Antioxidant capacity and characteristics of theaflavin catechins and ginger freeze-dried extract as affected by extraction techniques

ABSTRACT The aim of current research was to determine the effect of different extraction techniques on the antioxidant capacity of green tea, black tea and ginger polyphenols. Initially, the raw materials were subjected to compositional analysis. Afterward, the bioactive moieties from all the samples were extracted through ultrasound and conventional solvent extraction method using different solvents (i.e. ethanol, methanol and water). Furthermore, the functional ingredients, namely, catechins and theaflavins were isolated by solvent partition method from green and black tea, respectively. In addition, the extracts and isolates were analyzed for their total phenolic and antioxidant profile using various spectrophotometric assays. For the quantification of theaflavins and catechins, a reverse phase HPLC system was used. Results showed that ultrasound (P > .005) proved more effectual for the extraction and isolation of polyphenols (757.33 mg/100 g GAE) from the tested materials among the other extraction techniques (741.66 mg/100 g GAE). Likewise, green tea showed better performance than the rest and in solvents ethanol performed better as compared to their counter parts. Similarly, the isolates showed higher antioxidant potential as compared to their extracts and order of effectiveness was catechins > theaflavins > Ginger freeze dried extract (GFDE).


Introduction
Recently, diet-based therapy with special reference to polyphenols has been invigorated worldwide and people are using natural food materials as an intervention against various maladies. Among different dietary regimen tools, polyphenolic enriched functional and nutraceutical foods engrossed attention due to their acceptability, easy access, low cost, and long administration. In this scenario, green and black tea (Camellia sinensis) are the members of Theaceae family and are examples of plants containing bioactive molecules with unique nutraceutical potential. Likewise, the therapeutic worth of ginger is also well established. Extensive studies have suggested that tea consumption provides numerous health benefits mainly attributed to its polyphenols especially catechins, theaflavin, and thearubigins. Tea is mainly divided into three distinct types i.e., black, green and oolong differed in terms of processing method and chemical profile. Among all types, green tea holds a 20% share of total tea production and is regularly consumed in Asia especially in the south and East

Compositional analysis proximate analysis
The proximate composition of black tea, green tea, and ginger was evaluated through the estimation of moisture, fat, protein, fat, fiber and ash by adapting the guidelines of Kaur et al. [7] Whereas, NFE was estimated through the subtraction method.

Minerals analysis
The green tea, black tea, and ginger samples were evaluated for minerals like sodium (Na), potassium (K), calcium (Ca), zinc (Zn), magnesium (Mg) and iron (Fe) through the standard protocol of . [8] The first two were estimated through Flame Photometer-410 (Sherwood Scientific Ltd., Cambridge) however, remaining by Atomic Absorption Spectrophotometer (Varian AA240, Australia).

Polyphenols extraction
Bioactive molecules i.e. catechins, theaflavin and ginger freeze-dried (GFDE) extract were extracted from black tea, green tea, and ginger, respectively, by the solvent extraction and ultrasound extraction technique by selecting the protocol of Chen et al., [9] with some modifications. Purposely, using solvents including water, ethanol, and methanol at a constant temperature of 60°C and constant time (7 hrs for conventional extraction and 20 minutes for ultrasound extraction). The conventional extraction was carried out by using the orbital shaker (Model 780, thermos fisher, US), whereas the ultrasound added extraction was carried out by using the ultrasonic equipment ((VCX 750, Newtown). After the extraction from both conventional and sonication, the mixture was filtered by using a Whatman® filter paper (Whatman, United Kingdom) and final extracts were obtained). The obtained extracts were further concentrated and turn into powder form by using freeze-drying and stored at optimum condition. The adapted processing conditions (time and temperature) were selected based on earlier experiments results of polyphenol extraction through these modules by the same research group. Moreover, in preliminary trials, these conditions elucidated better results among the different tested processing combinations.

Isolation of theaflavin and catechins
The resultant extracts (Table 1) were utilized for the isolation of respective ingredients. In this context, the green tea (catechins) and black tea (theaflavins) extract were subjected to solvent partition method following the guidelines of Coda et al. . [10] Purposely, the respective tea samples were treated first with chloroform and then ethyl acetate to obtain catechins and theaflavins. Afterward, the ethyl acetate fraction was subjected to a freeze drier (Zirbus, Va Co 5, Germany) at −70 C shelf temperature, −80°C condenser temperature, 760 mm/Hg vacuum pressure for 24 hrs to obtain the powder form of respective nutraceutical. Whereas ginger extract was subjected to freeze-drying and its freeze-dried powder was further utilized. The resultant isolates and prepared ginger freeze-dried extracts were subjected to different antioxidant assays.

Phytochemical screening assays
Different antioxidant assays were executed through total phenolics, total flavonoids, DPPH, ABTS, and FRAP assay estimation.

Total phenolics (TPC)
The total phenolics (TPC) were the most common assay to estimate the antioxidant potential of the tested compound. Purposely, the equal amount of FC reagent and sample was taken alongside 500 µL of distilled water and provide stay for 5 minutes. Afterward, 4.5 mL of 7% Na 2 CO 3 was added and provided to stay for 90 minutes. Lastly, the absorbance was measured through a spectrophotometer (IRMECO, U2020) at 760 nm. The total phenolics were calculated as gallic acid equivalent (mg gallic acid/g) following the procedure of . [11] Antioxidant potential DPPH Radical Scavenging Assay: DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging ability estimation is the most common test to apprehend the antioxidant potential of the tested compound. In short, sample and DPPH solution (0.12 mM) were added to the test tube in the ratio of 4 and 1 mL, respectively and placed for 30 minutes in a dark place. Afterward, absorbance was measured at 520 recorded by using UV/Visible Spectrophotometer alongside control and blank . [12] ABTS (2,2ʹ-azino-bis, 3-ethylbenzothiazoline-6-sulfonic acid) Assay: ABTS assay of the ginger extract was estimated conferring to the method outlined by Piljac-Žegarac et al. . [13] For the preparation of ABTS radicals, % mL freshly prepared ABTS solution (7 mM) was mixed with 5 mL potassium persulfate solution (2.45 mM) to make 10 mL total volume. The mixture was transferred to an opaque bottle and allowed to stand for 16 hrs in a dark place to achieve the stable oxidized state. The mixture was diluted with ethanol and was adjusted to give 0.7 absorbances at 734 nm. Additionally, 10 µL ginger extract was added 1 mL ABTS solution, mixed thoroughly and subjected to spectrophotometry to measure the absorbance at 734 nm after 30 min stays time. The antioxidant activity using the Trolox standard curve was reported in µmol Trolox/g sample extract.
Ferric Reducing Antioxidant Power (FRAP): The metal ion chelation ability is another parameter to access the antioxidant capacity of the tested compounds under different challenges and for this ferric reducing antioxidant power is the most adapted test. For estimation of FRAP, 0.5 mL of sample was added in 125 mL of phosphate buffer prepared 0.2 M and pH of 6.6 and potassium ferricyanide solution (1%), placed in the water bath at 50°C for 15 min. In the resulted sample, an equal amount of 1.25 mL Trichloroacetic acid (10%) and distilled water alongside 0.25 mL of ferric chloride (1%) were added and provide stay time for 10 min and record the reading at 700 nm. [5,13]

HPLC quantification
For the quantification of theaflavins and catechins, a reverse phase HPLC system (PerkinElmer, Series 200, USA) was adapted with C18 column,10 μL sample size and column temperature of 40°C. Whereas, acetonitrile, ethyl acetate and phosphoric acid in ratio of 21:3:76 were utilized as mobile phase and quantification was done with UV/vis detector (model 481) at a wavelength of 278 nm . [5] Likewise, gingerol is the most abundant antioxidant in ginger and its estimation is important to impart positive health benefits to the ginger. Purposely, HPLC quantification was carried out for gingerol contents estimation in ginger extracts obtained through all extraction techniques. Briefly, reverse phase HPLC (PerkinElmer, Series 200, USA) with C18 column. The mobile phase was comprised of methanol/water, 65:35 (v/v), with a sample size of 1 mL, with 1.0 mL/min flow rate. For estimation, UV detection was made at 282 nm. The calculation was made by comparing the peak time and height with the gingerol standard . [14]

Statistical analysis
The data for each parameter was analyzed statistically to check the level of significance. For analysis of variance, one-way and two-way ANOVA were performed and for the estimation of significance among the means, Tukey's HSD test was applied. The values were represented in mean ± S.D.

Extract analysis
Among the extraction mode, ultrasound-assisted extraction exhibited a significantly (p ≤ 0. 001) better effect as compared to conventional solvent extraction. Likewise, in solvents, ethanol elucidated the highest yield followed by methanol and water in both ultrasound and conventional extraction modules. Amongst the tested compounds, green tea exhibited the richest antioxidant profile followed by black tea and ginger. From Figure 1, it was evident that the ultrasound extracted compounds exhibited higher antioxidant scores as compared to their conventional solvent-extracted extracts. Means for total phenolic contents (TPC) of GT extract showed the highest TPC in ethanolic extract of ultrasound and conventional extraction as 1005 ± 1.02 and 995 ± 2.43, mg/100 g GAE, respectively followed by methanol 890 ± 4.45 and 880 ± 2.34 mg/100 g GAE, respectively, while, the lowest 789 ± 4.84, 784 ± 1.98 mg/100 g GAE were detected in water extract, respectively. Likewise, the trend was observed for black tea (BT) maximum TPC value was exhibited by ethanol as 915 ± 6.42, 894 ± 1.02, mg/100 g GAE, respectively in Ultrasound and conventional extraction followed by methanol 788 ± 4.49, 767 ± 1.09 mg/100 g GAE, respectively, and water 649 ± 4.21, 635 ± 1.2 mg/ 100 g GAE for respective extraction techniques. Similarly, ginger freeze-dried extract (GFDE) also revealed the same trend; highest in ultrasound followed by conventional extraction and maximum in ethanol and minimum in water (Table 4). Means regarding DPPH activity of GT extracts ( Figure 2) indicated the highest DPPH in ethanolic extract of ultrasound and conventional extraction as 77 ± 4.65, 66.33 ± 2.51%, respectively. Whereas, the recorded concentration for methanol and water were 69.67 ± 1.68, 55 ± 1.98 and 62.67 ± 2.44 and 56 ± 4.55%, respectively for ultrasound and conventional extraction. Likewise, in BT the recorded DPPH values in ethanolic extract of ultrasound and conventional extraction were 75 ± 1.09 and 65 ± 1.09%. Similarly, in ginger, the DPPH was in the range of 49% to 61% with the highest in ultrasound ethanolic extract (Table 5).
The recorded ABTS concentrations of green tea in ultrasound added extracts of ethanol, methanol, and water were 22.52 ± 0.77 µM TE/g, 17.65 ± 0.61 µM TE/g, and 15.71 ± 0.47 µM TE/g, respectively. Similarly, black tea in ultrasound added extraction exhibited the highest ABTS 17.84 ± 0.55 µM TE/g in ethanol, followed by methanol 16.07 ± 0.47 µM TE/g, and the lowest 12.36 ± 0.67 µM TE/g in the water extract. In addition, the ginger extracts expounded the highest ABTS value (11.10 ± 0.14 µM TE/ g) in ethanol followed by methanol (9.56 ± 0.27 µM TE/g) and water (8.67 ± 0.25 µM TE/g). Likewise, in conventional extraction, the highest ABTS 0.678 ± 0.00768 µM TE/g were detected in ethanol followed by methanol 0.520 ± 0.006 µM TE/g, and water 0.458 ± 0.008 µM TE/g in GT. A similar trend was observed for black tea and ginger, maximum in ethanol, and minimum in water (Table 8).
Isolate analysis. The isolates like catechins, theaflavins, and ginger freeze-dried extract (GFDE) were also obtained from green tea, black tea, and ginger, respectively. The obtained isolates were subjected to their antioxidant activity estimation through DPPH, β carotene, and FRAP assay by dissolving in ethanol, methanol and water. Means for DPPH value of catechins, theaflavins and GFDE obtained through conventional extraction indicated the highest activity in ethanolic fractions as 85. 26    Values are mean ± SEM (n = 03): Three-way repeated measure ANOVA (Treatments, Solvent and Extraction technique) was applied to elaborate the effect of extraction techniques, solvents and treatments on antioxidant indices and showed significant effect of extraction techniques (P > 0.012), Solvent (P > 0.005) and treatment (P > 0.009) on β carotene contents. The results Means sharing similar letter in a row or in a column are statistically non-significant (P > 0.05) according to Tukey's HSD test The ethanolic fraction of catechins exhibited the maximum β carotene activity 71.86 ± 1.22% than methanol 67.27 ± 1.32% and water 64.24 ± 1.05%. Similarly, theaflavins also elucidated the same trend and recorded values for this trait were 75.51 ± 1.50, 71.79 ± 1.72 and 67.66 ± 2.32% in ethanolic, methanolic, and water fractions, respectively. The ß-carotene activity of GFDE in ethanol was (50 ± 0.71%), whereas 48 ± 1.09 and 41 ± 1.03% was observed in methanol and water extract, respectively. Likewise, trend was reflected in ultrasound-based fractions; maximum in ethanol followed by methanol and water (Table 10).
The FRAP values in Table 11

Discussion
The results regarding the compositional profile are in harmony with the observations of Coda et al.,; Hussain et al., [10,11] observed that both green and black tea contain appreciable amount protein and fiber. Likewise, [12] observed 6.12 to 8.25, 2.56 to 4.23, 13.24 to 19.21, 13 to 16.25, 2.21 to 6.12%, moisture, fat, protein, fiber, and ash, respectively. The findings regarding the minerals of the current investigation are corroborated with the observations of different researcher groups like [5,[13][14][15][16] narrated that green and black tea has rich mineral potential with special reference to Na, K, Ca, Mn, Zn and Fe. However, they ascribed the variations as the function of climate, agronomic practices and soil conditions.
The findings of present research regarding the higher polyphenolic yield of tea polyphenols through ethanol and methanol as compared to water are in line with the earlier investigation of [17] probed the effect of different solvents for the polyphenol extraction of tea. Purposely, they utilized ethanol, methanol, dimethylformamide (DMF), water, and acetone for the extraction and observed the highest polyphenols, DPPH in ethanol followed by methanol and water. The recorded values of TPC for ethanol, methanol and water were 1300.30 mg/100 g GAE, 820.30 mg/100 g GAE, 330.30 mg/100 g GAE, respectively, while 68.9, 58.3, and 29.9% DPPH.
Amongst the different extraction modes, solvent extraction has prime importance for the recovery of polyphenols owing to its ease, higher recovery, and easy application. However, to extract maximum Values are mean ± SEM (n = 03): Three-way repeated measure ANOVA (Treatments, Solvent and Extraction technique) was applied to elaborate the effect of extraction techniques, solvents and treatments on antioxidant indices and showed significant effect of extraction techniques (P > 0.019), Solvent (P > 0.001) and treatment (P > 0.015) on β carotene contents. The results Means sharing similar letter in a row or in a column are statistically non-significant (P > 0.05) according to Tukey's HSD test benefits, many factors must be taken into account like sample size, solvent-to-material ratio and other relevant parameters. All of these can actively influence the extraction yield. [18] Likewise, the trend was observed by Cabrera et al., [19] for DPPH inhibition; more in ethanolic solvent (97.70%) as compared to methanol (95.55%). The different researcher groups, kaur et al.,; chandini et al., [7,20] expressed that the time and type of solvents are the factors of prime consideration for the polyphenol extraction from tea. The findings of another scientific exploration conducted by Chen et al., [9] are corroborated with the outcomes of instant exploration. They recorded FRAP and TPC values of different types of black tea and observed variations from 357 to 927 µmol Fe 2+ /g, and 400 to 1200 mg/100 g GAE, respectively. Later, Costa et al. [8] investigated the antioxidant potential of different black tea samples through the total phenolic estimation, free radical inhibition through DPPH test, and observed values of respective traits were 3000.10 mg/100 g GAE and 75%. Likewise, the tea samples exhibited good free radical scavenging and ß-carotene binding ability. The observed variations in DPPH and ß-carotene were 42.01 to 51.36 and 40.02 to 48%, respectively [21] The higher antioxidant activity of tea flavonoids is attributed to the presence of a specific chemical structure dominated by vicinal dihydroxy and trihydroxy moieties that can quench metal ions and imparted electron delocalization thus ultimately proved helpful to reduce free radicals production. [22,23] The more extraction efficiency of ethanol for polyphenol as compared to other solvents are in harmony with the findings of Friedman et al.,; Ghasemzadeh et al.,; Ghasemzadeh et al., [24][25][26] they observed better tea polyphenol extraction through aqueous ethanol as compared to methanol and water. In this context, they elucidated solvent-to-material ratio, time and temperature can play a vital role. The existing results for the better free radical inhibiting activity of tea polyphenols through ethanol as compared to methanol and water are supported by the findings of Herrero et al., [27] observed higher DPPH radical scavenging activity (92.20 ± 0.52%) in ethanol as compared to water (88.36 ± 0.76%). Similar findings were documented by Asensio-Vegas et al., [28] observed higher DPPH free radical scavenging activity through ethanolic extracts elucidated (46.6%) as compared to water extract (41.1%).
The findings regarding the theaflavin extraction yield are coherent with the observations of Hromadkova et al., [29] noticed higher extraction yield (1.51 g/50 g of theaflavin) from black tea sample. They envisaged that the isolation recovery is dependent upon medium, solvent, time, and temperature; moreover, variety has also influenced this trait. In this contest, [30] observed good polyphenols recovery up to 70% in freeze-dried ribose tea f with special reference to theaflavins and catechins in comparison with their crude extract (13.2%). Likewise, observations were documented by the findings of Jayasekera et al., [31] recorded the 75 to 80% variations in the recovery of the same bioactive moieties. However, they utilized different time solvents variations to optimize the extraction efficiency. They were of the view that the time and temperature intervention exerted cardinal effect on the isolation.
The effect of different time-temperature interactions on tea polyphenol isolation is well reflected through the findings of Joubert et al. [32] they utilized different time intervals at fixed temperatures and observed the influence on the tea isolate recovery. Purposely, they utilized 10-120 min at 90°C. They observed optimum recovery at 40 min; however, after 60 min a diminishing pattern was observed and the highest degradation was noted at maximum temperature. They expressed that a higher solvent to material ratio and 40°C are the optimum conditions for the extraction of black tea polyphenols. The possible mechanism behind the polyphenol less recovery at high temperature may be associated with their vulnerability at a higher temperature. The higher antioxidant activity of tea isolates as compared to their extract in the current study is verified by the outcomes of Khan et al. [33] probed the antioxidant activity of theaflavins and their mixtures from Assam black tea extract (ASTE). They explicated more DPPH value for isolated theaflavins 60-97% as compared to their extracts 40-82%. The isolated components showed higher antioxidant and free radical scavenging activity owing to their purity, structural diversity and multiple mechanistic approaches. In this context, theaflavins and catechins exhibited higher antioxidant capacity owing to the presence of multiple functional groups in C rings. The high free radical tackling potential of isolated black tea polyphenols may ascribe this tendency. [34] Likewise, observations were documented by McAlpine et al., [35] also recorded the higher antioxidant profiling of isolated components as compared to their respective extracts. Furthermore, [36] expressed that the higher free radical quenching ability is owing to the presence of benzotropolone ring and its gallate structure. The difference in the extraction yield owing to the solvents may be ascribed to the polarity factor and property of the component to be extracted. [37] Ginger has strong antioxidant potential owing to its major polyphenols like gingerol and others. The promising antioxidant potential of ginger in current research is corroborated with the earlier findings of Masmali et al., [38] investigated the total phenolic contents and antioxidant activity of different ginger verities and observed better performance of methanol as compared to water. The recorded TPC were varied between 95.2 to 87.5 mg/g dry extract. Whereas, the observed DPPH values were 83.87% and 83.03%, respectively. Moreover, the appreciable ferric reducing antioxidant power (FRAP) has been observed. One of their peers, [39] also carried out the intensive antioxidant activity estimation of different parts of ginger-like leaves, stems and rhizomes of two different varieties i.e. Halia Bentong and Halia Bara. They examined the TPC, FRAP and DPPH of different parts of both varieties by utilizing the two solvents methanol and water. Among the varieties, Halia Bara exhibited a higher antioxidant profile and in solvents methanol performed better as compared to water. The highest antioxidants were detected in rhizomes and least in the stem.
The higher antioxidant potential of Ginger freeze-dried extract (GFDE) in the present research are in agreement with the findings of Fatima et al., [40] examined the antioxidant potential of ginger freezedried extract through different antioxidant indices and observed TPC and DPPH were varied from 670.1 to 725.25 mg/g dry and 80.25 to 901.25%. They were of the view that GFDE has the more concentrated form of polyphenols as compared to their raw extract thus exhibited higher antioxidant activity. Different researchers groups [41][42][43]also observed higher antioxidant profiling of ginger freezedried extract as compared to their crude extract.
It is deduced from the piled evidence that all the tested components i.e. green tea, black tea and ginger have promising antioxidant potential, Likewise, the isolated bioactive molecules catechins (green tea), theaflavin (black tea) and ginger freeze-dried extract (Ginger) exhibited more pronounced effect than their raw extract. Among the solvents highest performance was observed by ethanol followed by methanol and water. Amongst the tested compounds catechins lead over their counterparts. Conclusively, it was inferred that both nutraceutical (catechins and theaflavins) and designer ingredient (GFD) has merit to be incorporated in the preparation of dietary intervention against oxidative stress induce maladies owing to their higher antioxidant potential.

Conclusion
From the above findings, it is envisaged that green tea, black tea and ginger have an appreciable nutritional profile and exhibited strong antioxidant potential. Among the extraction techniques, ultrasound proved more effective for the extraction and isolation of polyphenols from the tested materials. Likewise, among the treatments, green tea showed better performance than the rest and in solvents ethanol performed better as compared to their counterparts. Likewise, the isolates showed higher antioxidant potential as compared to their extracts and the order of effectiveness was catechins>theaflavins>GFDE.