nosuitable place when attacked by dioxygen causing a new radicalreaction or a chain reaction which can be quickly oxidized.

At present, the measurement of the antioxidant activity of mostnatural products was estimated in their ability to scavenge organicradicals. The IC50, as a measure of a compound’s ability to scavengefree radicals, has some errors when the concentrations of free rad-icals are different. While AAU, as a novel index, is specially builtfrom the DPPH assay. It means that AAU is the property of the sam-ple and is not concerned with the experimental conditions.

3.1.1. Effect of temperature and time on the concentration of the DPPHsolution

Three initial concentrations of a DPPH methanol solution with-out buffer were randomly chosen. The solutions were stored in thedark at 25 �C. The absorbance of each solution was measured atregular intervals. The change in absorbance of the DPPH solutionsover time is shown in Fig. 1 (curves 1, 2 and 3). Absorbance of thethree curves rallentando decreased at a range of 0.01 units within90 min except for random fluctuates due to measurement errors.This suggests that the free radicals in the methanol solution ofDPPH decomposed with time so that the real concentration couldnot be obtained. Therefore, the experimental DPPH solution shouldbe used within 2 h of preparation. Remarkably, curve 4 from a ran-dom initial concentration of DPPH methanol solution showed theabsorbance of the DPPH changed as the temperature changed from4 to 25 �C. It had a sharp decrease in absorbance at a range of 0.03units within 90 min. This implied that the DPPH solution kept in a

0 20 40 60 80 1000.42

0.43

0.44

0.45

0.46

0.47

0.48

0.49

0.50

0.51

0.52

3

2

4

1

Abso

rban

ce

Time (min)

Fig. 1. Effect of temperature on free radicals of DPPH. (Curves 1, 2 and 3 were DPPHsolutions of different random initial concentrations stored in 25 �C. Curve 4 was aDPPH solution of a random initial concentration that was taken out from therefrigerator.)

0 20 40 60 80 1000.175

0.200

0.225

0.250

0.275

0.300

0.325

Abso

rban

ce

Time (min)

Fig. 2. Gallic acid finished the reaction in about 55 min.

1432 J. Deng et al. / Food Chemistry 125 (2011) 1430–1435

refrigerator underwent rapid decomposition of free radicals whenit was taken out from the refrigerator to carry out experiments at25 �C or higher. The effect of temperature is more dramatic thanthe effect of time on the concentration of DPPH. In contrast, a DPPHsolution may be kept for a short period of time at room tempera-ture. This is better than in refrigerating the solution.

15

20

25

Y = 1080.0X - 0.036R2=0.999

tion

rate

(%)

3.1.2. Confirmation of DPPH systemAccording to Beer’s law, the optimal detectable range of a spec-

trophotometer was fixed between 0.221 and 0.698 (Abs), whichcorresponds to a DPPH concentration between 25 and 70 lM(Sharma & Bhat, 2009). Three solution concentrations of the DPPHwere determined based on the relationship between concentrationand absorbance. Concentration 1 is 2.9 mg/250 mL(11.6 � 10�6 g mL�1, 30 lM); concentration 2 is 4.4 mg/250 mL(17.6 � 10�6 g mL�1, 45 lM); concentration 3 is 5.9 mg/250 mL(23.6 � 10�6 g mL�1, 60 lM).

Before measuring the samples, concentration 1 of DPPH, thelowest concentration to be prepared, was used to generate a testcurve regarding concentration and absorbance of the samples inorder to determine a suitable concentration to measure. Ten sam-ples used in this work are shown in Table 1. The concentrations ofeach solution were decided by this test.

The reactive rate constant of an antioxidant, that is the scaveng-ing rate constant of free radicals, is another method use to evaluatethe antioxidation ability of a sample (Rufino et al., 2009). We thinkthat the antioxidation effect of a substance should consider thereactive rate of scavenging DPPH free radicals, simultaneously withthe consumed amount of scavenger. Therefore, before the scaveng-ing effect of free radicals of a sample was measured, and theapproximate time needed to complete the reaction was investi-gated. For most of sample, the reaction was completed within

Table 1The initial concentration of test reagents.

Compounds GAa VCb Lignans

1 2 3 4 5 6 7 8

Conc. 10�3 g/mL

0.02 0.05 0.2 0.1 0.1 1.0 4.0 0.1 0.1 0.03

a Gallic acid.b Vitamin C.

40 min, except for vitamin C. For example, using gallic acid thescavenging reaction was finished in about 55 min as shown inFig. 2, but the rate of reaction using vitamin C was quick, onlyneeding 5–10 min. Hence 40 min was used as the time needed tocomplete the reaction.

3.1.3. Calculation and deduction of AAU equationThe fitting curve equation of scavenging ratio is y = Bx + D. The

equation curve should pass through the origin under ideal condi-tions. The DPPH will partially break down when it is subject tothe influence of light irradiation and temperature change in thecourse of preparation. The absorbance deviation of a known con-centration of DPPH solution fell within a range of 0.3. The closerto zero the intercept D value, the less the measurement errorwas. The absolute values of D among experiments were all lessthan 0.7, so this was omitted. For example, Fig. 3 shows the exper-imental data of the scavenging ratio for concentration 2 of gallicacid and the fitting curve. The fitting curve equation isy = 1080.0x � 0.036, the correlation coefficient R2 = 0.999, theintercept D = �0.036 whose absolute value was less than 0.7. TheD value was omitted close to zero, so the fitting equation may bewrote as y = 1080.0x.

Assuming that N mole sample may react with 1 mol DPPH com-pletely. The mass ratio of the sample and DPPH is N �Mr: 394.32(the molecular weight of 1 mol DPPH), and the volume (Vt,sample)

0.000 0.005 0.010 0.015 0.0200

5

10

Elim

ina

Concentration (mg/mL)

Fig. 3. The elimination ratio vs. the concentration 2 system of gallic acid.

J. Deng et al. / Food Chemistry 125 (2011) 1430–1435 1433

of reactive sample takes 1/d the volume (VTotal sample) of the pre-pared sample concentration:

Mt;DPPH ¼ 394:32� MTotal sample

d� N �Mrð3Þ

where Mt,DPPH is the DPPH mass which takes part in the reaction andMTotal sample is the mass in the prepared sample concentration.

Since the concentration (Mt/Vt) and the absorbance (At) of theDPPH solutions which join the reaction have a correlative equation(ideal state): y = Kx (x is the DPPH concentration, y is the absor-bance), and following equation was derived:

At ¼K �Mt

Vtð4Þ

To get from (3) and (4):

At ¼ 394:32� K �MTotal sample

d� Vt � N �Mrð5Þ

Setting the reactive volume (Vt) of DPPH solution takes 1/b thevolume (VTotal DPPH) of the prepared DPPH solution (C is the initialconcentration of DPPH).

Vt ¼VTotal DPPH

bMTotal sample

b� C

So, to get from (5):

At ¼ 394:32� K �MTotal sampleb� Cd�MTotal DPPH � N �Mr

ð6Þ

Also to get from the ideal state (y = Kx) of the initial concentra-tion (C) and absorbance (A0) of DPPH:

K ¼ yx¼ A0

Cð7Þ

To replace K into Eq. (6), getting:

At ¼ 394:32� A0 �MTotalbd�MTotal DPPH � N �Mr

ð8Þ

To gain from fitting equation y = Bx + D of the scavenging ratio(herein for ideal state that is to omit D value):

Scavenging ratio ¼ At=A0 ¼ B� CTotal sample ð9Þ

To get from Eqs. (8) and (9):

394:32�MTotal sample � bd�MTotal DPPH � N �Mr

¼ B� CTotal sample

N ¼ 394:32�MTotal sample � bd�MTotal DPPH �Mr � B� CTotal sample

N ¼ 394:32� VTotal sample � bd�MTotal DPPH � B�Mr

ð10Þ

If setting

R ¼ b� VTotal sample

d� VTotal DPPH

That is R ¼ Solution volume of the sample each reactionSolution volume of DPPH each reaction

� �

MTotal DPPH/VTotal DPPH is just the initial concentration of DPPH, C,thus Eq. (9) may gain the simplest expression as Eq. (1) to beobtained.

N ¼ R� 394:32� VTotal DPPH

MTotal DPPH � B�Mrð11Þ

AAU ¼ N ¼ R� 394:32B� C �Mr

It can be proved that AAU value and AAI value (Scherer & Godoy,2009) of an antioxidant are both related to B (the slope of fittingequation of the free radical scavenging ratio) and C (the initial con-centration of DPPH solution observed). They are proportionate tothe later, but inversely proportionate to the former.

3.2. Analysis of experimental data

The structures of these eight lignans extracted from Z. Planispi-num sieb.et Zucc are so similar that the relation between molecularstructure and their antioxidation abilities can be determined bycomparison and analysis.

From Table 2 it is seen that the IC50 (lg/mL) values of each re-agent are different for each of the three different concentrations ofDPPH solution, but AAU (mol) values have no significant difference.For examples, gallic acid has the strongest antioxidation ability,AAU values are 3.03, 3.12 and 2.98 respectively, with a relativestandard deviation (RSD) of 1.91%. The IC50 values for the three dif-ferent concentrations of DPPH solution are 2.18, 3.20 and 4.65respectively, with a RSD of 27.0%. For compound 4, which hasthe weakest antioxidation ability, AAU values are 385.84, 371.78and 362.49 respectively with a RSD of 2.57%, but the IC50 valuesare 194.93, 277.74 and 358.38 respectively, with a RSD of 24.1%.These results indicate that AAU is a more all-purpose index be-tween different concentrations in terms of its consistency anduniqueness.

It is generally acknowledged that the activate energy of a reac-tion involving free radicals joining is very low, and this reaction hasthe kinetic characteristic of a chain reaction. Antioxidants preventfree radicals from causing harm in different ways. The most effec-tive way is by the phenolic hydroxyl group of an antioxidant react-ing with a harmful free radical to produce a more stablehemiquinoid radical, consequently cutting off the chain reactionof harmful free radicals. Another method is that the antioxidant di-rectly delivers an electron, by self-reduction, to stabilize the radi-cal. The lignans tested in this study stabilize radicals using thelatter method. The molecular weights of the eight lignans testedare between 350 and 400 close to the molecular weight of organicfree radical DPPH. Three lignans, syringaresinol, epipinoresinol and30-O-demethylepipinoresinol are stronger antioxidants than thestandard antioxidant vitamin C, in their ability to scavenging freeradicals. Vitamin C has a very small molecular weight, 176.13,and the rate of scavenging radicals is very fast (the reactive systemfinishes in 10 min in our experiments).

3.3. Analysis of lignans structure

The eight lignans in Z. Planispinum sieb et Zucc all contain diphe-nyl bifuranidine rings, only differing in their connection to thebenzene ring.

From a structural point of view, the magnitude of the antioxi-dant ability for these compounds is directly related to the strengthof the O–H bond. When the ionization energy of O–H bond of thesecompounds becomes larger, the antioxidant ability reduces whenthe substituent is the electron-withdrawing group. On the con-trary, the antioxidant ability of a compound increases when thesubstituent is an electron-pushing group. Both compounds 4 and5 without the phenolic hydroxyl group, obviously have a lowerantioxidant activity. In contrast, compound 5 donates electronsmore easily than compound 4 (Wang, Run, & Jin, 2007). Thereare two possible reasons. Firstly, the two ortho-OCH3 groups ofcompound 5 have better electron-pushing ability than the 1,3-dioxacycle of compound 4. Secondly, compound 4 with a certainextent symmetry has a more stable structure than compound 5(Long, Gao, Chen, & Wang, 2006) thus reduces its antioxidantactivity.

Table 2Comparison of AAU and IC50 values of the standard and the lignans samples.

Code Compound Concentration 1 Concentration 2 Concentration 3 Mean AAU Mean IC50 Mr RSDAAUa (%) RSDIC50

b (%)

AAU IC50 AAU IC50 AAU IC50

Gallic acid 3.03 0.74 3.12 1.15 2.98 1.48 3.04 1.12 170 1.91 27.0Vitamin C 5.38 1.35 5.59 2.15 5.33 2.74 5.43 2.08 176 2.08 27.4

1 Pinoresinol mono-methyl ether 13.09 4.92 13.05 8.34 13.15 13.31 13.10 8.86 372 0.31 38.92 Syringaresinol 4.94 2.91 4.26 3.86 4.12 4.96 4.44 3.91 418 8.07 21.43 De-40-O-methyl-yangambin 6.94 4.27 6.13 5.82 6.51 8.00 6.53 6.03 432 5.07 25.44 Asarinin 385.84 194.93 371.78 277.74 362.49 358.38 373.37 277.02 354 2.57 24.15 Fargesin 98.49 54.30 98.49 58.99 97.62 89.92 98.20 67.74 365 0.42 23.36 Horsfieldin 6.74 3.43 6.65 5.08 7.26 7.58 6.88 5.36 356 3.91 31.87 Epipinoresinol 4.31 2.18 4.11 3.20 4.42 4.65 4.28 3.34 344 3.00 30.38 30-O-Demethyl-epipinoresinol 5.25 2.60 4.55 3.39 4.39 4.38 4.73 3.46 344 7.90 21.0

Each value is the mean value of three replicate experiments.a RSDAAU is the relative standard deviate of AAU.b RSDIC50 is the relative standard deviate of IC50.

1434 J. Deng et al. / Food Chemistry 125 (2011) 1430–1435

Compared with compounds 4 and 5, the antioxidant activity ofcompound 6 is nearly twofold stronger than compound 1; com-pounds 1 and 6 both have a phenol hydroxyl group, which maybe the reason the activities of compounds 1 and 6 are higher thancompounds 4 and 5. Moreover, the reason the antioxidant activityof compound 6 is twice as high as compound 1, may be one side ofthe benzene ring with ether ring lignans may enhance the antiox-idation of the side of the benzene ring containing a hydroxyl group.For compound 3, three adjacent OCH3 groups on one side enhanceits antioxidation ability, but two OCH3 groups adjacent to thehydroxyl group inhibit it on another side. The result is that com-pound 3 has an antioxidant activity close to compound 6.

The structures of compounds 2 and 3 are very similar. The onlydifference is the OCH3 of compound 3 is changed into a hydroxylgroup displaying symmetry with another hydroxyl group on thepara-position. This gives rise to an increase in antioxidation activ-ity. Comparing compounds 7, 8 and 2, compound 8 with an ortho-hydroxyl group beside a para-hydroxyl group has a lowerantioxidant activity than compounds 2 and 7. One could speculatethat the ortho-substituent relative to two para-hydroxyl groupsmay inhibit the antioxidant activity. The inhibition of the hydroxylgroup on the non-para-position is greater than the methoxyl,which causes a decline in electron delocalization. The non-para-hydroxyl in compound 8 may be defined as ‘‘inefficient group’’from Zhao’s group suggestion (Zhao, Liang, & Yan, 2001). Com-pound 2 compared to compound 7, has an ortho-methoxyl groupwhich may weaken the function of the adjacent hydroxyl group.Compound 7 has the highest antioxidant activity among the com-pounds tested. By comparison, the order of the oxidant activity is7 > 2 > 8 > 3 > 6 > 1 > 5 > 4.

4. Conclusion

1. Experiments demonstrate that the absorbance curves of ran-dom concentrations of DPPH radicals rallentando decreased ata range of 0.01 units at 25 �C. The absorbance curves sharplyreduce at a range of 0.03 units when the temperature isadjusted from 4 to 25 �C within 90 min. Temperature affectedon the concentration of DPPH more notable than time. Theantioxidation effect is manifested by the reactive rate of scav-enging DPPH free radicals and the consumed amount of lignans.

2. The hydroxyl groups at opposite positions in the structure oflignans have a predominant antioxidant activation ability, butthe greatest inhibition of the antioxidation ability of a com-pound is if a hydroxyl group is present at the ortho-position.If one side of the benzene ring contains an ether ring, this ismay enhance the antioxidation of the other side of the benzenering containing hydroxyl groups in lignans.

3. The antioxidant activity unit (AAU) which is being proposed,can better and more accurately evaluation the antioxidationability of compounds. Its definition is that 1 mol of DPPH freeradical was completely scavenged to consume an amount (molenumber) of a certain scavenger. It can be proved that AAU valueand AAI value (Scherer & Godoy, 2009) of an antioxidant areboth related with the slope of fitting equation of the free radicalscavenging ratio and the initial concentration of DPPH solutionobserved. The AAU values showed no significant differencebetween different concentrations of each compound. Its consis-tency and uniqueness indicates that it is a more all-purposeindex to compare different compounds.

Acknowledgements

Authors are thankful to Dr. Richard Mclaughlin, Professor ofBiology Department from Saint Mary’s University (Winona, Minne-sota, USA) for careful revision of our manuscript. This work wasfinancially supported by National Natural Science Foundation ofChina (No. 30670215) and National Support Science and TechnologyProject (2007BAI48B08).

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