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On-line HPLC-DPPH Screening Method for Evaluation of
Radical Scavenging Activity of Phenols Extracted from
Regional Agricultural Products
Ashok K. SARKER, Chizuko YUKIZAKI, Miho KOMURA,
Masato KOKUBO and Reiji OKABE
An on-line HPLC-DPPH screening method for phenolic antioxidants in Miyazaki regional agricultural
products such as sweet potato leaf, burdock root, carrot leaf, lettuce and lemon balm was applied in orderto estimate the contribution ratio of each phenol to total antioxidant activity for those samples. The determination of antioxidants was based on a decrease in the absorbance at 515 nm after postcolumn reactionof HPLC-separated antioxidants with the 2, 2’-diphenyl-1-picrylhydrazyl radicalDPPH.. The on-line
method was applied for quantitative analysis of the antioxidants. A linear dependence of negative peak area
on concentration of the reference antioxidants was observed. When five kinds of regional agricultural
products were applied to the method, several peaks of known phenols were recognized which gave negative
peaks corresponding to the DPPH radical scavenging activity. For example, burdock root contained caffeic
acid, chlorogenic acid, and dicaffeoylquinic acids and those contributions were 0, 62 and 38, respectively. For lettuce, caffeic acid, chlorogenic acid, dicaffeoyltartaric acid and other unknown active
components were detected ; those contribution ratios were 0, 26, 54, 8 and 12 respectively.
This information should be useful for application of those regional agricultural products to functional materials.
INTRODUCTION
Oxygen and reactive oxygen speciesROSare among the major sources of primary catalysts that initiate
oxidation in vivo and in vitro1, 2. Oxygen-derived free radicals such as superoxide anion radical andhydroxyl radical are thought to be linked to the onset of various pathological conditions. Many researchers
have shown that lipid peroxides and reactive oxygen species are involved in the development of a variety
of diseases, including cancer, and also accelerate aging3-8. A compound might exert antioxidant actions
in vivo or in food by inhibiting generation of ROS, or by directly scavenging free radicals9-12.
Antiradical antioxidants act by donating hydrogen atoms to lipid radicals. Radicals obtained from antioxidants with molecular structures such as phenols are stable species and then stop the oxidation chain
reaction4, 13. Furthermore, there is now growing evidence that polyphenols may possess inhibitory
effects against cancer14. Many anticarcinogens are naturally occurring non-nutrients, primarily of plantorigin, such as flavones and polyhydroxy compounds15, 16.
A multitude of natural antioxidants have already been isolated from different kinds of plant materials suchas oilseeds, cereal crops, vegetables, fruits, leaves, roots, spices, and herbs6, 9, 17. Plants contain adiverse group of phenolic compounds including simple phenolics, phenolic acids, anthocyanins,hydroxycinnamic acid derivatives, and flavonoids. All the phenolic classes have the structural requirementsof free radical scavengers and have potential as food antioxidants18, 19. Factors influencing the antioxidant activity of plant phenolics include position and degree of hydroxylation, polarity, solubility, reducing
potential, stability of the phenol to food processing operations, and stability of the phenolic radical20,
Miyazaki Prefectural Food Research & Development Center, Miyazaki, Japan.
Miyazaki Prefecture Agriculture Experiment Station, Miyazaki, Japan.

On-line HPLC-DPPH Screening Method for Evaluation of Radical Scavenging
Activity of Phenols Extracted from Regional Agricultural Products

21. Cuvelier et al.22studied the antioxidative efficiency of some phenolic acids, including caffeic acid,ferulic acid, rosmarinic acid, chlorogenic acid, and BHT. Chen et al.23investigated the antioxidative
potency of some hydroxycinnamic acid compounds and elucidated the relationship between their activities
and chemical structures. Koleva et al.24published a new rapid online method for screening complex
mixtures for radical scavenging components using a methanolic solution of the DPPH stable free radical,
and presented an optimized instrumental setup. The greatest benefit of this method is that it is immediately
clear which constituent possesses radical scavenging activity.
To the best of our knowledge, there is no information available about the contribution ratio of a phenol
compound to the total antioxidant activity present in a sample using an on-line HPLC-DPPH method.
From this purpose, the authors tried to apply the feasible on-line HPLC-DPPH screening method developed
by Koleva et al.24to some regional agricultural productssuch as sweet potato leaf, burdock root,
carrot leaf, lettuce and lemon balmin Miyazaki prefecture and estimate the contribution ratio of the antioxidant activity of a phenol compound which is contained in each sample.
MATERIALS AND METHOD
Materials : The root of burdockArctium lappa L., Sweet potato leafIpomoea batatas L., Carrot leaf
Daucus carota L. and Lettuce Lactuca sativa L. were supplied from Miyazaki Agricultural
Experimental StationMiyazaki, Japan. Lemon BalmMelissa Officinaliswas supplied from Miyazaki
herb research centerMiyazaki, Japan. The all samples were washed thoroughly in water, cut into small
species and freeze driedFTS SYSTEM, Dura-Top MP & Dura-Dry MP. The freeze-dried sample waspowderedMRK & RETSCH, EM-1 ; mesh size 0.5 mm. Chlorogenic acid and Caffeic acid were purchased from Sigma ChemicalsSt. Louis, MO, USA. Dicaffeoyl Tartaric acid was obtained from
ChromaDex Inc.Santa Ana, CA, USA. Rosmarinic acid was purchased from MP Biomedicals, Inc.
Germany. Gallic acid, formic acidHPLC gradeand acetic acid were obtained from Nakalai TesqueInc.Kyoto, Japan. DPPH2, 2-diphenyl-1-picrylhydrazyland LC grade methanol were obtained from
Wako Pure Chemical IndustriesOsaka, Japan. All other chemicals were of analytical reagent grade andused without further purification.
Sample preparation : Twenty millilitermlof 80 MeOH was added to freeze-dried sample powder
0.2 g. The slurry was stirred for 10 min at room temperature with a magnetic stirrer for extraction. The
solution was filtered with 0.45 m filter. The filtrate was analyzed by HPLC with proper dilution.
On-line HPLC-DPPH Analysis : An on-line method was described for the detection of radical scavenging
components. The on-line HPLC-DPPH method was developed using a methanolic solution of DPPH stable
free radical. A scheme of the instrumental setup is given in figure 1. The HPLC separated analytes reactedwith the DPPH at a concentration of 10 mgl in methanol in postcolumn. The flow of the reagent solution
was set to 1.0 mlmin and the induced bleaching was detected as a negative peak photometrically at 515
nm. The length of the capillary15 mused for the postcolumn reaction was adjusted to achieve a reaction
time of 50 seconds. The separation of antioxidative components was carried out by HPLCJASCO PU-980
intelligent HPLC pump ; JASCO DG-980 3-line degasser ; JASCO CO-1565 intelligent Column Oven
coupled with a photodiode array detectorPDA 195 - 650 nm
JASCO MD-9109and an auto-sampler
JASCO AS-950-10, operated by Borwin software. An ODS columnAtlantis dC18 ; 150  4.6 mm id; particle size 3 
m ; waters Corporation, USAwith a guard column containing the same stationary phase
was used. The mobile phase consisted of water containing 0.2v vformic acidAand 100
methanolB. The gradient elution was performed with 2 B from 0-15 min, a linear gradient of 2 to
45 of B from 15 to 50 min, and 45 B from 50 to 65 min. The wavelength of detection was set at 328
On-line HPLC-DPPH Screening Method for Evaluation of Radical Scavenging
Activity of Phenols Extracted from Regional Agricultural Products

Fig.1. Instrumental setup for the HPLC-DPPH on-line detection of radical scavenging compounds.

nm. The column was temperature controlled at 40 
C. The methanolic extracts were used directly for online HPLC analysis. For the stock solution of the standards, the polyphenolsChlorogenic acid, Caffeic
acid, dicaffeoyltartaric acid and rosmarinic acidwere dissolved in 80 methanol at a concentration of 1
mg ml. The concentrations of the reference substances used for calibration of the HPLC analysis were
0.02, 0.04, 0.06, 0.08 and 0.01 mgml.
LC-MS Analysis : Electro-spray mass spectrophotometric analyses were performed in the negative andpositive mode using a Finnigan LCQThermo Electron Co.LC-MS spectrophotometer. All the analyses
were performed using electrospray ionizationESIwith the following settings : Ion-spray voltage : 5 KV
; Capillary voltage : 10 V ; Capillary temperature : 270 
C. The chromatographic conditions were as sameas those used for the HPLC analysis. Only flow rate and gradient elution was changed as follows : flow rate
0.5 mlmin and gradient elution : 2 B0-15 min, 2-45 B15-55 min, 45 B55-75 min.
Full-scan data acquisition was performed, scanning from mz 150 to 1000.
RESULTS AND DISCUSSION
A novel screening method for the antioxidants is achieved by on-line HPLC using the DPPH free radical.
It is well known that DPPH absorbs at 517 nm, but upon reduction by an antioxidant or a radical species
the absorption disappears9. The more rapidly the absorbance decreases, the more potent the antioxidant
activity of the compound in terms of hydrogen donating ability25. The HPLC separated antioxidants
react with the DPPH in postcolumn. The induced bleaching is detected as a decrease in absorbance at 517
nm.
For validation of the on-line HPLC method, the calibration curves of negative peaks at concentration of 10,
20. 40, 60, 80 and 100 ppm of reference phenolscaffeic acid, chlorogenic acidwere obtained. A linear
relationship between the negative peak areas and injected sample concentrations was observed for tested
phenolsY4.436x1.08. The correlation factors obtained for the relations of all tested radical scavengers were very high0.998-0.999. Here reported results are in good agreement with those previously published26.
As an example of application to real samples, the determination of antioxidants in extracts from sweet
potato leaf, burdock root, carrot leaf, lettuce and lemon balm were carried out with the present on-line
HPLC method.
First of all, the radical scavenging activity, total polyphenol and chlorogenic acid contents of burdock root,
sweet potato leaf, carrot leaf, and lettuce were measured. These vegetables are respective representativesOn-line HPLC-DPPH Screening Method for Evaluation of Radical Scavenging
Activity of Phenols Extracted from Regional Agricultural Productsof root and leafy vegetables consumed frequently in Japan. Among them, sweet potato leaf had highest
radical scavenging activity290 
mol Trolox eqg dryand chlorogenic acid content9.95 mgg dry;
its radical scavenging activity was 2.5-, 2.7- and 6.5-fold higher than burdock root, carrot leaf and lettuce,
respectively. The highest polyphenol was found in sweet potato leaf64 mg gallic acid eqg dry. Sweet
potatoSuioleaves have contained highest polyphenol compare to other leafy vegetables.
The antioxidant active component of sweet potato leafFig.2was determined. Sweet potato leaf contained caffeic acidtrace amountchlorogenic acid and quinic acid derivatives. Peak 2 was identified as
chlorogenic acid with the comparison of authentic marker. In LC-MS spectra peak 2 gavepositive ion
peakmz at 354 and characteristic fragmentation at mz 163. The LC-MS chromatogram in the-ve
ion mode of bothpeak 3 and peak 4showing mz 516 and mz 180 which were dicaffeoylquinic acidand its fragmentation caffeic acid. Yoshimoto et al.27reported also dicaffeoylquinic acid, which ispresent in sweet potato leaf. In sweet potato leaf calculated contribution of chlorogenic acid to the antioxidant activity is 65. Sweet potato leaf contained 9.95 mggpowderof chlorogenic acid. Therefore thehigh activity in sweet potato leaf is derived from its high chlorogenic acid content.

The HPLC chromatographic pattern of burdock root is shown in Fig. 3. Among these peaks only 4 peaks
peak 2, peak 3, peak 4 & peak 5were reacted postcolumn with the DPPH. Peak 1 and peak 2Fig. 2.
were identified as caffeic acid and chlorogenic acid by the comparison of authentic marker. The LC-MS
chromatogram of peak 2 showed the characteristic fragments at m z 353chlorogenic acidand 191

Fig. 2. HPLC Chromatogram of sweet potato leaf80 methanol extractquinic acid. Peak 3, peak 4 and peak 5 were unidentified quinic acid derivatives with mz 515, 515 and630, respectively. Wang et al. 28 reported that burdock root contains chlorogenic acid, 1,5dicaffeoylquinic acid and 1,5-dicaffeoyl-3-succinylquinic acid, with the predominant polyphenols among
13 kinds of burdock root being chlorogenic acid. We also found the predominant polyphenol in burdock
was chlorogenic acid. The content of chlorogenic acid in burdock root was determined. Burdock root contained 3.98 mggpowderchlorogenic acid. The calculated contribution of chlorogenic acid to the antioxidant activity is 62 in burdock root. Therefore, chlorogenic acid is the major antioxidant active
component of burdock root. Burdock root have contained very small amount of caffeic acid.
The antioxidant active components of carrot leaf and lettuce were also determined. Both samples contained
chlorogenic acid, which was characterized by the comparison of authentic marker and LC-MS. In LC-MS
carrot leaf & lettuceshowed mz at 353chlorogenic acidand its fragmentation 191quinic acid.
On-line HPLC-DPPH Screening Method for Evaluation of Radical Scavenging
Activity of Phenols Extracted from Regional Agricultural Products

Fig. 3. HPLC Chromatogram of Burdock root80 methanol extract.

Carrot leaf contained unidentified two antioxidant components. We also characterized dicaffeoyltartaricacid from lettuce by the comparison of authentic marker. Hisaminato et al.29also reported that lettuce
contains dicaffeoyltartaric acid. This is the major antioxidant compound in lettuce. This compound showedparent peakmzat 473 by LC-MS. The contribution ratio of chlorogenic acid and other two unidentified
antioxidant components in carrot leaf is 58 32 and 10, respectively. In lettuce the contribution ratioof chlorogenic acid and dicaffeoyltartaric acid is 26, 54, 8 and 12, respectively.
Figure 4 shows a typical HPLC profile of the lemon balm. The antioxidant active componentonly oneof lemon balm was characterized by the comparison of authentic marker and LC-MS. Rosmarinic acid is
an important phenolic active compound of lemon balm. In LC-MS base peak was found atmz359
Rosmarinic acidand its characteristic fragmentationmzat 179Caffeic acid. In lemon balm contribution of rosmarinic acid to the antioxidant activity is 98.

Conclusions :The proposed screening method using on-line HPLC-DPPH seems to be useful for the detection of antioxidants because of its high sensitivity and ease of handling. The method is advantageous for the determination of individual antioxidants in complex mixtures with simple operation. The method was applied forFig. 4. HPLC Chromatogram of Lemon Balm80 methanol extract.

On-line HPLC-DPPH Screening Method for Evaluation of Radical Scavenging
Activity of Phenols Extracted from Regional Agricultural Products

quantitative analysis of the antioxidants. Active phenolic components, caffeic acid, chlorogenic acid,dicaffeoylquinic, dicaffeoyltartaric acid and Rosmarinic acid were identified and characterized from above
agricultural products cultivated in Miyazaki Prefecture. Burdock root, sweet potato leaf and carrot leaf possesses significant free radical scavenging activity both DPPH and SOSA method, which was attributed tochlorogenic acid and other antioxidant active components.
The greatest benefit of the method is that, besides the quantification by UV detection, the radical scavenging activity of a single substance can be measured and its contribution to the overall activity of a mixtureof antioxidants can be calculated.
The identification of other antioxidant components being conducted in our laboratory.
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