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# Open Chemistry

### formerly Central European Journal of Chemistry

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Volume 16, Issue 1

# Modeling of Total Phenolic contents in Various Tea samples by Experimental Design Methods

Nuraniye Eruygur
/ Nazire Gulsah Kutuk Dincel
/ Nursah Kutuk
• Department of Chemical Engineering, Faculty of Engineering, Cumhuriyet University, Sivas, Turkey
• Other articles by this author:
Published Online: 2018-08-20 | DOI: https://doi.org/10.1515/chem-2018-0082

## Abstract

Tea, from the old ages to the day, is widely consumed both for enjoyment and health care due to its positive effects. The consumption of these products is increasing day by day as a result of the clear presentation of the fact that tea contains high amount of antioxidant substances (such as phenolic compounds), which is important in prevention and treatment of diseases. Tea beverage is a very important source of polyphenols. In this study, phenolic content and antioxidant capacity of different tea species were calculated by modeling with experimental design method. In the experimental part, polyphenol content was determined using the Folin-Ciocalteu method. The total amount of phenolic substance content was examined by Box-Behnken design and response surface method on black tea, green tea and white tea on different extraction temperature, extraction time and solid / liquid ratio. Solid / liquid ratio was found to be the most important parameter in terms of polyphenol content extraction from different tea samples. The highest polyphenol amount (411.762 mg gallic acid / mL) was found in green tea. To the best of our knowledge, this is the first data presenting comparatively study the effect of extraction condition on amounts of phenolic compounds from different tea samples.

## 1 Introduction

Tea, the top leaves of the plant Camelia sinensis, is most widely consumed and useful drink in the world next to water [1]. Tea are containing polyphenolics, flavonoids and catechins in large amounts [2]. Besides, saponins, caffeine, and tannins are also present in tea [3]. Green and black tea is the most known tea in the world, while white tea is a rare tea type [1]. Depending on the processing treatment of the tea, the chemical compositions changes greatly, thus affecting the potential effect on health [4]. The major compounds presented in green tea are catechins and its derivatives while black tea have theaflavins, thearubigins, except for catechins [5]. The biological activities of tea catechins are related with the affinity for cell membranes and the hydrogen peroxide formation during oxidations [6]. The phenolic compounds present in tea are well known for their potential antioxidant activity, which are in turn, closely associated with a variety of chronic disease such as arteriosclerosis, diabetes mellitus, cancer and liver injury [7,8,9]. Epidemiologic studies and in vivo research findings on animals have shown that the chemoprotective potential of tea polyphenols in cancer [10]. Tea and tea polyphenols have shown inhibitory activity during every stage of carcinogenesis such as initiation, promotion and progression [5]. The possible anticarcinogenic effects of tea attributed to its polyphenolic compounds demonstrate the activity by several mechanisms such as enhancement of antioxidant enzyme activities, inhibition of lipid peroxidation induced by chemical factors, cellular proliferation, and cyclooxygenase activities as well as anti-inflammatory and detoxification activities [11,12,13]. Owing to this, increasing detailed literature in terms of the effect of tea polyphenols has been reported in recent years.

The World Health Organization (WHO) reports that most of the populations of developing countries still uses natural medicine from medicinal plants for maintaining good health [14]. In this regard, secondary metabolites presented in the herbal products are important due to their biological functions in the human body. Therefore, antioxidants (polyphenol, flavonoid) become important.

Extraction is the important step in preparing herbal products from plants. Extraction of natural products by different methods may yield various chemical components. There are many factors may affect the extraction efficiency, phenolic compounds and antioxidant activity. Therefore, it is need to optimize the extraction conditions for obtaining highest phenolic content and antioxidant activity [15,16,17]. In this study, the amount of polyphenols contained in black, green and white tea was examined. Many experiments are necessary to investigate optimum experimental conditions with conventional methods. It is also not possible to examine the influence of these variables on the experimental varieties. In order to solve these problems, the amount of polyphenols in tea was optimized according to time, temperature and solid / liquid ratio by using Response Surface Method (RSM) and Box-Behnken experiment design. The mathematical models obtained are evaluated by ANOVA (Analysis of variance) statistical method and the validity of the models is discussed.

## 2.1 Preparation of the extract

Tea samples obtained from herbal market were extracted with distilled water according to the design conditions of maceration method. Dry white, black and green tea leaves and leaf buds were used for extraction. All extracts were filtered through whatman filter paper No: 1.

## 2.2 Determination of total phenol content

Then the amount of polyphenol in extracts was determined according to Folin-Ciocalteu colorimetric method described by Ainsworth et al. [18]. In the method, the sample solutions were prepared using extract, sodium carbonate and Folin reactant. 200 μL of 10% Folin-Cioceltau reagent was added to the 100 μL of extracts and vortexed thoroughly. Then 800 μL of 700 mM Na2CO3 was added. The mixture was allowed to incubate at room temperature for 2 hs in the dark. Transfer 200 μL of sample from the assay to a 96-well microplate and read the absorbance at 765 nm with an Elisa instrument. The total phenolic contents were expressed as mg of gallic acid equivalents (GAE) per mL of tea water extracts. Experimental results were calculated by using the equation of gallic acid-absorbance graph obtained from absorbance measurements of gallic acid prepared at different concentrations at 765 nm.

## 2.3 Mathematical Modeling of Experimental System

In order to achieve the total phenolic content in the design, the effect of extraction conditions was investigated. The Box-Behnken experimental design was applied to investigate the effects of extraction time, solid/liquid ratio and temperature for the optimization of the total phenolic content of black, green and white tea. Table 2 lists the codes of these independent factors and the highest and lowest values (time: 5-30 minutes, solid / liquid ratio: 0.01-0.1 g / mL and temperature: 25-100°C). Design expert 10 computer program was used.

Ethical approval: The conducted research is not related to either human or animals use.

## 3 Results

Table 1 shows the actual experimental parameters equivalent to the designed levels, which were performed for generating the second order polynomial model.

Table 1

Box-Behnken design matrix containing experimental and model values of total polyphenol content (mg gallic acid/mL) of black, green and white tea.

Table 2

Level and code of independent factors.

## 3.1 Determination of Test Conditions for the Analysis of Polyphenol Amount in Black Tea

The graphical representations of the regression of Equation 1 are called response surfaces. Three dimensional response surfaces were obtained using design expert and are shown in Figure 1-a, b, c. Equation 1 represents the polyphenol content of black tea as a function of time, temperature and solid / liquid ratio. The statistical significance of Equation 1 was checked by the F test. Variance analysis (ANOVA) for the model response surface is given in Table 3.

Figure 1

3-D graph showing the total polyphenol content in the black tea extract a) solid / liquid ratio and variation with time, b) temperature and variation with solid/liquid ratio, c) temperature and variation with time.

Table 3

Analysis of varience (ANOVA) for fitted quadratic polynomial model of polyphenol amount in black tea.

$Y1=349.14+8.83x1+93.65x2+33.02x3−0.89x1.x2−23.12x1x3−15.14x2x3−10.78x12−49.75x22−34.63x32$(1)

From the model F value and the low probability value, the model seems to be significant. The R2 coefficient indicates the degree of adaptation of the model. The fact that the R2, R2Adj and R2Pred values of the second order model for the total polyphenol content of the black tea are close to 1 indicates that the values predicted by the model are in perfect agreement with the experimental values. The adequate precision ratio is found as 50.497 bigger than 4 as expected. The lack of fit in the model is also not significant.

The polyphenol content of the black tea extract is significantly affected by the change in solids / liquid ratio when the 3D surface plots of response surface (Y1) are examined, and the highest polyphenol values were found when the solid / liquid ratio was 0.071. As can be seen from the graph of Figure 1, the amount of polyphenol increased to 387.714 mg gallic acid / mL.

## 3.2 Determination of Test Conditions for the Analysis of Total Polyphenol Amount in Green Tea

The graphical representations of the regression of Equation 2 are called response surfaces. Three dimensional response surfaces were obtained using design expert and are shown in Figure 2-a, b, c. Equation 2 represents the polyphenol content of black tea as a function of time, temperature and solid / liquid ratio. The statistical significance of Equation 2 was checked by the F test. Variance analysis (ANOVA) for the model response surface is given in Table 4.

Figure 2

3-D graph showing the total polyphenol content in the green tea extracts a) solid / liquid ratio and variation with time, b) temperature and variation with time, c) temperature and variation with solid/liquid ratio.

Table 4

Analysis of varience (ANOVA) for fitted quadratic polynomial model of polyphenol amount in green tea.

$Y2=367.62+9.88x1+73.60x2+57.39x3+13.15x1x2−20.37x1x3−53.25x2x3+13.38x12−58.23x22−18.58x32$(2)

It is seen that the model is important when we look at the value of P›F. In this case R2 with experimental value of 0.9718 shows the conformity of the values predicted by the model. When viewed as a percentage, the 2.82% of R2 value shows that the total change cannot be expressed in the model. It is also seen that Adj-R2 (0.9356) and Pred-R2 (0.6558) are in agreement.

Response surface plots of change in polyphenol content of green tea with temperature, time and solid / liquid ratio are given in Figure 2. The highest value of polyphenol content is 411.762 mg gallic acid / mL. Solid / liquid ratio is again seen as the most important parameter.

## 3.3 Determination of Experimental Conditions for the Analysis of Total Polyphenol Amount of White Tea

The graphical representations of the regression of Equation 3 are called response surfaces. Three dimensional response surfaces were obtained using design expert and are shown in Figure 3-a, b, c. ANOVA analysis of black and green tea was also made for white tea. The statistical significance of Equation 3 was checked by the F test. Variance analysis (ANOVA) for the model response surface is given in Table 5.

Figure 3

3-D graph showing the total polyphenol content in the white tea extracts a) temperature and variation with time, b) solid/liquid ratio and variation with time, c) temperature and variation with solid/liquid ratio.

Table 5

Analysis of varience (ANOVA) for fitted quadratic polynomial model of polyphenol amount in white tea.

$Y3=416.40+6.55x1+42.17x2+11.82x3−8.21x1x2−6.37x1x3−3.39x2x3−0.65x12−24.46x22−6.55x32$(3)

Table 5 shows that the model is important. The lack of fit in the model is also not significant. It is good if R2, R2Adj and R2Pred values are close to 1.

Figure 3 shows that the ratio of solid / liquid ratio is the most effective parameter in 3D graphics. The highest value of polyphenol content is 325.452 mg gallic acid / mL in white tea.

## 4 Discussion

Regarding the chemical components, bioactivity of catechins as well as other phytochemical constituents, the water extraction was the ideal way to extract most of the phenolic compounds. However, the extraction method, the amount of liquid used, the duration of the retention, the temperature, etc. are influencing the chemical composition of the obtained extracts to a great extent. The researchers reported that hot water infusions of white teas and green teas not contain acylated flavonol glycosides, resulted in lower total phenol content levels and antioxidant activity [19].

Polyphenols, classified as non-nutrients, are biologically active compounds of plant origin. Many epidemiological studies have shown that food and beverages rich in polyphenolic compounds have been shown to affect people’s health and reduce the rate of cardiovascular disease and improve their life span [20]. Determination of total phenols is based on the quantification of the total concentration of hydroxyl groups present in the extract. The Folin-Ciocalteu is a yellow heteropolyacid solution containing a complex ion polymer is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric in vitro assay of phenolic and polyphenolic antioxidants. The total phenolic contents in the examined tea extracts using the Folin-Ciocalteu’s reagent is expressed in terms of gallic acid equivalent. The results suggested that, the solid/ liquid ratios is the most effective parameters in extraction of polyphenolic compounds regardless of tea type, highest amount of polyphenol content was found in 411.762 mg gallic acid / mL in green tea, followed by 387.714 mg gallic acid / mL in black tea, and lowest in 325.452 mg gallic acid / mL in white tea.

## 5 Conclusion

The response surface methodology was successfully employed for optimize the phenolic content extraction from three different tea leaves. Using Design Expert 10, it fully matched quadratic equality for the total amount of polyphenolic material in black, green and white tea. When the model is examined, the highest polyphenol values were reached with 411,762 mg gallic acid/mL for green tea. Solid / liquid ratio was found to be the most important parameter in terms of polyphenol content in all three models. As a result, total phenolic substance content and effective factors in different teas were optimized using the response surface method. These results indicated that the data will provide useful information for preparing polyphenol rich extracts or developing potential antioxidant nutraceuticals from different tea samples, for further application in food and pharmaceutical industries as natural valuable products.

## Acknowledgement

Experimental studies were carried out in the advanced technology research laboratory of Cumhuriyet University.

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Accepted: 2018-03-19

Published Online: 2018-08-20

Conflict of interest: Authors state no conflict of interest.

Citation Information: Open Chemistry, Volume 16, Issue 1, Pages 738–744, ISSN (Online) 2391-5420,

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