Three different types of herbal tea were categorized. Based on manufacturing condition using herbal plant material with C. sinensis shoots viz, green herbal tea (GHT), black herbal tea (BHT) and market herbal tea (MHT) is purchased from the local market Palampur. All these three types of teas were compared with green and black tea of Camellia sinensis. A total of 16 green and black herbal teas were manufactured using the Western Himalayan region herbs, such as sea-buckthorn (Hippophae rhamnoides), tulsi (Ocimum basilicum), lemongrass (Cymbopogon flexuosus), mint (Mentha piperita), timur (Zanthoxylum armatum), taxus (Taxus baccata), ginkgo (Ginkgo biloba) and kachnar (Bauhinia variegate). The manufactured GHT category include the kachnar green tea (KGT), timur green tea (TiGT), ginkgo green tea (GGT), mint green tea (MGT), lemongrass green tea (LGT), tulsi green tea (TuGT), taxus green tea (TGT) and sea-buckthorn green tea (SGT). The BHT manufactured category include the ginkgo black tea (GBT), kachnar black tea (KBT), timur black tea (TiBT), mint black tea (MBT), tulsi black tea (TuBT), taxus black tea (TBT), lemongrass black tea (LBT) and sea-buckthorn black tea (SBT). Rose tea (RT), mint tea (MT) and tulsi tea (TT) was purchased from the local market are in the MHT category.
Total polyphenols content (TPC)
Total polyphenol content was measured using Folin–Ciocalteu method which was ranged from 4.42 ± 0.53 to 13.37 ± 0.50%. Among all the teas, GT is the rich source of TPC (13.37 ± 0.50%) (Table 1). In green herbal tea (GHT), polyphenols content was ranged from 10.75 ± 0.14 to 13.37 ± 0.50%. From GHT, GT showed the highest 13.37 ± 0.50%, whereas KGT contain the lowest 10.75 ± 0.14% polyphenol content. In addition, there are some exceptions were observed in green herbal teas such as SGT (13.23 ± 0.10%) and TuGT (12.47 ± 0.19%) showed an almost similar level of TPC compared to GT. In BHT highest polyphenol content was observed in BT (10.05 ± 0.11%) and lowest was observed in GBT (5.61 ± 0.37%). The findings of this study suggested that the highest polyphenol content was observed in GHT followed by BHT and the least was observed in MHT.
Total catechin content
Catechins is the class of flavonoid commonly known as flavan-3-ols. Total catechin content in all the samples was ranged from 4.43 ± 0.28 to 15.17 ± 0.53% (Table 1). In GHT, GT showed the higher catechins content (15.17 ± 0.53%) and KGT show the lowest catechin content (9.30 ± 0.15%). While in BHT, BT showed the higher catechins content (12.25 ± 0.14%) which was almost equal to SBT 12.21 ± 0.12% and lowest was observed in TiBT (7.91 ± 0.76%). In MHT, RT contain the highest (9.59 ± 0.64%) and MT show the lowest catechin content (4.43 ± 0.28%).
Total flavonoid content
The total flavonoid content was ranged from 1.81 ± 0.67 to 4.68 ± 0.26% in all the tea samples. In GHT flavonoid content was ranged from 2.24 ± 0.27 to 3.10 ± 0.52% and in BHT it was ranged from 1.81 ± 0.67 to 4.68 ± 0.26% (Table 1). In MHT flavonoid content was ranged from (1.82 ± 0.63–2.67 ± 0.34%). SGT showed the highest flavonoid content (4.68 ± 0.26%) compared to GT (2.86 ± 0.21%), while GBT showed almost similar and lower amount of flavonoids (1.81 ± 0.67–1.82 ± 0.63%). In MHT, TT contain the highest amount of total flavonoid content (2.67 ± 0.34%), while in MT, it was lowest (1.82 ± 0.63%).
HPLC analysis of catechins
Total catechins content was ranged from 8.10 to 22.69% in all the tea samples. The highest amount of catechins was determined in GT (22.69%) in GHT, while in BHT, TuBT showed the highest (20.84%) and in MHT, MT show the highest catechin content (11.12%). In GHT, EGCG (8.08 ± 0.25%) content was highest in GT and lower in SGT (4.18 ± 0.17%). While in BHT, BT contain high (7.24 ± 0.16%) and GBT (1.21 ± 0.17%) contain low amount of EGCG. In MHT, MT contain high amount 1.45 ± 0.34% and RT contain low EGCG 0.87 ± 0.12% Caffeine content was also found higher in GT (5.92 ± 0.15%) in GHT, while BT (5.81 ± 0.21%) contain the hight caffeine content in BHT. The variation in the catechin content was shown in the heatmap (Fig. 1A). From the heatmap, it was observed that the catechins content was higher in GHT compared to BHT and the least was present in MHT.
Amino acids analysis
Amino acid profile was studied in all the herbal teas using UPLC which shows the variations in their chemical constituents. A total of 14 amino acids were determined ranged from 0.82 to 2.86%. A higher amount of l-theanine was found in GT (1.24%) in GHT samples. Principal component analysis (PCA) was executed for the comparison and classification of amino acids in herbal tea samples (Fig. 1B). Amino acids content showed variations in all the herbal tea samples. By applying PCA, the two components were extracted which explained the variation of amino acid content in all the herbal tea samples. Figure 1B shows the score plot of herbal tea for the component 1 and 2. GT, SGT, GGT, TGT, BT, TuBT, LBT and KBT were observed on the positive side of component 1. Whereas at the upper side KGT, TGT, TuGT, LGT, KGT, MGT, SBT, GBT and TBT appeared on component 2. Amino acids such as l-theanine, histidine, glycine and phenylalanine show the positive side of components 1, which contribute to the tea quality. RT, MT and TT are correlated to the negative side of component 1. In case of component 2, asparagine, serine, histidine and l-theanine were present on the positive side which contributes to the tea quality. Amino acids content shows the variation in a different class of herbal tea, the highest amount was identified in GHT followed by BHT and the least was observed in MHT.
The antioxidant activity of herbal tea samples was studied using DPPH and ABTS assays (Table 1).
DPPH free radical-scavenging activity
The antioxidant activity of 21 teas was studied using DPPH free radical. Which was ranged from 27.58 ± 4.74 to 226.28 ± 2.72 µg/mL GAE. In the studied GHT, GT (27.58 ± 4.74 µg/mL) showed the highest, while GGT (128.36 ± 4.24 µg/mL) showed the lowest antioxidant activity. Some exception was observed in the SGT (28.86 ± 6.37 µg/mL) which shows approximately similar antioxidant activity to GT (Table 1). In BHT, DPPH free radical-scavenging activity was ranged from 39.3 ± 2.74 to 144.8 ± 3.46 µg/mL. While in MHT, TT show the highest 145.65 ± 2.85 µg/mL and RT show the lowest 226.28 ± 2.72 µg/mL antioxidant activity.
ABTS free radical-scavenging activity
Among all the tea samples, antioxidant activity was ranged from 14.17 ± 4.09 to 117.62 ± 7.59 µg/mL using ABTS free radical (Table 1). In GHT, ABTS free radical-scavenging activity was ranged from 14.17 ± 4.09 to 71.81 ± 4.00 µg/mL. While, in BHT it was ranged from 21.48 ± 1.65 to 74.17 ± 3.96 µg/mL and in MHT, it was ranged from 74.21 ± 5.46 to 76.71 ± 3.32 µg/mL. The efficacy of antioxidants mainly depends on the polyphenolic compounds present in tea infusions. The high antioxidant activity in GT is due to the presence of high content of polyphenols.
The correlation was observed in GHT, BHT and MHT between the TPC, TFC and antioxidant activity of DPPH and ABTS. The best correlation was observed in GHT between the DPPH and ABTS (R2 = 0.99) (Fig. 2). In addition, correlation observed between TPC and TFC in GHT, BHT and MHT is positive (R2 = 0.77, 0.89, 0.98), respectively. Moreover, the correlation between DPPH and ABTS was positive in GHT (R2 = 0.99), BHT (R2 = 0.98) and MHT (R2 = 0.96).
Cytotoxic activity by SRB assay
Cytotoxic potential of tea samples was tested on three different cell lines, such as SW480, A549 and SiHa. The results revealed that TGT showed promising activity against SW480 cells (50.9 ± 0.7 at 200 µg/mL). Herbal tea samples exhibited remarkable cytotoxic potential against A549 cells in the SGT (87.01 ± 1.1 at 200 µg/mL), respectively. Whereas, TT did not show a considerable effect on A549 cells. However, herbal tea samples exhibited the highest activity against SiHa cells in the LGT (67.1 ± 0.4 at 200 µg/mL), respectively. Therefore, the tested sample of herbal tea showed dose-dependent activity against all the cells, except RT and TT on SW480 cells.
Volatile organic compound analysis
Herbal tea samples with the total composition ranged from 62.03 to 96.23%. Qualitative and quantitative analysis on VOCs in herbal teas were carried out. The VOCs were extracted using Liken–Nickerson apparatus and analysed by GC and GC–MS. A total of nine different classes of compounds were identified and quantified in herbal tea samples viz, aldehydes, ketones, alcohols, nitrogenous compounds, hydrocarbons, acids, esters and other compounds. Among all the classes of VOCs alcohols and aldehydes were the most important class. A total of 188 VOCs were characterized, the most commonly identified compounds were 2-hexenal, l-linalool, hotrienol and α-terpineol. Some substantial variations in the volatile profile of GHT, BHT and MHT were observed. The BHT contained the commonly present compound viz, 2-hexenal, geraniol, 2-pentanol. The variation in the volatile profile could be due to the processing of tea shoots. The highest number of VOCs were identified in LGT (96.23%) and the lowest was observed in TBT (62.03%). The unique aroma compounds identified in LGT are octenal (0.19), trans-chrysanthemal (1.68), levoverbenone (8.57), pulegone (5.03), borneol (0.56), citral (0.92), carane (11.22), dl-limonene (2.05), α-terpinolene (0.39), cyclopentene (0.21), cineole (0.56) and 6-methyl-6 heptone-2-one (6.95). It was observed that the BHT contains a large number of VOCs compared to GHT and MHT. In addition, ZGT contains unique aroma compounds, such as cryptone (0.19), β-phellandrene (2.84), limonene (1.01) and β-ocimene (0.18). The least number of VOCs were identified in TGT and TBT. This could be due to the presence of very complex molecules in taxus plant which cannot be detected, contain unique VOCs, such as 2-pentadecanone (0.31), neophytadiene (1.08), heptadecyl trifluoroacetate (1.93), di (2-ethylhexyl) adipate (0.33) and O,O′-biphenol, 4,4′-difluoro (0.96). MGT comprised of (E)-p-2,8-menthadien-1-ol (0.29), β-terpineol (0.96), menthol (9.69), thymol (0.36), 1-octadecene (0.23), methyl acetate (0.68), 2,5-dimethyl tetrahydrofuran (0.29), cis-2-cyclohexene-1-ol-1-methyl-4 (1-methylethyl) (1.52) and menthacamphor (5.81). The unique VOCs with sweet taste were observed in LBT, MBT, TuBT, LGT, MGT and TuGT. Therefore, these teas can be used as beverages with unique VOCs.
Aroma extract dilution analysis (AEDA)
Volatile organic compounds have a great influence on the flavour quality even when present in small amount which are due to their low threshold value. The odour threshold play an significant role for the determination of VOCs. These VOCs are present in very small amount and show a great influence on the quality.
A total of 90 odour active compounds were identified using AEDA in herbal tea samples (Tables 2, 3, 4). The commonly identified class of compounds are aldehydes and alcohols. AEDA was performed and compared between GHT, BHT and MHT which shows the variations in their volatile profile. cis-Linalool oxide, 1-dodecene, methyl salicylate and epoxylinalool show the sweet floral note in GT. Geranial (fresh, lemon-like), geranylacetone (faint woody floral), cis-linalool oxide (fruity), trans-linalool oxide (fruity and fresh), l-linalool (floral, fruity) and nerolidol (floral green) are the odour active compounds were identified in BT. While, LGT show the neral (lemon-like), geranial (fresh, lemon-like), levoverbenone (spicy, mint, camphor), pulegone (peppermint camphor fresh herbal), trans-linalool oxide (fruity and fresh), l-linalool (floral, fruity) and cineole (minty) odour active compounds. The most important aroma contributors present in MBT is the 2,4-heptadienal (green fruity), levoverbenone (spicy, mint, camphor), gernylacetone (faint woody floral), cineole (minty), menthol (minty) and citronella (floral, citrus). It was noticed that herbal tea samples such as LGT, MGT, TuGT, RT, GT and BT produce floral, fruity, green and sweet floral note. Fatty and roasted note were highly observed in TGT.
Sensory analysis was performed by the tea tasting panel. Six sensory terms were taken, including leaf appearance, infusion colour, taste, aroma, infused leaves and total score (Table 5). Sensory analysis was performed for all the herbal tea samples. The colour palate of the infusion of GHT showed a light brown colour with a greenish tint. This may be likely due to the presence of catechins which was extracted into tea infusions with a light green–brown colour. TQS for aroma, infusion colour, appearance, taste and infused leaves were observed by the tea-tasting panel were observed from 37 to 87 and averaged 65.85 (Table 5). The highest TQS was found in LGT (87) followed by TuGT (86) then TiGT (85) and least were observed in TBT (37). LGT infusion shows the sensory analysis for leaf appearance (15), infusion colour (8), taste (28), aroma (29) and infused leaves (7). Tasters showed blended flavours, with unique mouthfeel and sweet aftertaste for LGT, MGT and TuGT (Fig. 3). The astringency and bitter taste were observed in a large number of teas, which include GT. This is due to the presence of catechins in green tea and theaflavins in black tea. The bitter and astringent attributes are decreased in the herbal teas; therefore, these herbal teas are accepted by the consumer with better aroma and taste. The aromatic compounds of LGT, MGT, TuGT and TiGT dominate the characteristic compounds of GT and BT. The mild flavour was observed in the KGT, GGT, SBT, GBT and KBT. Herbal tea is accepted by all the tea tasting panels showing a unique and sweet taste.
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