Classification of Epigallocatechin and Catechin in Impedometric mode using PCA. Mopsy Dhiman a, Madan L.

Singla a, Pawan Kapur a, Abhijit

Ganguli b, a Central Scientific Instruments Organization(CSIO),CSIR, Sector-30C, Chandigarh 160030, India b Thapar University, Patiala 147004,Punjab,India c Panjab University, Chandigarh 160014,India

Corresponding author Tel.:+911722651787; fax: +911722657820.

E-mail addresses: mopsyd@gmail.com, (Mopsy Dhiman).

ABSTRACT

An Impedance study has been carried out at Epigallocatechin and Catechin levels present in black tea and its fingerprint mapping was done using Principal Component Analysis. Similar data has been generated from the known individual antioxidant compounds and the respective mixture. The antioxidant level has been also extracted from the complex structure of the other antioxidants present in black tea. It has been found that impedance data and their PCA have been able to clearly discriminate the presence of these two compounds. The reproducibility has been studied

continuously for about months time which lies within the + 2% of the output.

KEYWORDS

Antioxidants, Impedance, Principal Component Analysis.

PRACTICAL APPLICATIONS

In recent years, tea has become very attractive for consumers because of potentially beneficial antioxidants it contains. catechins are abundant in teas derived from the The

tea-plant

Camellia sinensis as well as in some cocoas and chocolates. Tea comprised nearly 90% of catechins. Several catechins are present in significant quantities; epicatechin (EC), epigallocatechin

(EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCg). Because the active compounds, the catechins, found in tea are known to possess potent antioxidant activity, they may provide beneficial health effects by protecting the body from the damaging effects of oxidative damage from free radicals. A number of chronic disease states have been associated with free radical induced oxidative damage, including cancer, heart

disease, and suppressed immune function and accelerated aging. Tea quality can be described by standard quality parameters and the analysis of antioxidant The capacity showed different influenced that by specific

related (EGC)

compounds. Catechin

results have

Epigallocatechin output in

and

(CAT)

impedance

selected frequency range than other compounds and PCA is able to differentiate.

INTRODUCTION

Tea is one of the worlds most popular beverages, second only to water, prepared by water infusion of dried leaves from Camellia sinensis. Consumption of teas may be associated with potential health benefits. The major groups of antioxidants in tea are flavonoids that appear to be digested, absorbed and metabolized by the body. Furthermore, as well as demonstrating antioxidant activity in vitro they also appear to have antioxidative

potential in vivo. Tea is a complex mixture of components presenting both volatile and non volatile compounds, responsible for aroma and flavor. Various kinds of polyphenols present in tea, are phenolic acids, flavonoids (catechin), tannins and other phenolics [Naczk and Shahidi, 2006]. Catechins, a group belongs to flavonoids, have been reported to be the most effective antioxidants in tea

[Hara,.Y,2001: Alves et al, 2005]. However, polyphenol content is varied with factors, like tea varieties, origins, plantation area, tea types, infusion conditions and degree of fermentation [Khan. And Mukhtar, 2007 ; Caffin et al , 2006].

Traditionally,

quality

of

beverage

was

measured

by

human

sense,which include vision, olfaction and gustation. In recent times ,chemical and physical analytical tools are being used for quality check and its control. Many researchers have worked in this field To identify the pharmacological and physiological

effects of various types of tea. [Amantana et al 2001], [Chen et al al, 2002], [Chuang et al, 2003], [Giri et al , 2001], [Hwang et 2008], [Juan et al , 2001] and [Baron et al, 2009]

Impedance techniques have been used as one of the important tool for quality assessment of various types of beverages. [Baner et al,

1999] Due to complex analysis several chemo metric technique are being applied (PCA), to data treatment like principal of component analogy

analysis

soft

independent

modeling

class

(SIMCA), hierarchical cluster analysis (HCA), canonical analysis (CA), discriminant analysis (DA), principal component regression (PCR) and partial least square analysis (PLS)[ Almeida et al , 2006]. In the present to study, Glassy Carbon (GC) electrode has been impedance of individual antioxidant

deployed

measure

the

compound dissolved in water and their mixture. PCA was applied for classification purpose of these antioxidants. It was

observed that EGC and CAT can be classified very clearly in the antioxidant mixture.

Materials and Methods

Electrodes and standards used

Glassy Carbon (GC) working electrode, Platinum (Pt) wire counter electrode and Ag/AgCl (4M KCl) reference electrode were supplied by CH Instruments (Austin, USA) whereas antioxidants used, viz. CAF, GA, ()-epigallocatechin gallate (EGCG), ()-epicatechin

gallate (ECG), ()-epigallocatechin (EGC), ()-epicatechin (EC) and (+)-catechin (C) were purchased from Sigma Chemicals Co. St. Louis, MO,USA. De-ionized (DI) water was used otherwise.

Experimental

An experimental set up of impedance Spectroscopy is shown in Fig. 1. All impedance GC measurements were carried out at room electrode was polished with alumina

temperature.

working

powder of particle size of 1.0, 0.3, and 0.05 m stepwise to get smooth surface. well by Each with reading DI water. was The taken data with after was the washing recorded the and

electrode displayed

the

software

provided

electrochemical

workstation which communicates with a personal computer using a RS232 link. Pt wire and Ag/AgCl (4MKCl) were used as counter and reference electrodes, respectively. All samples were of the 1mM

concentration were used in this study. Five observations per day were recorded for each sample continuously for 30 days. The

analysis was carried out after generating the mean data. [Bari et al, 2010]

Referen ce Electrode (AgCl)

Counter Electrode (Pt) Working Electrode

CH Instruments Electrochemical Workstation 660C

Pt/Ag/Au/Glass Carbon/PAN/PPY

Fig.1: Experimental Set up for AC impedance spectroscopy.

Data analysis and statistics

Statistical Analysis software, PAST was used for performing PCA. The PCA is a common pattern recognition algorithm used to

analyze data obtained from impedance system. [Alexandre et al, 2010] In particular, PCA is used to reduce the complexity of data by computing a new, much smaller set of uncorrelated variables

which best represent the original data. The application of PCA on the obtained impedance data transforms it into two or three coordinates, data set. which The yields an information-rich actually that representative projects maximize the the

transformation data into

multidimensional

coordinates

variance while minimizing the correlation in the data set.

RESULTS AND DISCUSSIONS

Impedance Response

The

impedance

study

was of

carried every

out

with was

the

open

circuit for 10

potential

(Eocp).

Eocp

sample

observed

minutes over a frequency range of 1Hz to 100 kHz. The electrical behavior of each antioxidant solution was measured repeatedly in the above frequency range at room temperature using AC technique of complex impedance spectroscopy.

Fig

shows

the

complex Gallic

impedance (GA), (ECG),

plots

(Nyquist

plots

of

Caffiene (EGCG),

(CAF),

Acid

Epigallocatechin Epigallocatechin

gallate (EGC),

Epicatechin

gallate

Epicatechin (EC) and Catechin (C) and electrode. Two compounds EGC and CAT

their mixture using GC showed only semicircle

formation while in other compounds straight line at an angle of 45 to the real axis has been observed. The CAT showed a semicircle both in high and low frequency

corresponding to electron transfer limiting process. There is a short linear part at low frequency which may be attributed to diffusion limiting step of electrochemical process. EGC Showed the first semicircle to low the occurring of in the high frequency region the

corresponds second in

behavior

bulk

EGC

compound to

while

frequency

region

corresponds

electrode

polarization or interfacial interactions. The exact cause for this behavior is not known, however it may be due to the

difference in chemical structures.

It has been found that there are short linear parts at low frequencies resulting (for from CAF, the GA, EGCG, ECG, EC and their step mixture) of the

diffusion

limiting

electrochemical process. The respective semicircles diameters at the high frequencies at the correspond to the The charge mixture transfer of these 8

resistance

electrode

surface.

compounds showed a straight line at an angle of 45, whereas the semicircular behavior has disappeared in this case which may be due to the domination of other compounds over, CAT and EGC. When the impedance is plotted in the complex plane, we obtained a semicircle combined with a straight line at an angle of 45 to the real axis, which may be due to Warburg impedance. The

physical model explaining this phenomenon is Randles equivalent electrical circuit [Fig. 3] and the behavior can be attributed to diffusion phenomenon occurring at the solutionelectrode

interface. The impedance data were simulated using the Randles equivalent circuit consisting of a parallel combination of the capacitance reactions resistance (C) (Rct) (Rs). and in The charge series transfer with of the resistance supporting to the by redox

electrolyte equivalent

fitting

spectras

circuit has indicated a good agreement between the circuit model and the real experimental data, especially at the high frequency values.

Fig. 2: Impedance plot for seven antioxidant compounds present in black tea from the known compounds along with their mixture.

10

Figure

3.

The

scheme

of

equivalent

circuit

simulating

the

impedance

spectra.

Rs-solution

(supporting

electrolyte)

resistance, Rct -charge transfer resistance, C -capacitance.

Data Analysis using PCA

Figure 4 shows the projections of normalized PCA results on the analysis of EGC, CAF, GA, EGCG and EC, CAT, ECG and their

mixture. The PCA shows the corresponding scores plot obtained after normalizing the impedance values. The data were plotted

in a two dimensional plane formed by the first two PCs, that is PC1 and PC2, which captured 99.96% of the data variance. They appear to provide the best discrimination of the samples. The scatter plot of PC1 (variability; 98.94%) vs. PC2 (variability; 1.02%) is shown in Fig. 4. The plot gives information about the patterns of species of antioxidant compounds present in black 11

tea and shows totally different patterns from the one obtained by CAT and EGC.

A single PCA model including the four training sets has been constructed. The score plot showed there is an overlapping of the different antioxidant compounds even when only two PCs were used. Besides, a good discrimination between the classes was observed and the different types of antioxidants were located in different quadrants on the score plot. Analyzing Fig. 4, EGC lies in 1st quadrant and lies at positive quadrant of PC1 and negative quadrant of PC2. It is evident that the CAF, GA, EGCG and EC lie in the same quadrant, i.e. in 2nd quadrant. This indicates that these compounds are positively correlated to one another. This can be attributed to structural similarity in the compounds. ECG and mixture of antioxidant compounds lie together in 3rd quadrant, of the which can be attributed to structural

similarity

compounds, with PC1 and PC2 both negative

while CAT lies in 4rth quadrant. Samples were grouped together into different clusters; EGC and CAT are clearly separated means that EGC and CAT share

relatively similar patterns. This means that CAF, GA, EGCG and EC share relatively comparable structures. Using PCA model five of antioxidant compounds cluster together (CAF, GA, ECG, EGCG, EC and mixture of antioxidant compounds) suggesting that they are similar and the other antioxidants (EGC and CAT) are 12

separated from the main cluster. PCA as shown has successfully been able to distinguish samples into different regions based on its unique characteristics as classified by PC1 and PC2 axis. PCA has become indispensible to multivariate analysis and was used on the impedance data obtained in black on tea scanning from the seven known

antioxidant

compounds

present

compounds along with their mixture. While in both the PCA and impedance plots the clusters corresponding to CAT and EGC are distinctly visible. Results obtained using PCA and AC Impedance study agree with each other.

Fig 4. PCA plot of Impedance readings of antioxidant compounds and their mixture. 13

Conclusion

An

Impedance and

study

has

been

carried

out

at in

Epigallocatechin known individual The out

(EGC)

Catechin

(CAT)

levels the

present

antioxidant

compounds impedance

and

respective (EIS)

mixture. was carried

electrochemical

spectroscopy

using CHI 660C with the open circuit potential (Eocp). This work proposes study of antioxidant compounds using impedance mode and PCA. The study of this rapid tea monitoring method will lead to

better quality control of tea tasting.

Acknowledgement The authors acknowledge number: CSIR for full financial Central for to support

(research

project

SIP-0022-06).The is are acknowledged also thankful

Scientific laboratory Kumar,

Instruments facilities.

Organization The authors

Manish

Abhishek kumar, Sanjeev Kumar and Rajiv Puri for their valuable inputs and suggestions.

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