Inhibition of gut bacterial β-glucuronidase by chemical components from black tea: Inhibition interactions and molecular mechanism

2.1 Chemicals and reagents

DDAO and DDAOG were chemically synthesized in our lab as previously reported (Feng et al., 2018). SN-38 was obtained from Dalian Meilun Biotech Co., Ltd (Dalian, China). SN-38G was purchased from Toronto Research Chemicals (Toronto, Canada). GUS (Escherichia coli) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Mentha haplocalyx Briq (1), Cannabis sativa L. (2), Myristica fragrans Houtt. (3), Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H.F.Chou (4), Sterculia Lychnophora Hance (5), Morus alba L. (6), Poria cocos (Schw.)Wolf (7), Houttuynia cordata Thunb. (8), Coix lacryma-jobi L (9), Gardenia jasminoides J.Ellis (10), Portulaca oleracea L. (11), Imperata cylindrical (L.) Raeusch (12), Hordeum vulgare L. (13), Nelumbo nucifera Gaertn. (14), Glycyrrhiza uralensis Fisch (15), Cirsium setosum (Willd.) MB. (16), Sesamum indicum L. (17), Citrus medica L. (18), Ginkgo biloba L. (19), Foeniculum vulgare Mill. (20), Keemun black tea (21), Angelica dahurica var. dahurica (22), Cassia obtusifolia L. (23), Perilla frutescens (L.) Britton. (24), Euryale ferox Salisb. (25), Allium macrostemon Bunge (26), Lonicera japonica Thunb. (27), Citrus medica L. (28), Chrysanthemum morifolium Ramat. (29), Crataegus pinnatifida Bunge (30), Taraxacum mongolicum Hand.-Mazz. (31), Raphanus sativus L. (32), Prunus mume (Siebold) Siebold & Zucc. (33), Phragmites communis Trin. (34), Prunus japonica Thunb. (35), Dioscorea oppositifolia L. (36), Dolichos lablab L. (37), Polygonatum odoratum (Mill.) Druce (38), Alpinia oxyphylla Miq. (39), Platycodon grandiflorum (Jacq.) A.DC (40), Polygonatum kingianum Collett & Hemsl. (41), and Pogostemon cablin (Blanco) Benth. (42) were purchased from Beijing Tongren Tang (Bejing, China). All other reagents were of analytical or HPLC grade.

2.2 Preparation of ethanol extracts of plants

The 42 commonly used dried plant materials (200 g) were crushed, grounded into powder, and transferred to 95% ethanol (50 mL), followed by ultrasonication extraction for three times in total 90 min. The supernatant of each extract was combined and concentrated in a rotary evaporator (EYELA, N-1300, Tokyo, Japan) yielding a total of 8–10 g of dry crude extract. Subsequently, the concentrated extracts (5 mg) were suspended in DMSO (250 μL) and the insoluble contents were removed by centrifugation for 5 min at 2000 g using eppendorf centrifuge 5804R (Hamburg, Germany). Then, the liquid supernatant was transferred to a 1.5 mL polyethylene tube (NEST Biotechnology) as stock solution (20 mg/mL) before EcGUS inhibition screening experiment using the high-throughput screening method.

2.3 Bioactivity-guided isolation of target compounds from black tea

Chemical fingerprinting and fraction collection procedures were the same as the previous publication (Tian et al., 2021), performed on a Waters Prominence UPLC system, equipped with a Waters sample manager FTN-R, a Waters quaternary solvent manager-R, and a Waters 2998 PDA Detector. Chromatograph column used was an Acquity UPLC HSS T3 C18 (2.1 × 100 mm, 1.8 µm). Mobile phase consisted of water/0.2% formic acid (A) and acetonitrile (B), and the effluent was monitored at 280 nm with LC-UV detection. After the establishment of the analytical method, the fractions were directly collected into the 96 well plates (Jet Biofil, Guangzhou) according to their retention time. After rotary evaporation, all the dried fractions were weighted and used for the assays of EcGUS inhibition. Fractions with potent inhibitory effects on EcGUS were further isolated and purified by the semi-preparative HPLC. Finally, the structures of purified compounds were identified by the spectral data (HRMS and NMR).

2.4 Inhibition assays using DDAOG hydrolyzation

DDAOG was used as a selective fluorescent probe substrate for detecting EcGUS activity. DDAOG was incubated with EcGUS (0.2 U/mL) in the presence or absence of different concentrations of molecules isolated from the procedures described above at 37 °C for 30 min. The negative controls (DMSO only) were also carried out under the same conditions. EcGUS activity was determined according to the previously published methods (Feng et al., 2018). In brief, supernatants were collected and 200 μL aliquots were loaded directly into the 96-well plate. Fluorescence was recorded using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, USA), under the excitation wavelength of 600 nm and the emission wavelength of 660 nm. DDAO standard curve was used to quantify the concentration of DDAO in the reaction system.

2.5 Inhibition assays based on SN-38G hydrolyzation

To determine whether the inhibitory effects caused by the identified constituents on EcGUS were substrate dependent, we examined the inhibitory effects of these constituents on EcGUS activity using SN-38G as probe substrate (a classical drug substrate for EcGUS). Inhibition effects of the tea polyphenols on SN-38G hydrolyzation mediated by EcGUS were preliminary screened. A typical incubation mixture (total volume 0.2 mL) consisted of PBS buffer (pH 6.5, 100 mM), SN-38G (1 μM for inhibition screening; 0.25–2.0 μM for inhibition constant determination), and EcGUS (0.5 U/mL) in the presence or absence of different concentrations of tea polyphenols (0.2–40 μM). Each reaction was started by addition of 10 μL SN-38G, and then terminated by addition of an equal volume of ice acetonitrile after 30 min incubation. The protein was removed by centrifugation at 20,000 g for 20 min. The supernatant was analyzed by LC-MS.

2.6 Molecular docking

The 3D crystal structure of EcGUS (PDB code: 3K4D) was downloaded from Protein Data Bank (http://www.rcsb.org/structure/3K4D, code: 3K4D) (Wallace et al., 2010). PyMOL 2.4 was used to add non-polar hydrogen and remove water molecules. The cavity of EcGUS was set based on the site of the intrinsic ligand in 3D crystal structure of EcGUS, and the interactions of CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG with EcGUS were analyzed by AutoDock 4.2 with default parameters.

2.7 Molecular dynamic simulation

The best pose of TFDG with EcGUS was used for molecular dynamics (MD) simulation by GROMACS 2018.6 package for further understanding of inhibitor-enzyme interaction mechanism. EcGUS and TFDG were prepared separately according to coordinate of the docked complex. The protein topology was defined based on the amber99sb force field (Hornak et al., 2006). The partial charge of TFDG was assigned according to AM1-BCC charges using antechamber module of AmberTool. The topology parameters were then generated by antechamber and ACPYPE programs. The topology of enzyme-inhibitor complex was assembled combining that of EcGUS and TFDG. A rectangular box with TIP3P water model was added around the complex with solvation thickness at least 1.2 nm. Na ions were added to ensure the neutrality. The production run was performed following a proper minimization to fully relax the prepared system. A total 20 ns molecular dynamic trajectory was sampled at isothermal–isobaric condition at 300 K. All the simulations were executed under periodic boundary conditions (PBC). The enzyme-inhibitor interaction at atomic-level was retrieved from the MD trajectory with analytic tools integrated in GROMACS.

2.8 Inhibition of EcGUS in living bacteria and fluorescence imaging

Escherichia coli DH5alpha BRL was cultured in nutrient broth (NB) at 37 °C. After the bacterial cells were grown to give an OD of 0.6–0.8, the bacterial cells were preincubated with CG, TF-3-G, TF-3′-G, and TFDG (10 μM, 20 μM, and 50 μM) for 2 h, and DDAOG (10 μM) was added to the incubation system for another 2 h. The final concentration of DMSO in the culture dishes was 0.45% (vol/vol). Fluorescence images of living bacteria after incubation with CG, TF-3-G, TF-3′-G, TFDG, and DDAOG were recorded in 96-well plates by an Amersham typhoon fluorescent image analyzer (GE Healthcare Bio-Sciences AB) (λex = 630 nm, λem = 655–685 nm).

2.9 Data analysis

IC₅₀ values (half maximal inhibitory concentration) and K_i values (inhibition constant) were calculated by the nonlinear regression analysis of Graphpad Prism 7.0 (San Diego, CA, USA). The inhibition kinetic types including competitive inhibition (Eq. (1)), noncompetitive inhibition (Eq. (2)), or mixed inhibition (Eq. (3)) were determined according to the following equations. (He et al., 2020; Tian et al., 2018)

3.1 Inhibition screening of medicinal and edible plants against EcGUS

The inhibitory effects of ethanol crude extracts of 42 medicinal and edible plants on gut bacterial GUS were firstly evaluated by using DDAOG as a specific fluorescent probe for EcGUS. As shown in Fig. 1a, upon addition of 100 μg/mL ethanol extracts, DDAOG hydrolysis by EcGUS was strongly inhibited (0–20% residual activity) by 5 edible plants including Nelumbinis Folium (B7), Glycyrrhiza uralensis Fisch (C1), Foeniculi Fructus (C6), black tea (C7) and Euryales Semen (D4), and the residual activities of EcGUS were 11.6%, 10.9%, 9.7%, 4.0% and 19.6%, respectively. Among all the edible plants investigated, the ethanol extract of black tea displayed the strongest inhibition towards EcGUS. As shown in Fig. 1b, the ethanol extract of black tea could inhibit EcGUS-mediated DDAOG hydrolysis in a dose-dependent manner with an IC₅₀ value as low as 4.60 μg/mL, which was much lower than that of silymarin (794.62 μg/mL), a well-known inhibitor of EcGUS (Karak et al., 2017). These results strongly demonstrated that the ethanol extract of black tea could significantly inhibit EcGUS activity, and this edible plant might contain naturally occurring potent EcGUS inhibitors.

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Fig. 1

Inhibitory effects of the ethanol extracts of 42 common edible plants (a) and black tea ethanol extract (b) on EcGUS-mediated DDAOG hydrolysis. Each grid represented an edible plant, and the color of each grid represented the inhibitory activity of each edible plant. A1-A7, 1–7; B1-B7, 8–14; C1-C7, 15–21; D1-D7, 22–28; E1-E7, 29–35; F1-F7, 36–42.

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3.2 Identification of the major EcGUS inhibitors in black tea

To identify the naturally occurring potent inhibitors in black tea, the ethanol extract of black tea was separated and fractioned using a simple LC-UV fingerprinting method. 45 eluted LC fractions were collected and dried for the evaluation of their inhibitory effects against EcGUS based on the high-throughput screening activity. Upon addition of each fraction, eleven major fractions (Fr. 17, 19, 24, 25, 26, 27, 28 and 30, 31, 32, 35) significantly inhibited EcGUS, while other fractions performed moderate or negligible inhibitory effects (Fig. 2). Combination the LC-UV fingerprinting with the EcGUS inhibition profile, seven bioactive components (structures are shown in Fig. 3) were isolated from the ethanol extract of black tea, and identified as CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG, respectively, according to their NMR data (Fig. S1-S14).

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Fig. 2

(a) UPLC-UV fingerprint of black tea extract monitored at 280 nm, and (b) the corresponding EcGUS inhibition profile of UPLC fractions collected at 60 s intervals.

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Fig. 3

Chemical structures of CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG.

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3.3 Inhibitory effects of the seven identified constituents from black tea against EcGUS

In order to further validate and explore the inhibitory effects of the seven isolated constituents against EcGUS, two selective probe substrates for EcGUS, DDAOG, a specific fluorescent probe substrate reported previously, and SN-38G, a classical drug substrate for EcGUS, were used in parallel for charactering the inhibition behaviors. Three concentrations (1, 10, and 100 μM) gradients were used in the preliminarily screening experiments. As shown in Fig. 4, all the seven major natural occurring compounds in black tea displayed evident inhibitory effects against EcGUS. Upon addition of 10 µM CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG, the residual activities of EcGUS-mediated deglucuronidation of DDAOG were reduced to be 16.0%, 37.2%, 41.4%, 58.6%, 5.8%, 5.6%, and 1.2% of the negative control, respectively. At 10 µM CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG, the remaining activities of EcGUS-mediated deglucuronidation of SN-38G were measured to be 9.1%, 28.2%, 22.7%, 56.0%, 4.9%, 2.8%, and 4.4% of negative control, respectively.

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Fig. 4

Inhibitory effects of CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG against EcGUS-mediated DDAOG (a) and SN-38G hydrolysis (b).

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To further investigate the inhibitory potentials of these polyphenol black tea constituents, their dose-dependent inhibition curves against EcGUS were depicted. As shown in Fig. 5, CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG could inhibit the deglucuronidation of DDAOG by EcGUS in a dose dependent manner. The IC₅₀ values of CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG against EcGUS-mediated deglucuronidation of DDAOG were evaluated as 1.48, 6.94, 6.84, 14.86, 1.92, 1.03 and 1.41 μM, respectively (Table 1). Meanwhile, the dose-dependent inhibition curves of these constituents against EcGUS-mediated deglucuronidation of SN-38G were depicted in Fig. S15, with the IC₅₀ values measured to be 0.77, 3.82, 2.64, 14.06, 1.39, 0.95 and 0.47 μM, respectively (Table 1). These results demonstrated that these seven inhibitors display evident concentration-dependent inhibitory behaviors against EcGUS mediated deconjugation reactions, and the IC₅₀ values of CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG against DDAOG hydrolyzation agree well with the IC₅₀ values of these constituents against SN-38G hydrolyzation. It is worth noting that these compounds are the major polyphenol constituents in black tea and all these compounds display either strong or moderate inhibitory effects against EcGUS. These results indicate that these constituents may block out SN-38G hydrolyzation, and could subsequently reduce exposure of SN-38 in gastrointestinal tract after administration of black tea.

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Fig. 5

Inhibitory effects of the seven identified constitutes in black tea against DDAOG hydrolysis in EcGUS. Left: the concentration-dependent inhibition curves of CG (a), GCG (c), ECG (e), EGCG (g), TF-3-G (i), TF-3′-G (k), and TFDG (m). Right: the representative Lineweaver-Burk plots of CG (b), GCG (d), ECG (f), EGCG (h), TF-3-G (j), TF-3′-G (l), and TFDG (n).

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3.4 Inhibition kinetics of the seven identified constituents against EcGUS

To further characterize the inhibition behaviors and define the K_i values of these identified constituents towards EcGUS, inhibition kinetics experiment were carried out. As shown in Fig. 5, the Lineweaver-Burk plots demonstrated that CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG followed the mixed inhibition behavior against EcGUS-mediated DDAOG hydrolysis, the K_i values for these molecules were determined as 0.59, 2.21, 2.42, 11.92, 0.87, 0.19, and 0.31 μM, respectively. Further kinetic analysis results showed that the inhibition types of CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG belong to competitive form in EcGUS-mediated SN-38G hydrolysis inhibition (Fig. S15). The K_i values for these molecules, in this scenario were calculated to be 0.77, 3.82, 2.64, 14.06, 1.39, 0.47, and 0.95 μM, respectively, agreed well with the K_i values measured from DDAOG hydrolyzation inhibition.

Notably, these seven identified compounds displayed different inhibition types against EcGUS-mediated DDAOG (mixed inhibition) and SN-38G (competitive inhibition) hydrolysis. In addition, there was less than 2.5-fold difference in the K_i values of these natural inhibitors (except TFDG) against EcGUS-mediated DDAOG and SN-38G hydrolyses. The substrate dependent inhibition of these natural constituents could be partially attributed to the large differences in their structures and different interactions with EcGUS. From the view of chemical structure, SN-38G was a camptothecin derivative with a large structure possessing two carboxyl group and a free hydroxyl group, while DDAOG was a simple compound with a DDAO skeleton. In terms of interactions, DDAOG and SN-38G might bind with different regions of EcGUS, and the binding site of these seven inhibitors was likely to be partially overlapped with the binding site of DDAOG in EcGUS but these seven polyphenolic compounds might occupy the same binding site of EcGUS with SN-38G. Such results further demonstrated that these seven natural compounds displayed inhibitory effects on EcGUS even if two different probe substrates were used. Moreover, CG, TF-3-G, TF-3-G, and TFDG are more potent inhibitors towards EcGUS compared with ECG, GCG, and EGCG.

3.5 Structure-activity relationships of tea polyphenols against EcGUS

CG, ECG, GCG, EGCG, TF-3-G, TF-3′-G, and TFDG have the same parent nucleus flavanols but demonstrated varied inhibitory potency against EcGUS. All these polyphenols possessed a galloyl ester substituted on hydroxyl group at the 3 or 3′ position of C ring (Fig. 3), suggesting that the galloyl esters substitutions at the flavanol moiety may be essential for the potent inhibition towards EcGUS. Furthermore, TFDG with two galloyl esters-substituted on hydroxyl group of C ring displayed stronger inhibitory effects than the compounds with only one galloyl ester-substituted on hydroxyl group at the same position. Furthermore, TF-3′-G with one galloyl ester attached to 3′-position of C ring showed better inhibitory effects on EcGUS than TF-3-G with one galloyl ester attached to 3 position of C ring. The results suggested that the inhibitory effects of theaflavins on EcGUS may depend on the position and the number of galloyl esters substitution in their structure. Moreover, comparing the difference in structure and inhibitory activity of CG, ECG, GCG, and EGCG, it could be easily found that non-epicatechins were more beneficial than their epimers that are epicatechins for EcGUS inhibition, such as CG (K_i = 0.59 μM) VS ECG (K_i = 2.21 μM), GCG (K_i = 2.42 μM) VS EGCG (K_i = 11.92 μM). Interestingly, two hydroxyl groups at C-3′ and 4′ of B ring is more beneficial than three hydroxyl groups at the C-3′, 4′, and 5′ of B ring (pyrogallol) for EcGUS inhibition, such as CG (K_i = 0.59 μM) VS GCG (K_i = 2.42 μM), ECG (K_i = 2.21 μM) VS EGCG (K_i = 11.92 μM). Nevertheless, further studies are still needed to be investigated about the essential of galloyl esters substitutions at the flavanol moiety for the potent inhibition towards EcGUS.

3.6 Molecular docking study

Docking is a technology for the investigation of the interaction of small molecules with target proteins, and it is widely used in the flied of drug design. In order to reveal the potential interaction of tea polyphenols with GUS, we performed molecular docking study. CG, GCG, ECG, EGCG, TF-3-G, TF-3′-G, and TFDG displayed significantly inhibitory effects towards EcGUS in the EcGUS-mediated DDAOG hydrolysis with IC₅₀ and K_i values ranging from 0.19 to 16.52 μM. The similar results were also observed in the EcGUS-mediated SN-38G hydrolysis (Table 1). As described in Fig. 5 and Fig. S15, they were all assigned as competitive or mixed competitive inhibitors, therefore, their interactions with EcGUS were analyzed based on the competitive-type strategy by AutoDock software in order to reveal the potential mechanism of CG, GCG, ECG, EGCG, TF-3-G, TF-3′-G, and TFDG with EcGUS. The aforementioned compounds could bond in the cavity of GUS and occupy this active pocket to prevent the EcGUS-mediated hydrolysis of DDAOG and SN-38G. As shown in Fig. S16A-G, CG, GCG, ECG, EGCG, TF-3-G, TF-3′-G, and TFDG could interact with EcGUS via the interaction forms of hydrogen bond, van der Waals, alkyl, π-π stacked and T-shaped, π-alkyl, and π-δ. More specifically, CG could form four hydrogen bonds with amino acid residues Glu413, Arg417, Met447, and Tyr472. GCG and ECG interacted with Glu413, Met447, and Tyr472 via hydrogen bonds, meanwhile GCG and ECG interacted with Ser360, and Ile363, respectively. EGCG and TFDG interacted with Glu413 and Met447 via hydrogen bonds. Except for the abovementioned compounds, TF-3-G could form three hydrogen bonds with Leu361, Met447, and Tyr469, and TF-3′-G interacted with Leu359, Asn369, and Arg417 via hydrogen bonds. These results help to explain the inhibition mechanisms of these molecules against EcGUS.

3.7 Molecular dynamics simulation

Among the investigated compounds, TFDG is a representative polyphenol derivative with two galloyl groups and exhibited the most potent inhibitory effect against EcGUS. Therefore, MD simulation was used for the investigation of the dynamical interaction of TFDG with EcGUS. In 20 ns of MD simulation, TFDG and EcGUS could form a stable complex with the potential energy of model system stabilized around − 1.38 × 10⁶ kJ/mol (Fig. 6A). Root mean square deviation (RMSD) is a crucial parameter to evaluate the stability of the simulated complex, and result of RMSD for GUS-TFDG was described in Fig. 6B. During 20 ns of MD simulation, RMSD remained under 2 Å indicating a stable simulation run of the tested system. Root mean square fluctuation (RMSF) for GUS residues was also acquired to figure out regions exhibiting higher flexibility (Fig. 6C). From the hydrogen bond analysis, it is worth noting that TFDG could form 1–6 hydrogen bonds, and interact with thirteen residues as Ser360, Gly362, Il363, Phe365, Glu366, Ala367, Glu413, Arg417, Met447, Trp471, Tyr472, Ser475, and Ile560 located in EcGUS during the simulation run. We further monitor the distances of these residues with TFDG. Results for this part are shown in Fig. 6D-G. From the time profiles of distances for the abforementioned residues with TFDG, an obvious variation was emerged at timestamp of 16 ns in the simulation. The distances of residue-TFDG pair for Gly362, Il363, Phe365, Glu366, Ala367, Arg417, Met447, Trp471, Tyr472, and Ser475 were measured from 4.5 Å to 14.5 Å, indicating that hydrogen bond interactions between these residues and TFDG were unreliable. On the other hand, Ser360, Glu413 and Ile560 kept close contact with TFDG as the measured distances converged under 3.5 Å in the whole simulation process, and their interaction with TFDG in the form of hydrogen bond are thus stable and reliable. After 20 ns MD run, the final frame of simulation has proved that TFDG still bonded in the GUS cavity, and interacted with Ser360, Glu413, and Ile560 through hydrogen bonds (Fig. 6H). These results clarify the role of residues Ser360, Glu413, and Ile560 in the inhibition of TFDG against EcGUS and suggest that TFDG could be considered as a potential agent for the development of EcGUS inhibitors.

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Fig. 6

(A) The potential energy of TFDG with EcGUS in 20 ns of MD simulation. (B) RMSD of TFDG with EcGUS in 20 ns of MD simulation. (C) RMSF of TFDG with EcGUS in 20 ns of MD simulation. (D) The numbers of hydrogen bond in 20 ns of MD simulation. (E) The distances of TFDG with Ser360, Gly362, Ile363, and Phe365 of EcGUS. (F) The distances of TFDG with Glu366, Ala367, Glu413, and Arg417 of GUS. (G) The distances of TFDG with Met447, Trp471, Tyr472, Ser475, and Ile560 of EcGUS. (H) 3D structure of the complex of TFDG bounded EcGUS and their interaction form in the final frame of 20 ns MD simulation.

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3.8 Inhibitory effects of the major EcGUS inhibitors isolated from black tea on endogenous EcGUS activity in living bacteria

To further evaluate the inhibition ability of the major EcGUS inhibitors in black tea on EcGUS in living bacteria, fluorescence imaging of living bacteria in the presence of each inhibitor was also performed. Escherichia coli DH5alpha BRL was selected in this experiment due to the high expression of GUS, and it is present in the human gastrointestinal tract. The fluorescence images of living bacteria after incubation with CG, TF-3-G, TF-3′-G, TFDG and DDAOG were recorded in 96-well plates by an Amersham typhoon fluorescent image analyzer. As shown in Fig. 7, there was an intense fluorescence emission after incubation with DDAOG (10 μM) at 37 °C for 2 h. In contrast, the fluorescence intensity reduction was observed in the presence of each tested compound at different concentrations, indicating that the endogenous GUS in Escherichia coli DH5alpha BRL were subject to inhibition by these natural components in black tea. Furthermore, increasing each inhibitor concentration led to a dose-dependent reduction of fluorescence intensity at 660 nm.

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Fig. 7

Inhibitory effects of CG, TF-3-G, TF-3′-G and TFDG on endogenous GUS activity in living bacteria Escherichia coli DH5alpha BRL. The bacterial cells were pre-incubated with each inhibitor (10, 20, and 50 μM) for 2 h, and DDAOG (10 μM) was added to the incubation system for another 2 h.

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