Comparative studies on the antioxidant, anticancer and anti-inflammatory activities of green tea, orthodox black tea and CTC black tea

Abstract

Tea is a natural dietary supplement rich in polyphenols and based on the manufacturing process, their polyphenol content also varies. In the present study, we have compared the in vitro antioxidant, anticancer and anti-inflammatory activities of green tea (GT), orthodox black tea (oBT) and CTC black tea (cBT). The analysis was carried out in 50:50 ethanol:water extracts. The total antioxidant capacity, total polyphenol content and free radical scavenging activity were found to be high in GT samples. HPLC profiling indicated a higher percentage of polyphenols like catechin, epicatechin, epigallocatechin and epigallocatechin-gallate in GT when compared to other samples. The comparison of the anticancer potential was done in breast cancer MDA MB-231 cells and it was found that GT has a higher percentage of cell growth inhibition than oBT and cBT. Anti-inflammatory effects were done in LPS stimulated RAW264.7 macrophage cells and here also GT showed maximum effects. This was confirmed by the lower production of iNOS, reduced level of ROS generation and proinflammatory cytokines such as MCP-1, IL-1ɑ, and IL-6 by GT. To conclude, the order for the biological effectiveness of different teas tested is in the order GT > oBT > cBT.

Introduction

Tea is the most prevalent and second most consumed beverage in the world. The freshly collected tea leaves, which are either hand-picked or mechanically picked, are typically utilized to make tea. Tea is typically split into three fundamental categories based on the various processing methods, especially the level of fermentation: green tea (non-fermented), oolong tea (semi-fermented), and black tea (fully fermented) (LIczbiński and Bukowska 2022). The process of fermentation involves the enzymatic oxidation of tea polyphenols, specifically colourless flavonols, which are then partially transformed into theaflavins and thearubigins, which give black and oolong teas their distinctive flavor and colour (Ho et al. 2018). The traditional rollers (orthodox) or machines (CTC: crush-tear-curl) are used to rupture the withered tea leaves, speeding up the oxidation process by releasing enzymes that react more quickly with oxygen. Due to the intense maceration processing, catechin oxidation and theaflavin production are more pronounced and quicker in CTC black teas. Due to reduced structural damage to rolled tea leaves, theaflavin formation is predicted to be lower in black orthodox teas. In unfermented teas, the endogenous enzymes are inactivated by steaming or heating using various techniques (pan frying, roasting, baking), which is done before rolling and drying. This prevents the fermentation of the withering leaves (Wong et al. 2022). Quality of tea depends on their polyphenol content which further depends on the plucking season, shade, growth altitude, soil, and climate. Metabolomic analysis using NMR, GC–MS, LC–MS etc. can be an effective tool in assessing the quality of tea products (Farag et al. 2023).

Research interest in tea has increased recently because of its possible health advantages for humans. Many published research attribute green tea’s protective qualities against several diseases due to its polyphenol content (Kochman et al. 2020). It has been demonstrated that oxidative damage is linked to a wide variety of clinical disorders. Tea polyphenols are strong antioxidants that can both treat and prevent diseases by scavenging free radicals and regulating the activity of different oxidases in the body (Zuo et al. 2018). In vivo investigations and numerous clinical trials have also revealed the antioxidant activity of tea polyphenols (Truong and Jeong 2021). Gut microbiota plays a major role in human health and food derived phenolics such as flavonoids, hydroxybenzoic acids etc. by producing functional metabolites that contribute to gut health. (Loo et al. 2020, Xiong et al. 2023). An increasing body of research from cellular, animal, clinical, and epidemiological studies has connected drinking tea with several health advantages, including the chemoprevention of cancers, diabetes, chronic inflammation, heart and liver diseases, neurodegenerative diseases etc. (Tomata et al. 2016; Cai et al. 2018). Green tea polyphenols exert spectrum of health benefits by interacting with the gut microbiota in experimental animals (Wang et al. 2018).

Tea leaves are processed differently to make green tea, CTC black tea, and orthodox black tea and it would be interesting to know which tea has the potential to be more advantageous in terms of its polyphenol content and their biological activity. In light of this, the purpose of the current study is to assess how differently tea processing affects the polyphenol content and specific biological properties such as antioxidant, anticancer and anti-inflammatory properties.

Materials and methods

Materials

Green tea (GT) BOPD grade, CTC black tea (cBT), SFD grade and orthodox black tea (oBT) GBOP grade, samples were collected from the tea factories located at top station area of Kanan Devan Hills Plantations Company Pvt. Ltd, Munnar, Kerala. Top station area of Munnar is the one of the highest tea growing regions in the world which is located in the mountain scopes of Western Ghat and lies at an altitude of 1800–2300 m (5900–7500 ft) from the sea level. The temperature in Munnar ranges between 5 and 25 °C (41–77 °F) in winter and 15–27 °C (59–80 °F) in summer. The above said geographical features and climatic conditions imparts specific quality attributes to the tea grown from Munnar region.

Reagents

The chemicals including Folin ciocalteu reagent, Na₂CO₃, Ascorbic acid, Disodium phosphate, Ammonium molybdate, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), Tris HCL, Nitro blue tetrazolium (NBT), Ferrozine, EDTA, Sodium diphosphate, Sodium monophosphate, Potassium ferricyanide were purchased from Sisco Research Laboratories Pvt. Ltd (SRL), India. Methanol, Trichloroacetic acid (TCA) were purchased from Merck Life Science Private Limited, Mumbai, India. Gallic acid, Phenazine methosulfate (PMS) Griess reagent was purchased from Sigma-Aldrich. Ferric chloride (FeCl₃·4H₂0), Dulbecco’s Modified Eagle Medium (DMEM), Fetal bovine serum (FBS), Roswell Park Memorial Institute (RPMI)-1640 media were purchased from Himedia, India. Potassium persulfate was purchased from S D Fine Chem. Ltd. NADH was purchased from Spectrochem Pvt. Ltd. Fecl₂ was purchased from Fluka.

Preparation of tea extracts

Tea extracts were extracted by using 50:50 ethanol and water as a solvent. In brief, 5 g of tea samples were taken in a conical flask and the extraction was carried using 100 ml of solvent for 24 h in a shaker at 100 rpm. After 24 h, the filtrate was collected and the extraction process continued for another 2 times. The filtrate collected were pooled and the excess solvent was evaporated using rotavapor and the lyophilized samples kept at 4 °C were used for different activity studies.

Determination of total phenolic content (TPC)

Total phenolic content was determined by using the Folin-Ciocalteu method (Aremu et al. 2019). 500 μL of the samples with different concentrations (125–500 μg/mL) and 2.5 ml Folin-Ciocalteu reagent (1:10 dilution with distilled water) were added and incubated for 6 min at room temperature. Then, 2 mL of 7.5% of Na₂CO₃ was added to the reaction mixture and incubated in the dark at room temperature for 30 min. After incubation, the blue colour developed was measured at 760 nm using a UV–visible spectrophotometer against the corresponding blank. Gallic acid was used as standard with different concentrations (5–25 μg/mL) and the total phenolic content in the extracts were estimated as mg GAE/g tea extract.

Determination of total antioxidant activity (TAC)

The total antioxidant activity was estimated by phosphomolybdenum assay (Aremu et al. 2019). This assay is based on the reduction of Mo(VI) to Mo(V) by the extract and subsequent formation of Green phosphate and Mo(V) complex at acidic pH. 100 μL of the sample with different concentrations (125–500 μg/mL), 1 mL of molybdate reagent (0.6 M H₂SO₄, 28 mM disodium phosphate, 4 mM ammonium molybdate) were added and incubated in 95 °C water bath for 90 min. After cooling, the absorbance was measured at 695 nm by using a multimode plate reader. Ascorbic acid was used as standard with different concentrations (25–125 μg/mL) and the total antioxidant content was expressed as mg AAE/g tea extract.

DPPH radical scavenging activity

The free radical scavenging activity of the extract was determined using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) method with some modifications (Aremu et al. 2019). DPPH working solution as prepared from the stock solution (100 μM) by diluting with methanol to generate an absorbance of 0.9–1.0 at 517 nm. 200 μL reaction mixture contained 100 μL of DPPH and 100 μL of extract at various concentrations (12.5–75 μg/mL). In control, 50% ethanol water was used instead of the sample. Antioxidants present in the sample reduced the DPPH and the resulting decrease in absorbance at 517 nm was recorded using a multimode reader. Gallic acid at various concentrations (10–20 μg/mL) was used as the standard.

$$\text{DPPH}\text{scavenging}\text{activity}=\left[\left({\text{A}}_0-{\text{A}}_1\right)/{\text{A}}_0\times 100\right]$$

where A₀ is the absorbance of the control and A₁ is the absorbance in the presence of extract/ standard.

DPPH scavenging activity was expressed as IC₅₀, defined as the amount of antioxidant material required to scavenge 50% of free radical in the assay system and it was plotted against the corresponding concentration of the extract to obtain this value.

ABTS radical scavenging activity

ABTS scavenging activity was evaluated by using a reference method with slight modification (Aremu et al. 2019). ABTS is oxidized to ABTS⁺ (coloured radical cation) in the presence of potassium persulphate. The scavenging of ABTS⁺ by antioxidants reduces the dark green colour to colourless and the absorbance was read at 734 nm. ABTS stock solution was prepared from 7 mM ABTS and 2.45 mM potassium persulfate in a volume ratio of 1:1 and then was incubated in the dark at room temperature for 12 h. Before use, the ABTS solution was diluted to get an absorbance of 0.675–0.725 at 734 nm. Sample was taken at different concentrations (25–125 μg/mL). 200 μL reaction mixture contained 150 μL of ABTS and 50 μL of extract. In control, 50% ethanol water was used instead of the sample. After 6 min of incubation at room temperature, the absorbance was read at 734 nm. Ascorbic acid was used as standard.

$$\text{ABTS}\text{scavenging}\text{activity}=\left[\left({\text{A}}_0-{\text{A}}_1\right)/{\text{A}}_0\times 100\right]$$

where A₀ is the absorbance of the control and A₁ is the absorbance in the presence of extract/ standard.

ABTS scavenging activity was also expressed as IC₅₀ value.

Superoxide radical scavenging activity

The scavenging activity of superoxide anion was determined by using a reference method with slight modifications (Hazra et al. 2020). Various concentrations of (100–300 μg/mL) of tea extracts and the standard, ascorbic acid (25–100 μg/mL) were taken and made upto 1 mL using Tris HCL buffer (100 mM) and to this was added 250 μL NBT (300 μM), 250μL NADH (936 μM) and 250 μL PMS (120 μM). In control, Tris HCL buffer was used in place of tea extracts. The addition of PMS induced the reaction to undergo NADH oxidation, producing superoxide radicals that decrease NBT. The absorbance was determined at 560 nm after 5 min of room temperature incubation.

$$\text{Superoxide}\text{scavenging}\text{activity}=\left[\left({\text{A}}_0-{\text{A}}_1\right)/{\text{A}}_0\times 100\right]$$

where A₀ is the absorbance of the control and A₁ is the absorbance in the presence of extract/ standard.

Superoxide scavenging activity was also expressed as IC₅₀ value.

HPLC analysis

Chromatography study of tea extracts were performed on Nexera X2 series (Shimadzu, Japan) HPLC–DAD system. The samples were prepared in 70:30 methanol–water solvent (1 mg/ml), sonicated, and filtered through a 0.22 μm Nylon filter, and, 10 μl of sample was injected using UHPLC system equipped with autosampler injector (SIL-30AC HT) and then separation happened on reverse phase, Shim pack GISS 5 μm C18 column (250 × 4.6 mm Shimadzu) kept in a column oven (CTO-20AC) maintained at 33 °C. A binary solvent system in gradient mode where mobile phase A is 100% Acetonitrile and mobile phase B is 0.1% TFA in HPLC grade water (Merck) was used as presented in Table 1 with a run time of 60 min. The sample absorbance were automaticaly detected by a diode array detector (SPD-M20A) at 280 nm. Gradient solvent system for HPLC analysis is as shown below.

Time/Minute	Solvent A (%)	Solvent B (%)
0–15	85	15
16–20	50	50
21–35	30	70
36–50	0	100

Sample components were identified by matching retention time and spectra of standards. External standards were used for the identificatoion and quantification of components in the samles. Results are expressed as mg/g of extract. Standards of six concentration (0.1–1.0 mg/ml) were prepared in methanol and calibration curve was prepared using concentration versus peak area. Slope of the regression line and corelation coefficient for indivisual standard were plotted by using MS Excel software.

Cytotoxic studies in breast cancer cells by MTT assay

The breast cancer MDA MB-231 cells was used to evaluate the cytotoxicity of the tea extracts. In brief, MDA MB-231 (1 × 10⁴ cells/well) and RAW 264.7 (5 × 10³ cells/well) cells were seeded in 96 well plates and cultured in DMEM and RPMI-1640 media respectively supplemented with 10% FBS and incubated for 24 h in a CO₂ incubator. Then cells were treated with extracts of concentrations and cultured for another 24 h. After the incubation, the medium containing extracts were removed and cells were supplemented with 100 μL of MTT (5 μg/mL) was mixed with each well and again incubated for 4 h at 37 °C. The media was discarded and 100 μL of DMSO was added to dissolve the formazan precipitate. Cells without treatment were used as control. Plate was read at an absorbance of 570 nm (Taniya et al. 2020). The percentage cytotoxicity/viability was calculated using the formula,

$$\%\text{cytotoxicity}/\text{viability}=\left[\left({\text{A}}_0-{\text{A}}_1\right)/{\text{A}}_0\times 100\right].$$

where A₀ is the absorbance of the control and A₁ is the absorbance in the presence of extract/ standard. For anticancer studies paclitaxel was used as the standard.

Anti-inflammatory screening in RAW264.7 cells

RAW264.7 cells were seeded in 96 well plates with a density of 5 × 10³ and cultured for 24 h. The cytotoxic effects of tea extracts were studied by MTT assay with different concentrations, 25 μg/mL, 50 μg/mL, 100 μg/mL and 200 μg/mL. The anti-inflammatory activity of the extracts were screened in LPS stimulated RAW264.7 cells. For this, tea extracts at two different concentrations (20 μg/mL and 40 μg/mL) and the standard drug dexamethasone (8 μg/mL) were pre-treated for 4 h. Then, LPS (1 μg/mL) was added to the culture for stimulation and incubated again at 37 °C in a CO₂ incubator for 24 h. The spent medium from the culture was collected and 50 μL of media was taken to a new 96 well plates. 50 μL of Griess reagent was added to each well and incubated for 20 min in dark at room temperature and measured absorbance at 540 nm (Hamsalakshmi et al. 2021). Sodium nitrite is used as a standard for measuring the NO released.

Determination of ROS production

The oxidant-sensitive fluorescent probe, DCFDA was used to measure the intracellular ROS levels. In brief, RAW264.7 cells were seeded at a density of 5 × 10³ cells/well in black 96 well plates and kept for 24 h. After 24 h incubation, tea extracts were treated with a concentration of 40 μg/ml and kept in a CO₂ incubator for 4 h. Then, LPS was added and incubate again at 37 °C in a CO₂ incubator for 24 h. After that, the medium was taken out followed by washing twice with 1X PBS. After PBS wash, 100 μl of DCFDA working solution (20 μM) was placed in each well and incubated in dark at 37 °C in a CO₂ incubator for 20 min. The stain was removed and the wells were gently washed twice with 1 × PBS. Then, cells are layered with 100 μl of 1 × PBS and visualized using a fluorescent microscope (FITC filter) (Tian et al. 2021).

ELISA

The levels of inflammatory cytokines were evaluated by indirect ELISA. Briefly, after the treatment, the spent culture media (100 μL per well) from control and different treatment were added to an ELISA plate and incubated at 4 °C overnight. Media was discarded and wells were washed one time with 1X PBST (1X PBS, 0.05% Tween-20). Then, 100 μl of blocking buffer (1X PBS, 0.5% Tween-20, 5% BSA) was added and again incubated for 45 min at 37 °C. After incubation, discarded the blocking buffer followed by three times wash with PBST. 100 μl of primary antibody (1:1000) against different cytokines were added and incubated for 2 h at 4 °C. After three PBST washes, the plates were treated with anti-rabbit secondary antibody (1:2000) for 2 h. After wash, 100μL of TMB substrate solution was added to each well and incubated for 15 min. 50% sulphuric acid was used to terminate the reaction by incubating for 5 min and the plates were read 490 nm.

Statistical analysis

Results are expressed as global mean ± standard deviation (SD) of three independent experiments containing technical triplicates per group. Data were analysed by one-way ANOVA using IBM SPSS 20.0 software (IBM, Armonk, NY, USA), and the differences were considered statistically significant at p ≤ 0.05.

Results and discussion

Many plants contain polyphenols and their extracts are used to make an overwhelming beverages and tea is one of them. The high polyphenol content in tea and the ease of high-dose consumption may help to deliver considerable amounts of antioxidants to the body. Tea polyphenols can reduce the risk of a diverse range of human disorders. Based on the manufacturing process, there are different types of tea in market and depending on the type their polyphenol content also varies. The present study aimed to compare the total phenolic content (TPC), antioxidant capacities, anticancer and anti-inflammatory activities of tea processed under different conditions ie. CTC black tea (cBT), orthodox black tea (oBT) and green tea extracts (GT). For this we have prepared the 50:50 ethanol water extracts of tea samples and percentage yield of extracts was found to be 29% for cBT, 23% for oBT and 34% for GT.

Total polyphenolic content as well as the antioxidant activity of the extracts were calculated as described in the methods. The total polyphenolic content of GT was found to be high (314.23 ± 8.51 mg GAE/g tea extract) followed by oBT (301.94 ± 20.69 mg GAE/g tea extract) and cBT (191.62 ± 7.74 mg GAE/g tea extract). Similarly, GT extract was having higher antioxidant capacity (729 ± 71.63 mg AAE/g tea extract) when compared to oBT (392.29 ± 83.58 mg AAE/g tea extract) and cBT (343.79 ± 60.53 mg AAE/g tea extract). The antioxidant activity of extracts can also be measured on their ability to scavenge free radicals. Previous literature also revealed that methanolic extracts of green tea have more polyphenol content (46.9 ± 3.404 mg of GAE/g extract) when compared to black tea (9.3 ± 0.818 mg of GAE/g extract) (Korkmaz et al. 2019). The tea samples we have tested contains higher amounts of polyphenol content than the previously reported samples. The geographical and climatic conditions of Munnar may have positive effects on the increased polyphenol content of the teas grown in this area. The reason that green tea has more polyphenols than black tea may be due to the fermentation process which transforms the polyphenols in black tea into theaflavins and thearubigins (Teshome 2019). According to earlier research by Dutta et al., the total antioxidant capacity and polyphenol content exhibited a high association, indicating that polyphenols have antioxidant properties that protect against oxidative stress (Dutta et al. 2013).

The free radical scavenging ability (measured in terms of DPPH and ABTS method) and superoxide radical scavenging activity of the tea extracts was found to be greater for GT when compared to oBT and cBT samples. In the present study, GT had an IC₅₀ value of 16.30 ± 0.36 μg/ml by DPPH assay followed by oBT (18.45 ± 0.27 μg/ml) and then cBT (21.29 ± 0.25 μg/ml). Previous report also indicated the lower IC₅₀ value for green tea methanol extract when compared to black tea methanol extract in terms for the free radical scavenging effects by DPPH method (Korkmaz et al. 2019). ABTS radical scavenging activity demonstrated that the GT extract showed and IC₅₀ values of 32.40 ± 0.20 μg/ml followed by oBT (54.24 ± 0.25 μg/ml) and cBT (99.25 ± 0.19 μg/ml). Another literature study indicated an IC₅₀ value of 14.17 ± 4.09 μg/ml for green tea and 21.48 ± 1.65 μg/ml for black tea for the ABTS free radical scavenging activity (Kumari and Kumar 2022). Superoxide radical was found to be a precursor of all other reactive radicals. We have also assessed the superoxide radical scavenging capabilities of the extracts and the IC₅₀ values for GT, oBT and cBT were observed to be 133.71 ± 0.12 μg/ml, 240.43 ± 0.39 μg/ml, and 278.52 ± 0.09 μg/ml respectively. A previous report indicated that ethanolic extract of green tea has more superoxide radical scavenging than black tea. They expressed their values in percentage inhibition, for green tea it was 90.68 ± 3.20 μg/ml, and for black tea 76.95 ± 8.20 μg/ml (Hu et al. 2020). For our tested extracts, GT have the maximum capacity to scavenge free radicals as indicted by the lower IC₅₀ values when compared to oBT and cBT. Table 1 gives a summary of the results of antioxidant, polyphenol content and free radical scavenging activity of different tea extracts tested along with values of their respective standards used.

Table 1.

Radical scavenging activity, total phenolic content and antioxidant capacity of different tea extracts

Sample/standard	Free radical scavenging activity (DPPH method) IC50 (µg/ml)	Free radical scavenging activity (ABTS method) IC50 (µg/ml)	Superoxide radical scavenging activity IC50 (µg/ml)	Total Polyphenol content (mg GAE/g tea extract)	Total antioxidant Capacity (mg GAE/g tea extract)
cBT	21.29 ± 0.25	99.25 ± 0.19	278.52 ± 0.09	191.62 ± 7.74	343.79 ± 60.53
oBT	18.45 ± 0.27	54.24 ± 0.25	240.43 ± 0.39	301.94 ± 20.69	392.29 ± 83.58
GT	16.30 ± 0.36	32.40 ± 0.20	133.71 ± 0.12	314.23 ± 8.51	729 ± 71.63
Gallic acid	9.09 ± 0.09	–	–	–	–
Ascorbic acid	–	27.24 ± 0.15	88.46 ± 0.48	–	–

Values given are the representatives of the means ± standard deviation of measurements made in experimental triplicates

HPLC analysis enables the proper separation and identification of polyphenols and caffeine in tea samples. The gradient binary solvent system with TFA (trifluroacetic acid) helps in proper resolution. Further, in order to characterize the extracts in terms of their polyphenols and caffeine contents, HPLC analysis was carried out using six polyphenols standards ie. epigallocatechin (EGC), catechin, epicatechin (EC), epigallocatechin gallate (EGCG), theaflavin and theaflavin-3 gallate and caffeine standard present in tea. Among all, GT contains more amount of EGCG content which is the predominant polyphenol found in green tea. Very less amount of EGCG is found in both oBT and cBT. This may be due to the oxidation of catechin during processing. The HPLC chromatogram was recorded at 280 nm and the amounts of individual polyphenols identified by HPLC in different tea extracts are summarised in Fig. 1. According to a previous study, green tea contains more amount of catechin and caffeine content than black tea which correlates with our study results (Nurul and Uthumporn 2015). Koch and colleagues showed that EGCG and EGC were the two main catechins in several green tea infusions from various production locations, but they obtained a higher concentration of EGC rather than EGCG in the green tea under investigation (Koch et al. 2018). But our present study implies that EGCG is a derivative of catechin, existing predominantly in green tea extracts. Integrated metabolomic and transcriptomic approaches are now used for revealing the key metabolites responsible for the quality of instant and flavoured tea (Jin et al. 2023, Zou et al. 2024).

Fig. 1.

Chromatogram showing the presence of individual polyphenols in the tea extracts. A–D represent the chromatogram of standards, cBT, oBT, and GT respectively. Table represent the quantitative analysis data of different polyphenols in the tea extracts. Values represent the means ± standard deviation of measurements made in triplicates. EGC- epigallocatechin, Catechin, EC- epicatechin, EGCG- epigallocatechin gallate, nd- not detected

To assess the anticancer potential of tea extracts, an in vitro cell line based cytotoxicity testing was carried out. Different concentrations of GT, oBT and cBT (50, 100 and 200 μg/ml) were treated to the breast cancer MDA-MB-231 cells for 24 h and cytotoxicity was measured by MTT assay as described in methods. The results given in Fig. 2A indicated that, upon increasing the concentrations of tea extracts more cytotoxicity was observed. At 50 μg/ml, there observed no effects, but at 100 μg/ml concentration, 50% of cells were found to be dead and the effect was more prominent in cells treated with GT. The morphological changes are shown in Fig. 2B which also indicated significant rounding up and detachment of cells due to cell death upon treatment. According to previous study, EGCG is the main compound in green tea polyphenol, successfully promoted apoptosis in T47D breast cancer cells by up-regulating pro-apoptotic genes like p53, p21, caspase3, caspase9, Bax, and PTEN and down-regulating survival genes like PI3K, AKT, and Bcl-2 (Moradzadeh et al. 2017). Theaflavins were reported to upregulate Fas expression in human breast cancer cells with mutant p53 by activating c-jun N terminal kinase while simultaneously downregulating the pAkt/p A Bad cell survival mechanism that results in apoptosis, increased cytochrome c release, and loss of mitochondrial transmembrane potential (Lahiry et al. 2010). Moreover, there is no study has been conducted for comparing the antibreast cancer potential of orthodox black tea, CTC black tea and green tea.

Fig. 2.

A MTT assay showing the percentage of cytotoxicity in MDA-MB-231 cells. Values given are the representatives of the means ± standard deviation of measurements made in experimental triplicates. B Phase contrast images showing the morphology of MDA-MB-231 cells treated with different concentrations of GT, oBT and cBT. Magnification-10 X

The comparison of the anti-inflammatory activities of GT, oBT and cBT were done in RAW 264.7 cells. In order to determine the effective concentrations for the study, a primary cytotoxicity study was done employing MTT assay by treating the cells with different concentrations (25 μg/ml, 50 μg/ml, 100 μg/ml, and 200 μg/ml) of the extracts. Up to 100 μg/ml, the extracts were found to safe and we have selected concentrations less than 100 μg/ml for the further anti-inflammatory evaluations. The results of MTT assay and the phase contrast images of the cells are shown in Fig. 3A, 3B respectively. According to previous reports, NO generation is a mediator of the inflammatory reactions caused by LPS in macrophages (Malayil et al. 2022). Proinflammatory cytokines can be produced in response to high levels of the inflammatory mediator NO, and vice versa. This creates a positive feedback loop that increases inflammation (Zhang et al. 2020). Here we have studied the anti-inflammatory effects of GT, oBT and cBT by means of production of NO, ROS and inflammatory cytokines in LPS induced RAW macrophage cells. The anti-inflammatory activities of GT, oBT and cBT were estimated by the ability to reduce the production of inducible notric oxide (iNOS) produced and the results are given in Fig. 3C. Theaflavine (TF) is a characteristic bioactive compound in fermented tea which helps in the activation of NRF-2 pathway, thereby reducing the liver inflammation (Shu et al. 2023). Theanine and γ-aminobutyric acid contents in the fermented tea suppressed the expression of inflammatory cytokines, as well as inhibited overexpression of apoptotic signals for the neuroprotective effects thorugh regulation of MAPK signaling (Lee et al. 2023).

Fig. 3.

A MTT assay showing the percentage of cell viability of tea extracts in RAW 264.7 cells. Values given are the representatives of the means ± standard deviation of measurements made in experimental triplicates. B Phase contrast images showing the morphology of RAW cells treated with different concentrations of GT, oBT and cBT. No significant toxicity observed till 100 μg/ml. Magnification-10X. C Amount of NO produced (μM) at different concentrations of extracts in LPS stimulated RAW cells. Significant downregulation of NO production was observed for oBT at high concentration and GT at both the concentrations tested similar to that of the positive control dexamethasone (DXM). Values given are the representatives of the means ± standard deviation of measurements made in experimental triplicates. *indicates the significant difference from the LPS treatment (p ≤ 0.05)

Intracellular ROS generation in RAW 264.7 as a result of LPS and their possible amelioration by the tea extracts were analysed by DCFDA staining and the results are given in Fig. 4A. The results indicated that GT is having the maximum capacity to reduce the ROS generation followed by oBT and cBT. Research reports are also there regarding the gut microbiome modulating effects of green tea polyphenols that may also indirectly connected to their anti-inflammatory potential (Chen et al. 2019, Choi et al. 2023). The fermented tea produced by mixing symbiotic bacteria and yeast (Kombucha tea) have increased immunomodulatory properties and gut microbiome protection (Fu et al. 2017). We have analysed the production of pro-inflammatory cytokines like MCP-1, IL-1α and IL-6 in the cells stimulated with LPS and treated with tea extracts and the results are given in Fig. 4B. In our study, we found that green tea has the highest potential to reduce NO production and proinflammatory cytokines such as MCP-1, IL-6, and IL-1α. Previous studies also indicated that the tea polyphenol EGCG induced anti-inflammatory activity by reducing nitric oxide production and by suppressing IL-6, COX-2, TNF-α, and IL-1β (Novilla et al. 2017). Till now, no reports are there on the comparative anti-inflammatory activities of green tea, orthodox black and CTC black tea collected from the same geographical locations.

Fig. 4.

A Inhibition of LPS-induced ROS production by tea extracts in RAW 264.7 cells. Images given are the representative images of experiments conducted in triplicates. Magnification-20X. B Indirect ELISA showing the activity of pro-inflammatory cytokines MCP-1, IL-1α and IL-6 in LPS stimulated RAW cells pretreated with 40 μg/ml tea extracts. Values given are the representatives of the means ± standard deviation of measurements made in experimental triplicates

Summary and conclusion

Tea is a natural beverage with less side effects. Our study clearly demonstrated the differences in the polyphenol content, antioxidant, anticancer and anti-inflammatory activities of green tea, orthodox black tea and CTC black tea. The differences in the activities are due to the difference in the polyphenol content retained in the tea after different manufacturing processes. Among different types of tea, green tea has the most predominant activities and will be the best option to take to reduce the oxidative stress and boost the immune potential of the body. If prefer black tea, it will be better to consume orthodox black tea instead of CTC black tea for more health beneficial polyphenols.

Author’s contributions

Arpan Singha Deo and Asheela Devi PJ did the extraction, antioxidant and anti-inflammatory studies and made the first draft of the paper. Anusha R did the anticancer studies. Sijisha KS and Tripti Mishra were involved in the HPLC analysis of the extracts. Siby Mathew, K Mathew Abraham and Jagadish R were involved in the sample collection from the industry, necessary documentation and correction of the manuscript. Priya S took the overall responsibility of conceptualization of idea, experimental planning and monitoring, fund generation, final correction and editing of the paper. All the authors have read and approved the final manuscript.

Funding

Authors thank Council of Scientific and Industrial Research (CSIR), India for the financial support for the project (Immunity mission project HCP-35).

Availability of data and material

Data will be available on request.

Code availability

Not applicable.

Declarations

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