Ultrasound-assisted extraction of some branded tea: Optimization based on polyphenol content, antioxidant potential and thermodynamic study.

Tea is one of the top beverages used around the world every day, which contains a high amount of polyphenols and antioxidants. The main aim of this research is to quantify some marketed black tea (Rabea, Lipton, Alkbous, Green gold and Haritham) for phenolic contents and antioxidant potential evaluation by ultrasound solvent extraction and was compared with conventional extraction. Ultrasonic extraction was optimized by considering frequencies (26 kHz, 40 kHz), temperature (30, 40 and 50 °C), and power (30, 40 and 50%) at a fixed time of 30 min. In both the ultrasonic frequencies, 40 °C temperature and 40% power combination exhibited highest cumulative yield (mg/100 g DW), total phenolic content (mg gallic acid/g DW), flavonoids (mg/g DW) and DPPH radical scavenging activity (%) in all branded tea. Within each brand of tea, at any temperature-power combination at particular frequency results were not significantly different. But, at a similar condition of temperature power results were found significantly different between two frequencies. Furthermore, ultrasonic extraction process was analyzed thermodynamically by selecting some basic parameters. Thermodynamics results showed the extraction process was feasible, spontaneous and irreversible. Also, 26 kHz ultrasonic probe is more appropriate for the extraction purpose and thermodynamically more acceptable as compared to 40 kHz ultrasonic bath. Moreover, Haritham was selected as the best tea brand due to its high polyphenol contents and antioxidant potential.

Introduction

Tea (Camellia sinensis, Theaceae) is the only beverage in the world which is consumed by all age group and it is just after water as a consumable substance. There is an increasing importance in the field of nutrition and food industry of plant-derived products (Food and agriculture organization of the united nation). There is a dramatic growth rate from 6% to 15% higher predicted of plant-based food and pharmaceutical (Martin, 2012). Treatment of plant materials for the extraction of valuable components are the top priority steps in any food/ beverage processing. Preclinical and epidemiological studies suggest the value of tea and its health benefits (Banerjee and Chatterjee, 2015). Tea extract and tea biomolecules have to gain demand in pharmaceutical and food industries as a natural antioxidant and for other purposes. Depending on the processing technique tea is classified as Black tea, Green tea, White tea, Yellow tea, Dark tea, etc. Each variety of tea is known for the amount of bioactive/antioxidant components present in it (Banerjee and Chatterjee, 2015). Tea biomolecules preferentially contained non-protein amino acid (theanine), free sugars (Unachukwu et al., 2010), methylxanthine, alkaloid like caffeine, theobromine, theophylline, phenolic acids like gallic acid and different catechins (Unachukwu et al., 2010, Fukino et al., 2005, Seeley et al., 2005). Since major bioactive of tea are polyphenols and methylxanthines and different extraction procedure and solvent largely affect its concentration in extracts. Various researchers used different extraction techniques and solvents for the evaluation of potential bioactive (Jun et al., 2009, Lin et al., 2008) such as supercritical carbon dioxide (Chang et al., 2000), Microwave extraction (Xuejun et al., 2003, Nikhil et al., 2009), high hydrostatic pressure (Jun et al., 2009, Jun et al., 2010) and ultrasound (Mason and Zhao, 1994, Tao et al., 2006, Simon et al., 2014).

Ultrasound technology was explored to enhance the food quality by reducing the process time, energy and increasing shelf life (Altemimi et al., 2015). It has been reported that ultrasound energy can be used to increase extraction yield by disrupting cell tissues (Miracea, 2001). There are different ways to utilize ultrasonic frequency for the extraction of value-added substances. Water bath shaker, Ultrasonic bath & probe were used as extraction techniques at different temperatures and adopting different solvents in order to optimize the methods and solvents (Banerjee and Chatterjee, 2015, Altemimi et al., 2015). A mechanical and thermal effect of ultrasound is used for the extraction of plant materials (Tao et al., 2006, Simon et al., 2014). Thermal effects were observed for the extraction, as a result of sonic waves converted to heat which was absorbed by plant tissues, while mechanical effects generate acoustic cavitation which leads to cell disruption to enhance extraction (Miracea, 2001, Horžić et al., 2012). Extensive studies have been carried out in the present research using the ultrasonic treatment on different branded tea in order to improve the yield of polyphenol contents as compared to conventional method. The main aim of present study is to compare important components such as yield of total phenol, total flavonoids and antioxidant activity, extracted from the ultrasonic probe (26 kHz), ultrasonic bath (40 kHz) by the treatments of various temperature-power combinations for five branded tea. Additionally, the data obtained as a result of ultrasonic treatment were used to establish the thermodynamics of the extraction process.

Materials and methods

Chemicals and reagents

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical, Folin-Ciocalteu reagent, butylated hydroxytoluene (BHT) and gallic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rest of the chemicals and reagents (analytical grade) used were also purchased from Sigma Aldrich unless stated otherwise.

Sample preparation

Five different brands (sample) of black tea (Camellia sinensis) were purchased from local market of Al-Kharj (South Riyadh), Saudi Arabia.

Conventional extraction

In conventional extraction, the tea samples were extracted using 80% aqueous methanol. Tea samples (10 g) were mixed with 200 mL of aqueous methanolic solvent and stirred with boiling at 80 °C up to 30 min. The extracts obtained were freed of excess solvents under reduced pressure. All the extractions were performed in triplicate. The extracts produced were evaluated for yield, total phenolic contents, total flavonoids and DPPH scavenging activity calorimetrically.

Ultrasonic extraction (probe)

Tea samples (10 g) for ultrasonic (26 kHz) extraction were placed in 500 mL beaker with same the amount of solvent (80% aqueous methanol) as discussed in conventional method using similar time of frame (30 min) with power ultrasound, low frequency, and high intensity, 26 kHz probe (Ultrasonic processor-200Ht, power 200 W, made in Germany). The probe used for extraction has vibrating horn diameter of 14 mm which is directly immersed in the solvent containing sample which is irradiated with ultrasonic waves generated from the tip of the probe. Amplitude applied for this experiment was 30%, 40% and 50% with a corresponding temperature 30 °C, 40 °C and 50 °C. Each experiment was repeated three times.

Ultrasonic extraction (Bath)

Digital ultrasonic bath of Branson (Model, 5800) was used for the extraction purpose of the present study with the frequency of 40 kHz. Ultrasonic bath dimension was length16, width 15.5 and depth 14.5 in. respectively. During the extraction, the processed amplitude was set at 30%, 40% and 50% with the corresponding temperature was 30 °C, 40 °C, and 50 °C. Before the start of the experiment, the bath was filled with 3 L of distilled water. Rest of the procedure were followed same as ultrasonic Probe.

Estimation of polyphenol and antioxidant potential

Effect of ultrasonic extraction and conventional extraction performance was estimated in terms of extract yield, total phenols, total flavonoids and antioxidant potential (DPPH assay).

Estimation of extract yield

Extract yield was calculated as a reported method (Altemimi et al., 2015) as follows

Estimation of total phenol

Total phenolics in branded tea extracts was evaluated by a reported method (Hussain et al., 2008) spectrophotometrically using Folin-Ciocalteu's reagent. Briefly, In a test tube, 1 g of the tea extract, 10 mL of methanol, 7.5 mL distilled water, 0.5 mL Folin-Ciocalteu's reagent and 1.5 mL sodium carbonate (20%) were taken. This mixture incubated for 30 min at room temperature and then reading was measured at 755 nm on spectrophotometer against blank. Results are expressed as mg/liter of gallic acid equivalents (GAE).

Estimation of total flavonoids

The total amount of flavanoid was estimated calorimetrically, with the support of previously published work (Altemimi et al., 2015). Nearly, 0.3 mL of extracts was taken in a test tube with the further addition of 6 mL of cinnamaldehyde with manual shaking for 30 min at room temp. Absorbance was determined at 640 nm. The standard calibration curve was established using a known amount of catechin. Total flavonoid was calculated with reference to the standard calibration curve of catechin, expressed as mg CE/g of dry matter.

Estimation of DPPH radical scavenging

The antioxidant activity of all the branded tea samples was also assessed by neutralizing purple colored 2,2′-diphenyl-1-picrylhydrazyl free radicals (DPPH°). This assay was performed by the method as described earlier (Hussain et al., 2008). Approximately, 10 µg/mL methanolic extract solution of branded tea was allowed to mix with 1 mL of freshly prepared DPPH solution (90 μM). This mixture was made up to 4 mL with 95% MeOH. After vigorous shaking, the reaction mixture was kept under ambient conditions in the darkness for 30 min for any reaction to accomplish. The absorbance of the incubated final mixture and the blank (control) was noted at 515 nm using UV/VIS spectrophotometer (V-630, JASCO International Co. Ltd., Hachioji, Tokyo, Japan). A well-known equation for calculating percent inhibition of oxidation is as follows:

Thermodynamic analysis

In present research ultrasonically extraction of branded tea samples was thermodynamically analyzed to determine some basic thermodynamic quantities such as Gibbs free energy (ΔG°), Enthalpy change (ΔH°), Entropy change (ΔS°), activation energy (Ea) to investigate spontaneity of extraction, heat of reaction, reversibility and excitation energy for the extraction process respectively. Following equations are used to generate these important parameters-where ke is the reaction rate constant for the extraction; A is the Arrhenius constant; Ea is the activation energy; R is the universal gas constant and T is absolute temperature.

Slope resulted from the plot of lnk vs 1/T gives the values of activation energy (Ea).

Determination of phenolic contents in both ultrasonic frequencies (26 kHz and 40 kHz) assisted extraction and Gibbs free energy (ΔG°), Enthalpy change (ΔH°), Entropy change (ΔS°) was calculated using following equations:where Ct is the concentration of total phenolic contents in the methanolic extracts at a given temperature and Cmax is the concentration in the saturated form of extract.

The values of Gibbs free energy change (ΔG°) (kJ/mol) gives an idea of the spontaneity of the extraction process. A negative and positive value of ΔG° is an indicator of spontaneous and non-spontaneous extraction process (Meziane and Kadi, 2008). Similarly, enthalpy change (ΔH°) (kJ/mol) postulates whether the reaction is exothermic or endothermic having value negative and positive respectively. Disorder or randomness of an extraction process was calculated from values of ΔS°. A reaction of values of ΔS° higher than zero is indicative of irreversible reaction (Meziane and Kadi, 2008).

Statistical analysis

Variation in the amount of polyphenol content and antioxidant activity mainly depends on frequency, temperature and power as independent variables, which were analyzed by repeated measures ANOVA within the samples of tea to test overall differences between means after repeated interaction of temperature and power. When the effect of the main comparison was found significant, simple comparison effects were analyzed for differences in both frequencies by Tukey-Kramer multiple comparison tests, when p < 0.05. Graph pad Instat version 3 was used for all statistical analysis.

Results and discussion

In the present research, we have selected five brands of black tea and extraction was performed conventionally (control) and using an ultrasonic bath (40 kHz) and probe (26 kHz) in order to calculate and compare the extract yield and antioxidant potential of samples. Further, the yield of samples and major antioxidant components such as total phenolics, flavonoids contents with DPPH radical scavenging potential were evaluated and accessed for both ultrasonic treatments (bath, probe). To optimize the two ultrasonic techniques different temperature-power combination were used and results were analyzed to identify the superiority of two ultrasonic techniques in terms of percent yield and antioxidant components results. Additionally, some thermodynamic parameters were also calculated to judge the merits of both ultrasonic techniques.

Impact of extraction parameters on the yield of branded tea samples using ultrasound technology

Extraction yield largely depended on the different variables such as frequency, temperature, and power with an exposure time of 30 min are statistically significant (P < 0.0001).

Effect of each temperature power pairing showed significant results among the selected treatment for 26 kHz and 40 kHz frequencies. In both of the chosen frequencies, temperature 40 °C and power 40% resulted in higher extraction yield as compared to the rest of the temperature-power groupings (Table 5). The yield of polyphenols (mg/100 g DW) at this temperature power pairing was found to be 18.61 ± 0.27, 15.65 ± 0.10, 22.43 ± 0.12, 19.36 ± 0.13, 32.68 ± 0.14 for Rabea, Lipton, Alkbous, Green gold and Haritham brands of tea respectively at 26 kHz (Table 1, Table 5).

Table 5

Effect of ultrasonic treatment at each temperature power combination to extract yield and antioxidant potential at frequency 26 kHz.

Sample (Brand)	Temperature –power	Extract yield* (mg/100 g DW)	Total Phenolics* GAE mg gallic/g DW	Flavanoids* (mg/g DW)	DPPH radical scavenging* (%)
Rabea	30 °C-30%	9.65ad	18.61acd	15.71acd	37.95ad
	30 °C-40%	10.78abd	19.57ad	17.15abd	38.19ad
	30 °C-50%	11.68 ad	23.29cd	21.81ad	39.22abd
	40 °C-30%	15.58 abd	26.59acd	24.56abcd	43.87abcd
	40 °C-40%	18.61ad	33.76ac	27.77acd	51.22ab
	40 °C-50%	16.88acd	27.17acd	21.43abd	48.71abc
	50 °C-30%	15.98abcd	24.88acd	18.96ad	46.48abcd
	50 °C-40%	14.89abcd	25.77acd	19.89ad	42.66ad
	50 °C-50%	13.71abcd	22.61acd	16.12abcd	41.18abd
Lipton	30 °C-30%	9.56ad	20.92acd	14.78acd	36.13ad
	30 °C-40%	10.19abd	21.89ad	15.71abd	38.82ad
	30 °C-50%	11.62ad	25.29cd	18.98ad	40.80abd
	40 °C-30%	13.42abd	28.82acd	21.18abcd	44.52abcd
	40 °C-40%	15.65ad	38.13ac	24.00acd	53.78ab
	40 °C-50%	14.42acd	27.97acd	22.92abd	49.82abc
	50 °C-30%	12.37abcd	24.12acd	18.75acd	44.76abcd
	50 °C-40%	12.86acd	25.91acd	19.08ad	42.85ad
	50 °C-50%	11.66abd	21.78acd	16.45abcd	40.98abd
Alkbous	30 °C-30%	16.31ad	14.98acd	8.24acd	34.45ad
	30 °C-40%	17.33abd	15.66ad	9.09abd	37.28ad
	30 °C-50%	18.17ad	17.49cd	11.39ad	40.16abd
	40 °C-30%	19.22abd	20.92acd	14.69abcd	42.56abcd
	40 °C-40%	22.43ad	24.71ac	17.50acd	56.38ab
	40 °C-50%	21.76acd	19.87acd	13.90abd	49.89abc
	50 °C-30%	17.88abcd	16.14acd	9.88acd	46.08abcd
	50 °C-40%	18.21ad	15.68acd	10.13ad	42.81ad
	50 °C-50%	13.51abcd	13.42acd	8.98abcd	40.61abd
	30 °C-30%	11.98ad	23.87acd	16.05acd	46.17ad
Green Gold	30 °C-40%	12.20abd	24.43ad	17.33abd	47.49ad
	30 °C-50%	13.24ad	27.40cd	19.81ad	44.98abd
	40 °C-30%	15.57abd	29.77acd	22.39abcd	55.39abcd
	40 °C-40%	19.36ad	32.73ac	25.11acd	65.30ab
	40 °C-50%	18.33acd	26.90acd	21.29abd	57.86abc
	50 °C-30%	15.35abcd	24.45acd	18.61acd	53.41abcd
	50 °C-40%	16.88acd	26.20acd	17.93ad	51.85ad
	50 °C-50%	14.29abcd	22.65acd	15.89abcd	48.94abd
Haritham	30 °C-30%	22.11ad	48.76acd	39.59acd	56.96ad
	30 °C-40%	23.34ab	51.61ad	41.84abd	59.22ad
	30 °C-50%	25.42ad	55.92cd	42.18ad	61.56abd
	40 °C-30%	27.49abd	59.97acd	46.08abcd	64.82abcd
	40 °C-40%	32.68ad	71.13ac	58.25acd	71.29ab
	40 °C-50%	29.72acd	63.12acd	55.69abd	65.91abc
	50 °C-30%	26.44abcd	54.58acd	51.38acd	60.11abcd
	50 °C-40%	27.78acd	56.88acd	48.78ad	58.79ad
	50 °C-50%	24.45abcd	49.30acd	47.23abcd	56.88abd

DW = Dry weight.

Means with different superscript letters (with the same column) indicate significantly different.

Table 1

Extract yield (mg/100 g DW) from different brands of tea using conventional and ultrasonic treatment.

Sample (Brand)		Extract Yield (mg/100 g DW)
	Conventional	Ultrasonic (26 kHz)	Ultrasonic (40 kHz)
Rabea	12.35 ± 0.15	18.61 ± 0.27	15.71 ± 0.10
Lipton	7.46 ± 0.19	15.65 ± 0.10	8.98 ± 0.08
Alkbous	12.83 ± 0.14	22.43 ± 0.12	14.79 ± 0.04
Green Gold	11.71 ± 0.11	19.36 ± 0.13	11.95 ± 0.07
Haritham	17.68 ± 0.14	32.68 ± 0.14	21.93 ± 0.04

Using 40 kHz ultrasonic frequency, it was 15.71 ± 0.10, 8.98 ± 0.08, 14.79 ± 0.04, 11.95 ± 0.07, 21.93 ± 0.04 (Table 1). Combination of the temperature of 30 °C and power at 30% were found lowest extract yield in both ultrasonic frequencies. The frequency, temperature, and power of ultrasound influence the efficiency of extraction. In these results, lower operating frequency of 26 kHz was more effective than 40 kHz with regard to producing extract yield. This was supported by the almost similar outcomes of few reported work (Altemimi et al., 2015, Zhou, 2010).

At 40% power combination with temperature in both frequencies, the results in terms of extract yield were in similar fashion (Table 5, Table 6, and Table 7). Considering 40% powers setting in both the frequencies the results were not significantly differenced at any temperature (p < 0.001). It was also evident that the yield was increased almost 1.5 to 2-fold from 30 °C to 40 °C in all temperature-power coupling but fall at 5 °C.

Table 6

Effect of ultrasonic treatment at each temperature power combination to extract yield and antioxidant potential at frequency 40 kHz.

Sample (Brand)	Temperature power combination	Extract yield* (mg/100 g DW)	Total Phenolics* GAE mg gallic/g DW	Flavanoids* (mg/g DW)	DPPH radical scavenging* (%)
Rabea	30 °C-30%	6.58abd	11.66abd	5.91acd	21.78ad
	30 °C-40%	7.78acd	12.12ad	6.37ad	22.16abd
	30 °C-50%	9.93acd	14.29abd	8.84acd	24.79abd
	40 °C-30%	11.12bcd	16.98abd	10.06abcd	29.44abcd
	40 °C-40%	15.71abd	21.29ab	14.85ad	36.74abd
	40 °C-50%	12.17acd	19.17abd	12.13abd	32.93acd
	50 °C-30%	11.44bcd	17.70ad	9.84abd	29.15abd
	50 °C-40%	14.64bd	15.51abd	8.11ad	27.07acd
	50 °C-50%	13.56ad	13.54ad	6.98abd	24.88abcd
Lipton	30 °C-30%	3.66abd	16.54abd	9.73acd	21.06ad
	30 °C-40%	4.10acd	17.78ad	10.33ad	22.58abd
	30 °C-50%	5.89acd	19.90abd	12.42acd	24.09abd
	40 °C-30%	6.75bcd	21.91ad	14.08abcd	27.11abcd
	40 °C-40%	8.98abd	24.71ab	17.82ad	39.12abd
	40 °C-50%	7.59acd	21.72abd	16.09abd	35.76acd
	50 °C-30%	6.77bcd	19.66abd	15.02abd	32.19abd
	50 °C-40%	7.91bd	20.14abd	14.21ad	30.17abcd
	50 °C-50%	6.28ad	17.95ad	12.24abd	29.49abcd
Alkbous	30 °C-30%	7.73abd	6.82abd	3.44acd	24.95ad
	30 °C-40%	8.64acd	7.88ad	4.95ad	25.11abd
	30 °C-50%	9.80acd	9.39abd	5.59acd	27.66abd
	40 °C-30%	11.91bcd	11.71abd	8.22abcd	29.49abcd
	40 °C-40%	14.79abd	14.34a	11.19ad	36.38abd
	40 °C-50%	13.51acd	12.17abd	10.39abd	34.89acd
	50 °C-30%	9.86bcd	10.10abd	9.15abd	32.60abd
	50 °C-40%	7.27bd	9.99abd	7.04ad	30.20abcd
	50 °C-50%	5.57ad	9.12ad	6.08abd	35.75abcd
Green Gold	30 °C-30%	4.92abd	12.49abd	7.77acd	34.50ad
	30 °C-40%	4.89acd	13.68ad	8.80ad	35.13abd
	30 °C-50%	5.96acd	14.54abd	9.13acd	37.19abd
	40 °C-30%	7.97bcd	16.89abd	12.83abcd	39.04abcd
	40 °C-40%	11.95abd	20.48ab	17.86ad	45.23abd
	40 °C-50%	9.17acd	18.83abd	15.31abd	42.78acd
	50 °C-30%	8.41bcd	17.49abd	13.06abd	39.09abd
	50 °C-40%	7.54bd	16.05abd	11.16ad	37.81abcd
	50 °C-50%	6.32ad	14.81ad	9.01abd	36.72abcd
Haritham	30 °C-30%	11.91abd	29.91abd	23.56acd	30.64ad
	30 °C-40%	12.76acd	30.78ad	24.19ad	33.42abd
	30 °C-50%	13.33acd	31.99abd	26.09acd	37.16abd
	40 °C-30%	16.19bcd	36.17abd	28.68abcd	42.79abcd
	40 °C-40%	21.93abd	40.88ab	34.14ad	50.86abd
	40 °C-50%	18.79acd	38.14abd	32.96abd	47.18acd
	50 °C-30%	17.18bcd	36.72abd	31.18abd	44.13abd
	50 °C-40%	16.12bd	35.89abd	30.04ad	42.69abcd
	50 °C-50%	15.66ad	31.59ad	27.36abd	39.14abcd

DW = Dry weight.

Means with different superscript letters (with the same column) indicate significantly different.

Table 7

Effect of ultrasonic treatment measured by temperature at 40% power for each frequency.

Sample (Brand)	Temperature (°C)	Extract yield* (mg/100 g DW)	Total Phenolics* GAE mg gallic/g DW	Flavanoids* (mg/g DW)	DPPH radical Scavenging* (%)
Rabea	Frequency =
	26 kHz
	30	10.78c	19.57c	17.15c	38.19c
	40	18.61ab	33.76ab	27.77ab	51.22ab
	50	14.89b	25.77b	19.89b	42.66b
	Frequency =
	40 kHz
	30	7.78bd	12.12bd	6.37bd	22.16bd
	40	15.71bc	21.29bc	14.85bc	36.74bc
	50	14.64cd	15.51cd	8.11cd	27.07cd
	Frequency =
	26 kHz
	30	10.19bd	21.89bd	15.71bd	38.82bd
	40	15.65bc	38.13bc	24.00bc	53.78bc
	50	12.86cd	25.91cd	19.08cd	42.85cd
Lipton	Frequency =
	40 kHz
	30	4.10bd	17.78bd	10.33bd	22.58bd
	40	8.98bd	24.71bd	17.82bd	39.12bd
	50	7.91d	20.14d	14.21d	30.17d
	Frequency =
	26 kHz
	30	17.33bd	15.66bd	9.09bd	37.28bd
	40	22.43bc	24.71bc	17.50bc	56.38bc
	50	18.21cd	15.68cd	10.13cd	42.81cd
Alkbous	Frequency =
	40 kHz
	30	8.64bd	7.88bd	4.95bd	25.11bd
	40	14.79b	14.34b	11.19b	36.38b
	50	7.27bd	9.99bd	7.04bd	30.20bd
	Frequency =
	26 kHz
	30	12.20bd	24.43bd	17.33bd	47.49bd
	40	19.36bc	32.73bc	25.11bc	65.30bc
			26
	50	16.88cd	26.20cd	17.93cd	51.85cd
Green gold	Frequency =
	40 kHz
	30	4.89bd	13.68bd	8.80bd	35.13bd
	40	11.95bc	20.48bc	17.86bc	45.23bc
	50	7.54cd	16.05cd	11.16cd	37.81cd
	Frequency =
	26 kHz
	30	23.34ad	51.61ad	41.84ad	59.22ad
	40	32.68ab	71.13ab	58.25ab	71.29ab
	50	27.78bd	56.88bd	48.78bd	58.79bd
Haritham	Frequency =
	40 kHz
	30	12.76ac	30.78ac	24.19ac	33.42a
	40	21.93ac	40.88ac	34.14ac	50.86ac
	50	16.12c	35.89c	30.04c	42.69c

DW = Dry weight

Means with different superscript letters (with the same column) indicate significantly different.

At 40 °C, with chosen power there is no significant difference (p < 0.001) were observed in both frequencies. With a 40% power setting and 40 °C was the highest extract yield and are significant, but no variation at 30% and 50% power in all samples at both frequencies (Table 8). The optimal temperature in the ultrasonic extraction of tea was discussed by some researcher (Milica et al., 2015).

Table 8

Effect of ultrasonic treatment measured by power setting at 40 °C temperature for each frequency.

Sample (Brand)	Power setting (%) (mg/100 g DW)	Extract yield	Total Phenolics GAE mg gallic/g DW	Flavanoids (mg/g DW)	DPPH radical scavenging (%)
Rabea	Frequency = 26 kHz
	30	15.58cd	26.95cd	24.56cd	43.87cd
	40	18.61cd	33.76cd	27.77cd	51.22cd
	50	16.88d	27.17d	21.43d	48.71d
	Frequency = 40 kHz
	30	11.12a	16.98a	10.06a	29.44a
	40	15.71abc	21.29abc	14.85abc	36.74abc
	50	12.17bc	19.17bc	12.13bc	32.93bc
Lipton	Frequency = 26 kHz
	30	13.42d	28.82d	21.18d	44.52d
	40	15.65d	38.13d	24.00d	53.78d
	50	14.42d	25.91d	22.92d	49.82d
	Frequency = 40 kHz
	30	6.75d	21.91d	14.08d	27.11d
	40	8.98d	24.71d	17.82d	39.12d
	50	7.59d	21.72d	16.09d	35.76d
	Frequency = 26 kHz
	30	19.22d	20.92d	14.69d	42.56d
	40	22.43d	24.71d	17.50d	56.38d
	50	21.76d	19.87d	13.90d	49.89d
Alkbous	Frequency = 40 kHz
	30	11.91cd	11.71cd	8.22cd	29.49cd
	40	14.79cd	14.34cd	11.19cd	36.38cd
	50	13.51d	12.17d	10.39d	34.89d
	Frequency =
	26 kHz
	30	15.57cd	29.77cd	22.39cd	55.39cd
	40	19.36c	32.73c	25.11c	65.30c
	50	18.33d	26.90d	21.29d	57.86d
Green Gnold	Frequency = 40 kHz
	30	7.97ab	16.89ab	12.83ab	39.04ab
	40	11.95ab	20.48ab	17.86ab	45.23ab
	50	9.17b	18.83b	15.31b	42.78b
	Frequency = 26 kHz
	30	27.49bd	59.97bd	46.08bd	64.82bd
	40	32.68bd	71.13bd	58.25bd	71.29bd
	50	29.72d	63.12d	55.69d	65.91d
Haritham	Frequency = 40 kHz
	30	16.19ab	36.17ab	28.68ab	42.79ab
	40	21.93a	40.88a	34.14a	50.86a
	50	18.79bc	38.14bc	32.96bc	47.18bc

DW = dry weight.

*Means with different superscript letters (with the same column) indicate significantly different.

Moreover, the temperature at 40 °C and 40% power setting was results in good extraction in both the ultrasonic frequencies. But results were found better in case of ultrasonic probe of frequency 26 kHz than ultrasonic bath of frequency 40 kHz which might be due to cavitational growth, collapse was better in lower frequency (Ashley, 1998) and thus good rarefaction time to grow bubble to sufficient size leads to disruption of cells resulting in increased in extraction yield.

Impact of extraction parameters on total phenol (GAE mg gallic/g DW) of branded tea samples using ultrasound technology

Results of total phenol content of different brands of tea sample were obtained quite significant (p < 0.0001). There was also found significant results among all parameters in both frequencies (Table 2). Considering the temperature-power combination there was a significant difference between the total phenol content of each brand of tea, but there is no difference were found within respective samples for a particular temperature power combination for 26 kHz and 40 kHz frequencies. Maximum total phenol content observed at 40 °C and 40% power as compared to other alliances was 33.76 ± 0.12, 38.13 ± 0.12, 24.71 ± 0.14, 32.76 ± 0.10& 71.13 ± 0.13 at 26 kHz and 22.29 ± 0.08, 24.71 ± 0.14, 14.34 ± 0.12, 20.48 ± 0.13 & 40.88 + 0.14 at 40 kHz for Rabea, Lipton, Alkbous, Green gold and Haritham brands of tea respectively (Tables 5 and 6).

Table 2

Total Phenolics content of different brands of tea using conventional and ultrasonic treatment.

Sample (Brand)		GAE mg gallic/g DW
	Conventional	Ultrasonic (26 kHz)	Ultrasonic (40 kHz)
Rabea	20.26 ± 0.10	33.76 ± 0.12	21.29 ± 0.08
Lipton	22.78 ± 0.11	38.13 ± 0.12	24.71 ± 0.14
Alkbous	13.58 ± 0.11	24.71 ± 0.14	14.34 ± 0.12
Green Gold	19.73 ± 0.12	32.73 ± 0.10	20.48 ± 0.13
Haritham	36.23 ± 0.12	71.13 ± 0.13	40.88 + 0.14

Thus, this study showed that an optimal temperature-power (40 °C-40%) was needed to obtain good results in terms of total phenol content (TPC) in either of the utilized ultrasound frequencies.

The results of TPC also indicated that at low temperature-power (30 °C-30%) and high temperature-power (50 °C-50%) combination was quite similar (Tables 5 and 6) with regard to obtained results. Consequently, the extraction of total phenol depends on both variable factors i.e temperature and power. Ignorance of any single factor might be unsatisfactory in relation to results of total phenol content. Hence, our study and some previous reports pointed out similar facts that combined effects of temperature-power variables was more valuable in the extraction of total phenolic content (TPC) (Horžić et al., 2012).

At a power setting of 40%, there was a significant difference (p < 0.0001) between temperatures (Table 7). There were also significant differences were found between 30 °C and 50 °C, but the highest total phenol was achieved at 40 °C for 26 kHz and 40 kHz frequencies. TPC of studied tea samples increased significantly with increasing temperature, attained maximum at 40 °C then declined. It has been reported that to the extraction of TPC from soybean temperature affects the mass transfer, solubility and in turn stability of phenolics (Lien et al., 2015).

For two frequencies (26 kHz and 40 kHz) all chosen power settings at 40 °C, total phenol content was significant (p < 0.0001) (Table 8). Power of 40% at 40 °C temperature exhibited a maximum total phenol outcome. Statistical analysis also revealed that no differences were found at power treatments of 30% and 50% at a constant temperature of 40 °C for both targeted frequencies. But results of TPC among different brands of tea using the same treatments of power temperature were found differently. In the present series, Haritham brand of tea was found maximum TPC at 40 °C temperature and 40% power was 71.13 and 40.88 mg equivalent to gallic/g DW respectively for 26 and 40 kHz. Likewise, extraction of multi bioactive compounds of Hypericum perforatum L. was different with similar ultrasonic conditions (Zou et al., 2004).

Impact of extraction parameters on total flavonoids (mg/g DW) of branded tea samples using ultrasound technology

Results of total flavonoid content of branded samples of tea were found significant (p < 0.0001) utilizing frequency, temperature, and power as independent variables. There were significant results were observed for both 26 and 40 kHz frequencies within each optimizing condition (Tables 3, 5 and 6). Total flavonoid concentration was maximum at both the treated frequencies at 40 °C and power setting 40% compared to the rest of the temperature power amalgamation. A close examination revealed that almost similar results were observed at other temperature power combination of all samples except Haritham, where the flavanoid content observed 2–3 folds higher in concentration (Tables 5 and 6).

Table 3

Total flavonoids content of different brands of tea using conventional and ultrasonic treatment.

Sample (Brand)	Conventional	(mg/g DW)
		Ultrasonic (26 kHz)	Ultrasonic (40 kHz)
Rabea	8.12 ± 0.30	27.77 ± 0.01	14.85 ± 0.45
Lipton	9.0 ± 0.33	24.00 ± 0.01	17.82 ± 0.30
Alkbous	6.97 ± 0.37	17.50 ± 0.28	11.19 ± 0.27
Green Gold	12.02 ± 0.32	25.11 ± 0.24	17.86 ± 0.45
Haritham	39.87 ± 0.48	58.25 ± 0.41	34.14 ± 0.33

For 40% power, observed a significant difference (p < 0.0001) for 26 and 40 kHz frequencies between treated temperatures. A fair combination of 40% power with 40 °C temperature achieved the highest total flavonoid content (TFC) at both the frequencies and each tea samples. In addition, there were different TFC results were found at 30 °C and 50 °C temperatures at both frequencies (Table 7). Similar findings were reported in the ultrasonic extraction of polysaccharides of Boletus edulis mycelia and concluded that an optimal temperature (below 50 °C) and power (below 50%) were needed to increase antioxidant potential (Chen et al., 2011).

At 40 °C treatment paired with different power (30, 40, 50%), flavanoid content results were not much significant difference within each frequency of 26 kHz and 40 kHz, but it was significantly different (p < 0.0001) at two different frequencies. At similar power, with a temperature of 40°, C TFC results were observed in similar fashion (Table 8). Highest cumulative results of TFC was 58.25 ± 0.41 and 34.14 ± 0.33 mg/g at 40 °C temperature paired with 40% power of Haritham brand tea for 26 kHz and 40 kHz respectively. A quantitative variation of flavanoid content of two frequencies was might have been being due to slow mass transfer and cavitational performance in case of the ultrasonic bath (40 kHz) in comparison to ultrasonic probe (26 kHz) (Wang et al., 2013).

Impact of extraction parameters on DPPH radical scavenging (%) activity of branded tea samples using ultrasound technology

The effect of optimization parameters (temperature and power) on the rate of DPPH radical scavenging activity of all the samples of tea at two ultrasonic frequencies (26 kHz and ‘40 kHz) was significant (p < 0.0001).

DPPH radical scavenging capacity of all the tea sample with each treatment of temperature-power was significantly (p < 0.0001) difference between ultrasonic frequencies. In another way, adopting similar conditions of optimization it was not significantly (p < 0.0001) difference within each frequency. The antioxidant potential of each brand of tea for both the frequencies was highest at 40 °C-40% temperature-power combination. The maximum mean results of DPPH radical scavenging was 71.29 ± 0.11 and 50.86 ± 0.10 for Haritham brand of tea at 26 kHz and 40 kHz ultrasonic frequencies respectively (Table 4). Rest of the temperature-power grouping showed almost similar DPPH radical scavenging within each sample at both the frequencies. Results also pointed out that as the operating frequency increased from 26 kHz to 40 kHz the rate of DPPH radical scavenging was decreased which was in agreement with some parallel research reports (Altemimi et al., 2015, Wang et al., 2013).

Table 4

DPPH radical scavenging (%) activity from different brands of tea using conventional and ultrasonic treatment.

Sample (Brand)		DPPH radical scavenging (%)
	Conventional	Ultrasonic (26 kHz)	Ultrasonic (40 kHz)
Rabea	24.49 ± 0.46	51.22 ± 0.10	36.74 ± 0.13
Lipton	29.21 ± 0.65	53.78 ± 0.90	39.12 ± 0.01
Alkbous	36.79 ± 0.90	56.38 ± 0.42	36.38 ± 0.90
Green Gold	41.95 ± 0.92	65.30 ± 0.80	45.23 ± 0.39
Haritham	46.61 ± 0.87	71.29 ± 0.11	50.86 ± 0.10

Considering 40% power, there was a significant difference (p < 0.0001) between temperatures for 40 kHz frequency, but no difference was obtained for 26 kHz. Maximum DPPH radical scavenging was exhibited at 40 °C for both the operating frequencies (Table 7). Also, for both operating frequencies, the DPPH radical rate was suddenly dropping at 50 °C, possibly due to some heat-sensitive bioactive present in the samples.

Statistical analysis revealed that at 40 °C, no difference in DPPH scavenging was observed at 30% and 50% power when exposed to ultrasonic frequency of 40 kHz. But at similar operating temperature and power with 26 kHz frequency, results were differing around 5–10 digits in a row for each brand of tea sample (Table 8). In the case of 26 kHz ultrasonic frequency, direct probe immersed in the reaction medium, therefore results might have been changes even alteration in power 5–10%. On the other hand, in case of the ultrasonic bath (40 kHz), no any significant changes was observed as it was not in direct contact with the medium and hence slight tuning of temperature or power wouldn’t affect results.

Appraisal of ultrasonic methods for yield, total phenols, total flavonoids and DPPH radical scavenging activities with conventional (control) extraction of branded tea samples

Performance evaluation of two ultrasonic techniques i.e. ultrasonic probe (26 kHz) and ultrasonic bath (40 kHz) in terms of extract percent yield and antioxidant potential was compared with experiments conducted by a conventional method (Fig. 1).

Fig. 1

Comparison of ultrasonic extraction with conventional (control) in terms of polyphenol content and antioxidant potential.

All the branded tea samples went through extraction, conducted at 40 °C temperature and 40% power for 30 min by both ultrasonic frequencies. A parallel experiment was also run by a conventional method at 40 °C temperature for 30 min. significant results were obtained in both ultrasonic set-ups. The polyphenolic extract was significantly (p < 0.0001) higher for 26 kHz frequency compared to 40 kHz and conventional (control) method (Table 1). The highest yield was 32.68 ± 0.14, 21.93 ± 0.04 and 17.68 ± 0.14 exhibited by Haritham brand of tea for 26 kHz, 40 kHz, and control respectively. Total phenol content (TPC) (Table 2) and total flavonoid content (TFC) (Table 3) produce significant (p < 0.0001) results in the order ultrasonic (26 kHz) > ultrasonic (40 kHz) > control. Many satisfied results of percent DPPH radical scavenging were also observed by the ultrasonic probe (26 kHz) and bath (40 kHz) with respect to control (Table 4). Furthermore, results of presents study were in agreements with various recent work reported on tea extraction, mentioned the efficiency of ultrasonication to the enhancement of polyphenolic compounds extract yield compared to conventional technique at low-temperature power decreasing the threats of degradation of valuable compounds (Horžić et al., 2012, Pasrija and Krishnan, 2015).

Impact of the thermodynamic parameter on the extraction of polyphenolic compounds using ultrasound technology

In the present study, a process of extraction for a branded tea samples was analyzed thermodynamically under the influence of two ultrasound frequencies i.e ultrasound probe (26 kHz) and ultrasound bath (40 kHz). All the basic thermodynamic parameters were calculated from Eqs. (2), (3), (4), (5) in material method section and results are summarized in Tables 9 and 10.

Table 9

Thermodynamic parameters at ultrasonic frequency 26 kHz.

Sample (Brand)	Temp, K	ΔG0, kJmol−1	Ea, kJmol−1	ΔH0, kJ	ΔS0, Jmol−1
Rabea	303.15	−0.72
	313.15	−0.78	3.64	1.04	5.83
	323.15	−0.84
Lipton	303.15	−0.60
	313.15	−0.61	2.42	−0.18	−1.31
	323.15	−0.62
Alkbous	303.15	−0.53
	313.15	−0.58	3.44	0.84	4.55
	323.15	−1.29
Green Gold	303.15	−1.00	4.01	1.41	7.98
	313.15	−1.08
	323.15	−1.16
Haritham	303.15	−1.52	4.75	2.15	12.12
	313.15	−1.64
	323.15	−1.76

Table 10

Thermodynamic parameters at ultrasonic frequency 40 kHz.

Sample (Brand)	Temp, K	ΔG0, kJmol−1	Ea, kJmol−1	ΔH0, kJ	ΔS0, Jmol−1
Rabea	303.15	−0.92
	313.15	−0.99	3.96	1.36	7.53
	323.15	−1.06
Lipton	303.15	−2.96
	313.15	−3.19	11.43	4.12	23.36
	323.15	−3.42
Alkbous	303.15	−2.07
	313.15	−2.24	5.57	2.97	16.65
	323.15	−2.41
Green Gold	303.15	−3.50	7.48	4.88	27.67
	313.15	−3.78
	323.15	−4.06
Haritham	303.15	−3.62	7.64	5.04	28.59
	313.15	−3.91
	323.15	−4.19

The values of ke for all brands of samples were found decreased as the temperature increased during the extraction in both ultrasonic frequencies (Tables 9 and 10) but at temperature 303.15 K it was lowest. Total phenolic contents (TPC) also found maximum at this temperature. 303.15 K (40 °C) temperature yielded the highest TPC in all brand of tea under the influence of both ultrasound frequencies. The values of Gibbs free energy (ΔG°) were found negative which confirmed the extraction process was spontaneous and feasible in both utilized ultrasound frequencies (Tables 9 and 10). Also, the degree of spontaneity was proportionally increased with increased in extraction temperature (Kim and Kim, 2016).

The heat of reaction (ΔH°) was obtained positive values in all brands of tea under the influence of both ultrasound technologies except Lipton, which exhibited the negative value (ΔH° = −0.18) using 26 kHz ultrasound (Tables 9 and 10). Therefore, the negative values indicating, that the extraction of tea samples requires energy and process were endothermic. The heat of reaction (ΔH°) during the extraction of Rabea, Lipton, Alkbous, Green gold and Haritham were found 1.04, −0.18, 0.84, 1.41 and 2.15 utilizing 26 kHz, whereas, 1.36, 4.12, 2.97, 4.88 and 5.04 kJ/mol with 40 kHz frequency. Interestingly, heats of reaction in our results were far less than some recent reports. The value of ΔH° = 8.040 kJ/ mol was recorded by the extraction of paclitaxel from biomass (Kim and Kim, 2016). Extraction of gossypol from defatted cottonseed meal (Saxena et al., 2012), extraction of oil from sunflower seeds (Topallar and Geçgel, 2000) and the extraction of oil from caster cake (Amarante et al., 2014) were found ΔH° = 27.749–28.093, 11.200 and 12.270 kJ/mol which is more than that of our present results as shown in Tables 9 and 10. Moreover, comparing values of heat of reaction (ΔH0) of each brand and under the influence of two frequencies was quite variable. Amount of polyphenolic contents using 26 kHz ultrasonic probe were found more with the expense of less energy but in case of 40 kHz ultrasonic bath, comparatively less amount of polyphenolic contents received with more energy dissipation considering any brand of tea sample (Tables 9 and 10). In both ultrasonic frequencies, Haritham brand of tea used maximum energy, ΔH° = 2.15 and ΔH° = 5.04 kJ/mol with 26 kHz and 40 kHz respectively yielded highest polyphenolic contents.

Further, the value of activation energy (Ea) should be more than their corresponding heat of reaction (ΔH°) in an endothermic reaction (Kim and Kim, 2016) and thus in our results indicated that all Ea values in either of the frequency were higher than ΔH°. Utilizing 26 kHz and 40 kHz frequencies, Ea values range from 2.42 to 4.75 and 3.96 to 11.43 kJ/mol respectively (Tables 9 and 10). These values also pointed out that relatively less activation energy (Ea) needed when 26 kHz ultrasound technique was employed compared to 40 kHz for the extraction process. Also, the Ea values in all brand of tea were less than that of most of the reported results. During the extraction of flavanols from green tea Ea was used 30–50 kJ/mol (Price and Spitzer, 1994). In another research, Ea = 8.56 kJ/mol was utilized for the oil extraction from the olive cake (Meziane and Kadi, 2008). All the positive value of ΔS° in both ultrasound extraction methods was irreversible.

Conclusion

In conclusion, ultrasound-assisted extraction was found to be the most reliable method for the extraction of polyphenol compounds from branded tea. Results of ultrasonic treatment were found statistically significant at temperature of 40 °C and power setting of 40% for both utilized frequencies. Total yield, total phenolic content, total flavonoid content and antioxidant potential (%DPPH) were found highest when used 26 kHz ultrasound probe as an extraction technique than 40 kHz bath. Thermodynamic analysis of branded tea sample for both ultrasonic treatments showed that the extraction process was spontaneous, endothermic and irreversible in the ranges of temperature employed. Under similar thermodynamic set up 26 kHz ultrasonic probe was found superior over 40 kHz ultrasonic bath. The futuristic study could be employed for the different food and beverages under the effect of ultrasound by varying parameters to access antioxidant potential as a techno-economical application.