Evaluation of seven different drying treatments in respect to total flavonoid, phenolic, vitamin C content, chlorophyll, antioxidant activity and color of green tea (Camellia sinensis or C. assamica) leaves (2024)

Plant material

Green tea leaves obtained from tea farms in Lahijan City in the northern region of Iran in the fall growing season. In order to preserve them from enzymatic browning, the leaves were blanched with steam at 90°C for 1min.

Drying methods

The leaves were dried using one of the following methods: (a) air drying at shade and room temperature (25°C); (b) sun drying; (c) drying in a hot air oven at 60°C; (d) drying in a hot air oven at 80°C; (e) drying in a hot air oven at 100°C; (f) drying in a microwave oven at 800W; (g) freeze drying. Shade drying was carried out under natural air flow and surrounding’s temperature (mean temperature = 25°C) for 36h. In the case of sun drying, leaves were dried into trays under direct sunlight at temperatures between 30 and 35°C for 7.5h in November in Isfahan, Iran. Oven drying was conducted in a ventilated oven (Osk, Japan) at three different temperatures (60, 80 and 100°C). Microwave drying was performed in a domestic digital microwave oven (Nikai, NMO-518N, Japan) with technical features of 230V, 800W. The samples were dried for 240s. Freeze-drying was carried out in HeltoHolten DW8 freeze dryer. The plant materials were frozen in −80°C for 24h and dried under vacuum and the condenser temperature at −15°C for 24h.

Color measurement

Five grams of each method were ground in a coffee grinder for 10s to produce a powder of a uniform color. The samples were transferred to a 10cm petri dish and subsequently read by a spectrophotometer Texflash (Datacolor, Swiss). The colorimeter was calibrated against a standard calibration plate of a white surface and set to CIE Standard Illuminant C. The L*, a* and b* values were determined and averaged from three readings. The color brightness, L* measures the whiteness value of a color and ranges from black at 0 to white at 100. The chromaticity coordinate a* measures red when positive and green when negative, and the chromaticity coordinate b* measures yellow when positive and blue when negative (Arslan and Ozcan 2008).

Chlorophyll content

One hundred mg of material was ground in a mortar with 4mL of 80% acetone. The ground material was collected in a 15mL falcon tube. The mortar was rinsed with 4ml of acetone. The volume of the falcon was adjusted to 10ml with acetone. The falcons were kept in ice and dark environment. The samples were centrifuged at 3000rpm (Sigma, Germany) for 10min at 4°C. Immediately after centrifugation, the falcons were transferred to ice before rapid measurement with spectrophotometer. The absorbance of the solution was read at three wavelengths including 663.2, 646.8 and 470nm. Chlorophyll concentrations were calculated in mg/l of acetone according to the formula:

Vitamin C content

Ascorbic acid (AA) was determined by titration method using 2,6-dichlorophenol–indophenols according to AOAC method No. 967.21 (AOAC, 2000). The vitamin C content in fresh and dried samples was expressed as mg AA/100g wet basis.

Total phenolic content

2.5 g of the finely ground sample was extracted with 50ml methanol 80% in falcon and was shacked in 240rpm for 24h. The sample was filtered twice with Whatman 0.2μm. Total phenolic content (TPC) was determined using Folin-Ciocalteu method (Pinelo et al. 2004). 500μl of diluted extraction (500μl primary extraction was adjusted in the volume of 50ml methanol 80% for producing 1/250 dilution), 2.5ml Folin-Ciocalteu reagent and 2ml of 7.5% sodium carbonate were also mixed. After heating at 45°C for 15min, the absorbance was measured at 765nm against methanol 80% as blank. TPC was expressed as Gallic acid equivalent/g dry weight of sample. The calibration equation for Gallic acid was obtained as y = 17.422x + 0.0872 (R2 = 0.998), where x is the absorbance and y is the concentration of Gallic acid in mg/l.

Total flavonoid content of the extracts

Total flavonoid content was determined spectrophotometrically using a method based on the formation of a complex flavonoid–aluminum, with some modifications. An aliquot (0.5ml) of the extract solution was mixed with distilled water (2ml) and subsequently, with NaNO₂ solution (5%, 0.15ml). After 6min, AlCl₃ solution (10%, 0.15ml) was added and allowed to stand for further 6min; thereafter, NaOH solution (4%, 2ml) was added to the mixture. Immediately, distilled water was added to bring the final volume to 5ml. Then, the mixture was properly mixed and allowed to stand for 15min. The intensity of pink color was measured at 510nm.

DPPH radical-scavenging activity

The ability of the plant extract and reference substance to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals was assessed using the method described by Stankovic et al.(2012). The stock solution of the plant extract was prepared in methanol 80% to achieve the Phenolic concentration of 5, 3, 1 and 0.5mg/ml. Dilutions were made to obtain Phenolic concentrations of 500, 300, 100 and 50μg/ml (ppm). 100μl of diluted solutions was mixed with 5ml of DPPH methanolic solution (0.1mM) and was adjusted to the volume of 10ml with methanol 80%. After 30min in darkness at room temperature (23°C), the absorbance was recorded at 517nm. The control samples contained all the reagents except the extract. The percentage inhibition was calculated using equation: % inhibition = 100 × (A of control − A of sample)/A of control, while concentrations of extracts required to inhibit radical by 50% (IC₅₀) values were estimated from the percentage of inhibition versus concentration of sigmoid curve, using a non-linear regression analysis. The data were presented as mean values ± standard deviation (N = 2) (Arslan and Ozcan 2012).

Reducing power

The extracts (2.5ml) and Butylated hydroxytoluene (BHT) were mixed with 2.5ml of 1% potassium ferricyanide and 2.5ml of 200mM sodium phosphate buffer (pH6.6) and were incubated at 50oC for 20min. Then, 2.5ml of 10% trichloroacetic acid was added, and the mixture was centrifuged at 200g for 10min. The upper layer (2.5ml) was mixed with 2.5ml of deionized water and 0.5ml of 0.1% ferric chloride. The absorbance at 700nm was measured against a blank. The increased absorbance of the reaction mixture correlates with greater reducing power (Ardestani and Yazdanparast 2007).

Statistical analysis

Cluster analysis and calculation of correlations among the compounds were performed using SPSS ver.11 based on Ward’s minimum variance method (Ward 1963). Cluster analysis was conducted to classify the drying methods based on their similarity of the major compounds. Statistic ver. 8 was also used to evaluate. The significance of data was calculated using SAS ver. 8.

Total flavonoid and Phenolic content

The antioxidant (phenolic compounds, vitamins, etc.) contents of fresh plant materials were higher than those of dried plant materials due to their degradation during drying, while some recent studies have shown that dried plant materials contain higher antioxidants such as polyphenolics, and antioxidant activity as compared to fresh plant materials (Pinela et al. 2012). Drying process can break down the cellular constituents leading to accelerate in releasing phenolic compounds from the food matrix (Chang et al. 2006; Arslan and Ozcan 2010). Another possibility concerning the low level of phenolic compounds in the fresh sample was the presence of an active enzyme in the sample that probably causes these compounds to be depredated. In the dried samples, due to the low water activity, destructive enzymes were inactivated and high levels of phenolic compounds remained in the extract (Hassain et al. 2010). In the present study, the highest total phenolic content (TPC) was obtained in oven 60°C (216.16mg Gallic acid/gdw), while the lowest one belonged to shade drying (109.85mg Gallic acid/gdw) (Fig.1). Similar to the present study, oven drying method possessed the highest amount of phenolic compounds among other methods as reported by Pinela et al. (2012). Rabeta and Lai were also reported that vacuum dried samples had the highest TPC, followed by freeze dried and fresh samples in Ocimum tenuiflorum (Rabeta and Lai 2013). TPC decreased in higher temperatures (80 and 100°C). Total flavonoid content (TFC) also ranged from 14.3mg Quercitin/gdw in microwave drying to 38.18mg Quercitin/gdw in oven 100°C.

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Fig. 1

Total phenolic content (TPC) variation in green tea using seven drying treatments. Bars with different letters are significantly different

Previous studies also reported the high amount of TFC in green tea compared to other types (Fig.2). This can be due to more oxidation and polymerization by enzymes derived from tea leaves during the production of oolong and black tea from green tea by fermentation (Bae et al. 2015). Furthermore, previous reports have shown that temperature more than 50°C leads to a decrease in essential oil yield and total phenolic content of different plant species (Braga et al. 2005; Rezaeinodehi and Khangholo 2008; Rabeta and Lai 2013). Prathapan et al. (2009) studied the effect of heat treatment (60–100°C) on TPC, color value (yellowishness and brightness), polyphenol oxidase (PPO) activity and curcuminoid of fresh turmeric rhizome. In their study, TPC values increased gradually when samples were heated from 60 to 80°C. Moreover, PPO was completely inactivated at 80°C (Prathapan et al. 2009).

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Fig. 2

Total flavonoid content (TFC) variation in green tea using seven drying treatments. Bars with different letters are significantly different

See Also

vikaspedia Domains

Generally, plants are often dried and stored for a long time before being used in manufacturing various types of products. Dehydration is an important preservation method for plant materials, as it inhibits enzymatic degradation and minimizes microbial growth of plants (Pinela et al. 2012). Freeze-drying also maintained a high amount of total phenolics. In most previous studies, freeze-drying was considered as one of the appropriate methods to preserve the quality of plants during processing (Arslan and Ozcan 2008, 2012; Annegowda et al. 2014). Chan et al. (2009) studied the effects of five different drying methods (microwave, oven, and sun drying) on antioxidant properties of Alpinia zerumbet, Etlinger aelatior, Curcuma longa and Kaempferia galangal. Air drying of ginger leaves resulted in significant losses in total phenolic content and antioxidant activity for all four species (Chan et al. 2009). Drying process can break down the cellular constituents leading to acceleration in releasing phenolic compounds from the food matrix (Chang et al. 2006; Arslan and Ozcan 2010). Another possibility about the low level of phenolic compounds in the fresh sample is the presence of an active enzyme in the sample that probably causes these compounds to be depredated. In the dried samples, due to the low water activity, destructive enzymes are inactivated and high levels of phenolic compounds will remain in the extract (Hassain et al. 2010).

During drying, metabolically active plants would slowly lose their moisture and might sense the moisture loss as stress. Plants, in general, produce phenolic compounds as a defense mechanism in response to stress. It is reported that the synthesis of several phenylpropanoid compounds (flavonoids, isoflavonoids, psoralens, coumarins, phenolic acids, lignin and suberin) was induced in plants in response to biotic and abiotic stress, such as wounding, low or high temperature and by attacks pathogens (Dixon and Paiva 1995).

DPPH assay

The ability of different extracts of tea drying treatments to quench DPPH free radical was measured. The extracts and BHT demonstrated a dose-dependent scavenging activity by reducing DPPH radical (Fig.3). By plotting the graph of extract concentrations against the scavenging activity, a specific concentration of the sample that needed to provide 50% inhibition (IC50) was calculated. Among treatments, oven drying at 60°C revealed the highest radical scavenging activity (the lowest IC ₅₀ = 167.166μg/ml), while the microwave treatment showed the lowest scavenging activity (IC ₅₀ = 505.5μg/ml). Previous researches showed that the radical scavenging activity was elevated at higher drying temperatures using oven drying treatments (Lee Mei Ling et al. 2013; Rodriguzer et al. 2014). It is believed that the high total phenolic content might contribute to the high antioxidant activity by high temperature drying (Lou et al. 2015).

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Fig. 3

DPPH radical scavenging activity of different dried treatments in green tea. Bars with different letters are significantly different

Reducing power

The ability of the antioxidant fraction to reduce Fe⁺³ to Fe⁺² represents the reductive power of the antioxidant (Ardestani and Yazdanparast 2007). Color changes of the solution in the presence of the methanolic extract of green tea and control antioxidant were evaluated. Reducing capacities of methanolic extract of green tea are shown in Fig.4. The reducing power of the extracts and the standard was elevated by increasing the concentration of the samples. Similar to DPPH assay, in FTC model system, the strongest reducing power was obtained in microwave drying method, while the weakest activity was observed in oven drying at 60°C.

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Fig. 4

Reducing power of different dried treatments in green tea. Bars with different letters are significantly different

Reducing capacity of an extract can serve as an indicator of its potential antioxidant activity. However, the antioxidant activity of antioxidants could be attributed to different mechanisms (Yildirım et al. 2000). For example, DPPH assay is mostly attributed to such phenolic compounds that participate in scavenging free radicals, while in reducing power the inactivation of the metal ions such as ferric ions is evaluated by phenolics (Gharibi et al. 2013).

Vitamin C

Besides phenolic and flavonoid compounds, tea provides a significant dietary source of polysaccharides, vitamin B complex, vitamin C, vitamin E and fluoride, as well as trace elements such as K, Mn, Cr, Ni and Zn (Komes et al. 2010). As shown in Fig.2, the results revealed that different drying methods led to a decrease in vitamin C in the green tea leaves. Ascorbic acid (vitamin C) is considered an important nutrient in fruits and vegetables. It is usually selected as a parameter of nutritional value because of its labile nature compared to other nutrients in foods. The degree of vitamin C loss during drying vegetables depends on the physical properties of the product and the type of process (Goula and Adamopoulos 2010). In the fresh leaves, vitamin C content was 17.72mg/100gDM. During drying, a significant decrease was observed in the ascorbic acid content (Fig.5). The highest vitamin C in dried leaves was obtained in freeze drying (16.36mg/100gDM) followed by microwave drying (15.99mg/100gDM) (P < 0.05). Two factors affect the vitamin C content in final products. Vitamin C oxidizes and disappears in high temperatures and long drying times (Vega-Galveza et al. 2009; Jin et al. 2014). In microwave method the amount of vitamin C was notably high. This might be due to the short drying time. Previous reports also illustrated that short drying time and lower temperature can maintain ascorbic acid (Negi and Roy 2000).

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Fig. 5

Vitamin C content of green tea in different drying treatments. Bars with different letters are significantly different

Chlorophyll content

The color of green vegetables is mainly determined by the chlorophyll pigments available in plant materials, which to catch the energy from the sun light. In most cases, color changes are reflected in the chemical, biochemical and microbiological reactions. Temperature and relative humidity during drying are among the reasons leading to color degradation in dehydrated products. However, the visual appearance of dehydrated product is very important for shopping (Shahabi et al. 2014). Furthermore, previous reports highlighted that in green tea leaves and non-fermented teas, chlorophyll is considered an important pigment as its amount determines thefinal color of green tea infusion (Ostadalova et al. 2014).

Results revealed that different methods of drying had a significant effect on the amount of chlorophyll in the leaves (Fig.6). They showed that chlorophyll content in dried samples was higher than that in the fresh sample based on wet basis. During drying, chlorophyll content was significantly increased (Fig.6). It is believed that chlorophyll is sensitive to heat and its retention is dependent on temperature and duration of heat treatment. For this reason, the sample dried by oven drying in 100°C method contained the lowest chlorophyll content (6.2904 Ca mg/l and 3.2004 Cb mg/l in leaf, respectively). The results were in agreement with those obtained by Negi and Roy (Negi and Roy 2000).

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Fig. 6

Changes in chlorophyll a and b of green tea as affected by different drying treatments

The highest and the lowest chlorophyll content were obtained in freeze drying (17.35mg/l) and oven 60°C (5.73mg/l), respectively. Chlorophyll is much susceptible to degradation during food processing and storage. Degradation of chlorophylls a and b to pheophytin a and b, respectively, followed first-order kinetics and chlorophyll a was more heat sensitive than chlorophyll b (Arslan and Ozcan 2012; Rahimmalek and Goli 2013). During heat treatment, the magnesium in chlorophyll is readily replaced by two hydrogen atoms in the presence of mild acids such as oxalic or acetic acid to produce pheophytin (Arslan and Ozcan 2010). Since this degradation is considered an index of loss of vegetables quality by consumers, food processors are trying to prevent or minimize the chlorophyll degradation (King et al. 2001).

Color

The color data in terms of the Hunter L*, a* and b* values of fresh and dried green tea leaves are illustrated in Table ​Table1.1. The L* value of freeze drying treatment was the highest among all dried leaves (25.2), while sun drying revealed the lowest L* (20.1) value among dried treatments (P < 0.05). High temperature and long drying time is the most effective factors in color damage during drying (Arslan and Ozcan 2008; Arabhosseini et al. 2011; Rahimmalek and Goli 2013). Similar results were also obtained for b*(Table ​*(Table1).1). Freeze drying treatment showed the highest b* values in comparison to others. So, freeze drying and sun drying possessed the highest and the lowest yellow color, respectively (b* value). For a* value (greenness) freeze drying had the highest value (more negative), while oven 80°C showed the lowest green color. Overall, sun and freeze drying treatments were considered the least and the most desirable drying methods regarding the final color of green tea leaves.

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