Antioxidant potential of biotransformed green tea catechin metabolites and their impact on peripheral blood mononuclear cells

2.1 Chemicals

Catechin (Tea polyphenol), ABTS: 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), DPPH; 1,1-diphenyl-2-picrylhydrazyl. Ascorbic acid, potassium persulphate, and Trolox; (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid were purchased from Sigma Aldrich (St. Louis, Mo., USA). Histopaque-1077 (Sigma Aldrich) and RPMI-1640 medium (gibco, life technologies, USA), Obtained Ultra purity water was from Milli-Q system (Millipore Laboratory Bedford, MA). All other chemicals and reagents used were of analytical grade.

2.2 Biotransformed catechin metabolites

In our previous research, we performed In vitro catechin biotransformation. The biotransformed crude (Cr) catechin metabolites were collected and further partially purified by preparative thin-layer chromatography and analyzed by Gas chromatography-mass spectrometry and High-performance liquid chromatography-mass spectrometry (Krishnamoorthy et al., 2019). We obtained two sets of partially purified metabolites (PPM); PPMA consists of dehydroquinic acid, 4-ethylphenol, 4-methoxyphenyl propan-2-ol, 3-phenyl propionic acid, 2-phenoxyethanol, benzene tricarboxylic acid,1,2-dimethyl ester, catechol-1,4-benzenediol, benzene-1,3,5-tris1-methylpropyl, 3,5,7-trihydroxy-2H-chromen-2-one, and 4-hydroxyphenylpropionic acid. PPMB consist dimethoxycinnamic acid.

2.3 DPPH radical scavenging assay

The catechin biotransformed metabolites' effect on DPPH radicals was evaluated as previously reported with some modifications (Kumari et al., 2016). Briefly, the reaction mixture was prepared by adding DPPH (0.1 mM) in 3.9 mL methanol and 0.1 mL of different concentration (5, 10, 20, 40 and 80 μg/mL) of biotransformed metabolites. The mixture was vortexed and then incubated in the darkroom at room temperature for 30 mins, and after incubation, absorbance was recorded at 517 nm. All the assays were performed in triplicates, for standard Ascorbic acid and Trolox were used. The percentage of DPPH scavenging activity of tested samples and standard was calculated using the bellow equation. $$\text{Inhibition}\left(\%\right)=\left[\left({\text{A}}_0-{\text{A}}_{\text{s}}\right)/{\text{A}}_0\right]\times 100$$

A0 - absorbance of the control, As - absorbance of the test samples.

The IC50 (required concentration to scavenge 50% free radicals) for of the test samples and the standard was estimated using CalcuSyn1 software.

2.4 ABTS⁺ radical cation decolorization assay

The ABTS (2,2′- azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) diammoniumsalt) radical cation scavenging ability was analysed as previously described with some modifications (Re et al., 1999). Briefly, the radical solution was prepared by mixing an equal quantity of 7 mM ABTS aqueous solutions and 2.45 mM potassium persulfate solution. This mixture was incubated 16 h in dark conditions at room temperature. The assay was carried out with freshly prepared working solutions by mixing 3 mL of ABTS⁺ solution with 0.2 mL of different concentrations of pure and partially purified catechin biotransformed metabolites. After 6 min, the absorbance was measured at 734 nm. All the assays were performed in triplicates. Inhibition (%) = ODc-ODs/ODc × 100

ODc – absorbance of the control, ODs- absorbance of the samples.

2.5 Hydrogen peroxide (H₂O₂) radical scavenging activity

The method, as previously described, determined the hydrogen peroxide radical scavenging activity of the catechin and partially purified biotransformed metabolites (Sarma et al., 2016). Briefly, the reaction mixture was prepared by mixing different concentrations of pure and partially purified catechin biotransformed metabolites and 0.6 mL 40 mM hydrogen peroxide prepared with phosphate buffer (pH 7.4) and incubated this reaction mixture at 37°Cin for 10 min. After incubation, the absorbance was measured at 230 nm against the blank. All the assays were performed in triplicates. Inhibition (%) = (OD1-OD2)/OD1 × 100

OD1 - absorbance of hydrogen peroxide, OD2 - absorbance of reaction mixture along with test samples.

2.6 Cytotoxicity effects on human mesenchymal stem cells

The cytotoxicity of the test samples was analyzed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. At the density of 10,000 cells/mL human mesenchymal stem cells (hMSCs) were seeded in the respective wells in 96 well plates. The pure catechin and partially purified biotransformed metabolites were treated at different doses and incubated for 24 and 48 h at 37 °C in a humidified environment maintained with 5% CO₂. After incubation of MTT 20 µL/well was added and incubated overnight. After incubation, the supernatant was removed from the wells by centrifugation. The formazan crystal was dissolved by adding dimethyl sulfoxide (100%). Using a microplate reader (Promega, Madison, WI, USA) optical density of the samples was measured at 570 nm and a reference filter (630 nm). The cell viability was calculated as percentage from the obtained values by comparing survival of control cells. All the analyses were performed in triplicates/dose, and data are presented as the percentage mean ± SD.

2.7 Peripheral blood mononuclear cells (PBMC)

The experiment was performed in conformity with the Declaration of Helsinki, and the protocol was approved by the Institutional Review Board of King Saud University College of Medicine (E-19–3703). The volunteers were educated with the purpose and the implications of the study, and informed consent was obtained. The venous blood 100 mL was collected from a healthy volunteer, who was accepted and not consumed antioxidant-rich foods for 24 h before blood collection. Using density gradient method, the collected fresh blood was immediately treated with Histopaque-1077 (Sigma, St Louis, MO), and PBMC were separated by centrifugation for 15 min at 2500 g (Carrie et al., 2005). The PMBC concentration was diluted to 1 × 10⁶ cells/mL in RPMI 1630 culture medium and maintained at 37 °C for 24 h in a humidified environment supported with 5% CO₂.

2.8 Quantitative real-time reverse transcription-polymerase chain reaction (qPCR)

RT-qPCR experiment was performed using Applied Biosystems 7500 Fast real-time PCR system. Cell to cDNA synthesis kit (FastLane Cell cDNA Kit (Qiagen, Germany) was used to synthesis cDNA. The cDNA quantification and purity were carried out using the NanoDrop 2000 UV-spectrophotometer (Thermo Fisher, USA). qPCR reaction was carried out with the SYBR Green PCR Master Mix (Qiagen, Chatsworth, CA, USA) and Quantitect primer assays in an ABI 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA) as per the manufacturer’s instruction (Table 1). Reaction mixtures and thermal-cycling conditions were followed as per the manufacturer’s instructions. Relative changes in the GPX1 and POR gene expression were calculated using ΔΔCt method, and reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization. Data were presented as mean fold change over control with standard deviations.

2.9 Statistical analyses

All the experiments were carried out in triplicates, and the results were presented as mean ± SD. The data were analyzed using the SPSS software, version 22.0 (Chicago, IL, USA) and Microsoft excel. One-way analysis of variance (ANOVA) was carried performed. The statistically significant differences were expressed as *p ≤ 0.05.

3.1 DPPH radical scavenging activity

Pure catechin and partially purified biotransformed catechin metabolites reduction potential on DPPH radicals were assessed by the decreased absorbance at 517 nm and expressed as a percentage of inhibition. The free radical scavenging potential is based on the hydrogen donating ability of the compound to convert unpaired electrons to paired electrons (Ozsoy et al., 2008). Fig. 1 represents the scavenging activity of ascorbic acid, Trolox, catechin, and catechin biotransformed metabolites PPMB, crude and PPMA inhibition at a concentration 80 µg/mL were 79.66 ± 0.50, 70.45 ± 0.70, 63.24 ± 0.32, 57.31 ± 0.73, 50.40 ± 0.50, and 43.59 ± 0.71% respectively. The obtained results were statistically significant (p < 0.05). The partially purified biotransformed catechin metabolites exhibited low inhibition potential compared to pure catechin. The IC₅₀ of positive control ascorbic acid and Trolox, catechin and their biotransformed metabolites PPMB, crude, and PPMA were 15.60, 23.58, 40.60, 86.32, 111.25, and 137.64 µg/mL, respectively. The current study demonstrated that the IC₅₀ value of partially purified catechin biotransformed metabolites was twofold higher than pure catechin.

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Fig. 1

2–2- Diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging activity of pure catechin and partially purified colonic bacterial transformed catechin metabolites at different concentrations. Data represented as mean ± SD of triplicates along with the error bar, ***, **, * statistically significant differences at P < 0.001, P < 0.01, and P < 0.05. Partially purified biotransformed catechin metabolites A - PPMA, Partially purified biotransformed catechin metabolites B - PPMB, Crude metabolites - Cr, Catechin, Ascorbic acid -AA, Trolox - TE.

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3.2 ABTS⁺ radical cation decolorization assay

The ABTS⁺ radical scavenging assay was employed as an index that revealed the antioxidant activity of chain-breaking antioxidants (Leong and Shui, 2002). Compounds with higher reduction potential to neutralize the active pronated radicals via accepting or donating electrons capable of terminating radical chain reactions (Khan et al., 2013). The cationic radical inhibition potential was high in ascorbic acid followed by Trolox, catechin, crude, PPMB, and PPMA; 75.40 ± 0.64, 69.72 ± 1.02, 68.00 ± 0.56, 66.61 ± 1.11, 56.70 ± 0.90 and 53.81 ± 0.82% respectively (Fig. 2). The IC₅₀ value of ascorbic acid, Trolox, catechin, crude, PPMB, and PPMA was 15.61, 19.39, 30.74, 55.27, and 69.09 µg/mL, respectively. The IC₅₀ values indicate that partially purified catechin biotransformed metabolites were demonstrated significant scavenging capacity at high concentrations compared to pure green tea catechin.

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Fig. 2

ABTS⁺ radical scavenging activity catechin and partially purified colonic bacterial transformed catechin metabolites at different concentrations. Data represented as mean ± SD of triplicates along with the error bar, ***, **, * statistically significant differences at P < 0.001, P < 0.01, and P < 0.05.

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3.3 Hydrogen peroxide (H₂O₂) radical scavenging activity

Free hydroxyl radicals are highly reactive and formed during the cellular metabolic process in the biological system. However, in humans metabolic enzymes cannot act to neutralize them (Liu et al., 2005). The scavenging ability of hydroxyl radical was shown in Fig. 3. At high concentration inhibition percentage of ascorbic acid, Trolox, catechin, biotransformed metabolites crude, PPMA and PPMB were exhibited 79.23 ± 0.73, 77.64 ± 0.70, 74.16 ± 0.34, 76.77 ± 1.06, 73.37 ± 0.83, and 63.60 ± 0.89% respectively, and the obtained values are statistically significant. The IC₅₀ value of ascorbic acid, Trolox, catechin, crude, PPMA, and PPMB biotransformed metabolites was 13.23, 16.68, 22.18, 23.94, 41.22, and 29.03 µg/mL, respectively. The present study found that pure catechin, partially purified catechin biotransformed metabolites B, and positive control exhibits a similar hydroxyl radical scavenging activity at high concentration, with varying IC₅₀ values.

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Fig. 3

Hydrogen peroxide (H₂O₂) radical scavenging activity catechin and partially purified colonic bacterial transformed catechin metabolites at different concentrations. Data represented as mean ± SD of triplicates along with the error bar, ***, **, * statistically significant differences at P < 0.001, P < 0.01, and P < 0.05.

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3.4 Cytotoxic effect on human mesenchymal stem cells

The pure catechin, catechin biotransformed metabolites crude, PPMA and PPMB were treated at different concentrations ranging from 10 to 80 µg/mL on hMSCs, and showed viability percentage after 24 and 48 h in Figs. 4 and 5. The pure catechin showed a statistical difference (p < 0.05) cytotoxic effect in a dose-dependent manner. In contrast, catechin biotransformed metabolites PPMA showed mild proliferative activity at the lower concentrations at 24 and 48 h treatment. However, crude, PPMA, and PPMB metabolites exhibit non-cytotoxic impact on hMMSc even at higher concentrations (80 µg/mL) and longer treatment times.

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Fig. 4

Cytotoxic effect of pure catechin and partially purified colonic bacterial transformed catechin metabolites at the different concentrations on human mesenchymal stem cells at 24 h. Data represented as mean ± SD of triplicates along with the error bar. * statistically significant differences at P < 0.05.

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Fig. 5

Cytotoxic effect of pure catechin and partially purified colonic bacterial transformed catechin metabolites at the different concentrations on human mesenchymal stem cells at 48 h. Data represented as mean ± SD of triplicates along with the error bar. * statistically significant differences at P < 0.05.

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3.5 Gene expression

The role of pure catechin and partially purified biotransformed metabolites on antioxidant gene expression on PBMC was analyzed. After the treatment with crude PPMA, PPMB, and pure catechin, the GPX1 and POR gene expression results were depicted in Figs. 6 and 7. The GPX1 and POR gene expression was significantly increased four folds in a dose-dependent manner in pure catechin-treated cells. In contrast, crude, PPMA, and PPMB downregulated the GPX1 expression and upregulated the POR gene expression in a dose-dependent manner.

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Fig. 6

Antioxidant gene GPX1 expression in treated peripheral blood mononuclear cells at different concentrations of pure catechin and partially purified colonic bacterial transformed catechin metabolites.

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Fig. 7

Antioxidant gene POR expression in treated peripheral blood mononuclear cells at different concentrations of pure catechin and partially purified colonic bacterial transformed catechin metabolites.

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