Identification of Escherichia coli β-glucuronidase inhibitors from Polygonum cuspidatum Siebold & Zucc.

Abstract

Gut bacterial β-glucuronidase (GUS) can reactivate xenobiotics that exert enterohepatic circulation- triggered gastrointestinal tract toxicity. GUS inhibitors can alleviate drug-induced enteropathy and improve treatment outcomes. We evaluated the inhibitory effect of Polygonum cuspidatum Siebold & Zucc. and its major constituents against Escherichia coli GUS (EcGUS), and characterized the inhibitory mechanism of each of the components. Trans-resveratrol 4’-O-β-D-glucopyranoside (HZ-1) and (-)-epicatechin gallate (HZ-2) isolated from P. cuspidatum were identified as the key components and potent inhibitors. These two components displayed strong to moderate inhibitory effects on EcGUS, with Ki values of 9.95 and 1.95 μM, respectively. Results from molecular docking indicated that HZ-1 and HZ-2 could interact with the key residues Asp163, Ser360, Ile 363, Glu413, Glu504, and Lys 568 of EcGUS via hydrogen bonding. Our findings demonstrate the inhibitory effect of P. cuspidatum and its two components on EcGUS, which supported the further evaluation and development of P. cuspidatum and its two active components as novel candidates for alleviating drug-induced damage in the mammalian gut.

Keywords:
β-glucuronidase; Polygonum cuspidatum Siebold & Zucc.; Trans-resveratrol 4’-O-β-D-glucopyranoside; (-)-Epicatechin gallate; Inhibitory mechanism

INTRODUCTION

Gut bacterial β-glucuronidase (GUS) is an acid glycoside hydrolase residing in the gastrointestinal tract, that catalyzes the hydrolysis of glucuronide conjugates and produces the corresponding aglycone (Pellock, Redinbo, 2017; Pollet et al., 2017; Wang et al., 2019). The released aglycone can be absorbed and accumulate in the intestinal tract, causing dose-limited gastrointestinal toxicities exemplified by the chemotherapy drugs (Clarke et al., 2019; Ervin et al., 2019). Irinotecan (CPT-11), an anti-cancer drug mainly used to treat colorectal cancer, can lead to severe delayed diarrhea and neutropenia, limiting its clinical applications (Bailly, 2019; Hahn et al., 2019; Shi et al., 2021). CPT-11 is hydrolyzed in vivo by carboxylesterase in the liver to its active as well as toxic metabolite SN-38 (Hicks et al., 2009; Tobin et al., 2006). After exerting its antitumor effect, SN-38 is primarily metabolized in the liver by uridine diphosphate glucuronyltransferase 1A1 to its inactive form SN-38- glucuronide (SN-38G), which is then excreted through the urine and bile (Iyer et al., 1998). Nevertheless, the highly expressed GUS in the gut can hydrolyze SN-38G to SN- 38, leading to an excessive SN-38 present in the gut that can cause delayed diarrhea and neutropenia (Jariwala et al., 2020). Recently, GUS inhibitors have been shown to alleviate the gastrointestinal toxicity of CPT-11 and other anticancer drugs (Awolade et al., 2020; Bhatt et al., 2020; Chamseddine et al., 2019; Wallace et al., 2010).

Based on their source, existing GUS inhibitors can be divided into mainly two types including synthetic and natural inhibitors (Awolade et al., 2020; Cheng et al., 2017; Zhou et al., 2020). Natural products-based inhibitors of GUS have attracted considerable attention owing to their physiological tolerance, satisfying safety and favorable pharmacodynamic profiles (Li, 2020; Sun, et al., 2020; Weng et al., 2017; Zhong et al., 2020). Therefore, it is important to discover novel GUS inhibitors with improved efficacy and safety to alleviate the side effects caused by CPT-11 and anti-cancer drugs as well as assist the cancer treatment. Polygonum cuspidatum Siebold & Zucc., known as Huzhang in China, is often used to treat inflammation, damp-heat jaundice, rheumatoid or rheumatoid arthritis and other diseases (Bralley et al., 2008; Hu et al., 2018; Liu et al., 2014; S. Liu et al., 2016; Lu et al., 2012). Despite extensive pharmacological studies, there are currently no reports investigating the interactions between P. cuspidatum and the gut microbiota. In a preliminary experiment, P. cuspidatum displayed strong inhibitory effects on gut bacterial GUS, which elicited our interest in exploring the main components responsible for its inhibitory effects.

The study aimed to identify the main components of the ethanolic extract of P. cuspidatum as it exhibited significant inhibitory effects on Escherichia coli GUS (EcGUS), and characterize the inhibitory mechanism and determine the inhibition constant of each inhibitory component. The chemical fingerprint of P. cuspidatum and EcGUS inhibition profile were combined to identify the components responsible for the inhibitory effect. Inhibition kinetic assays were performed to characterize the inhibitory behavior and obtain the kinetic constants (IC50, Ki) of the components that were active against EcGUS. Furthermore, molecular docking was carried out for evaluating the potential molecular determinants responsible for the potent inhibitory effects of the identified components of P. cuspidatum towards EcGUS.

MATERIAL AND METHODS

Chemicals and reagents

Dried rhizomes of P. cuspidatum were purchased from Beijing Tongrentang Co., Ltd. (Dalian, China) in January 2020, and identified by Prof. Jing-Ming Jia (Shenyang Pharmaceutical University). A voucher specimen (MO202001) has been deposited in the Department of Medicinal Chemistry, Dalian Medical University. DDAO was chemically synthesized and DDAOG was biosynthesized in our laboratory as reported previously (Feng et al., 2018). GUS from E. coli was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical or HPLC grade.

Preparation of P. cuspidatum ethanol extract

The dried plant materials (500 g) were crushed, powered, and then extracted by 95% ethanol (100 mL) for three times in total 90 min. P. cuspidatum ethanol extract was afforded after removing the solvent. Subsequently, the extract (5 mg) was suspended in DMSO (100 μL), and stored at 4 °C.

Bioactivity-guided isolation and identification of active compounds of P. cuspidatum

Chemical fingerprinting and fraction collection were performed using a Waters Prominence HPLC system, equipped with a Waters 2767 sample manager, Waters 2545 binary gradient module, and Waters 2489 UV/visible detector. A Waters XBridge C18 (19 mm × 250 mm, 5 µm) chromatographic column was used. The mobile phase consisted of methanol (A) and water (B), and the following gradient condition was used: 0.0-10.0 min, 20% B; 10.0-15.0 min, 20%-40% B; 15.0-45.00 min, 40%-100% B; 45.0-46.0 min, 100% B; 46.0-60.0 min, 100% B; 60.0-65.0 min, 100%-20% B; 65.0-80.0 min, 20% B. The flow rate was set to 10 mL/min and the injection volume of the crude extract (50 mg/mL) was 300 µL. The effluent was monitored at 270 and 220 nm with LC-UV detection. After establishing the analytical method, the fractions were automatically collected into sample tubes based on chromatographic peaks or retention time. After rotary evaporation, all dried fractions were weighed and used subsequently for EcGUS inhibition assays. The fractions displaying potent inhibitory effects on EcGUS were further isolated and purified using semi-preparative high- performance liquid chromatography (HPLC) (Singh et al., 2020). Finally, the structures of the purified compounds were elucidated using nuclear magnetic resonance spectroscopy.

DDAOG hydrolysis-based inhibition assays

The inhibitory effects of P. cuspidatum extract and its constituents on EcGUS were investigated using DDAOG as a specific fluorescent probe for EcGUS (Feng et al., 2018). The incubation system consisted of PBS buffer (pH 6.5, 100 mM), EcGUS (0.2 U/mL), DDAOG (10 μM for inhibition screening; 5-40 μM to determine the inhibition constants) in the presence or absence of the inhibitor. Each reaction was started by the addition of 10 μL DDAOG. After incubation at 37 °C for 30 min, the reaction was terminated by addition of an equal volume of ice cold acetonitrile. Then, 200 μL aliquots of the supernatants were diverted into a 96-well plate for fluorescence detection, at excitation and emission wavelengths of 600 and 660 nm, respectively.

Molecular Docking

The 3D crystal structure of EcGUS (PDB code: 3K4D) was downloaded from Protein Data Bank (http:// www.rcsb.org/, code: 3K4D). 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 HZ-1 and HZ-2 with EcGUS were analyzed by AutoDock 4.2 with default parameters.

Data analysis

IC₅₀ (half maximal inhibitory concentration) and K _i values were calculated by the nonlinear regression analysis of Graphpad Prism 7.0 (San Diego, CA, USA) (He et al., 2020; Song et al., 2019; Sun et al., 2020). The inhibition kinetic types including competitive inhibition, noncompetitive inhibition, uncompetitive type, or mixed inhibition were determined based on the goodness-of-fit parameters (Hou et al., 2020; Yi et al., 2019; Zhang et al., 2018).

RESULTS AND DISCUSSION

Inhibitory effects of P. cuspidatum extract on DDAOG hydrolysis

As shown in Figure 1a, at 10 μg/mL ethanolic extract of P. cuspidatum, the residual activity of DDAOG hydrolysis was markedly reduced to be less than 5% of the negative control. Moreover, the crude ethanolic extract of P. cuspidatum inhibited EcGUS-mediated DDAOG hydrolysis in a dose-dependent manner with an IC₅₀ value as low as 0.79 μg/mL, as depicted in Figure 1b. These results demonstrate that the ethanolic extract of P. cuspidatum exhibited a strong inhibitory effect on EcGUS, indicating that the herb may contain potent inhibitors of EcGUS.

FIGURE 1
(a) Inhibitory effects of P. cuspidatum ethanol extract (0, 1, 10, 100 μg/mL) on EcGUS-mediated DDAOG hydrolysis, and (b) the corresponding dose-dependent inhibition curve of different concentrations of P. cuspidatum ethanol extract against EcGUS-mediated DDAOG hydrolysis. All data represent the mean of triplicate determinations.

Identification of the major EcGUS inhibitors in P. cuspidatum

After establishing the chemical fingerprint of P. cuspidatum, the 20 eluted fractions were automatically collected into sample tubes and evaporated to dryness (Figure 2a). Then, the dried fractions were evaluated for their inhibitory effects on EcGUS based on the high- throughput screening method. As shown in Figure 2b, two fractions (No. 4 and 5) significantly inhibited EcGUS with the residual activities less than 25%, while the other fractions displayed moderate or negligible inhibitory effect on EcGUS. Subsequently, fractions 4 and 5 were isolated using preparative HPLC, and two compounds were purified and identified as trans-resveratrol 4’-O-β- D-glucopyranoside (HZ-1) and (-)-epicatechin gallate (HZ-2) with purity > 95% (Figure S1-S4).

FIGURE 2
(a) HPLC-UV fingerprint of P. cuspidatum ethanol extract (50 mg/mL) monitored at 270 nm, Fr.1-Fr.20 represent eluted fraction numbered from 1 to 20, and (b) the corresponding EcGUS inhibition profile of each eluted fraction (2 μg/mL) towards EcGUS-mediated DDAOG hydrolysis. Note: trans-resveratrol 4’-O-β-D-glucopyranoside (HZ-1) and (-)-Epicatechin gallate (HZ-2) were purified from Fr. 4 and Fr. 5, respectively.

Inhibitory effects of the two constituents isolated from P. cuspidatum against EcGUS

In order to further validate and explore the inhibitory effects of the two isolated constituents against EcGUS, the preliminarily screening experiments were performed. The residual activities of EcGUS-mediated DDAOG hydrolysis were reduced to be 65.7% and 16.9% of the negative control, respectively, when 10 µM HZ-1 or HZ-2 were used. (Figure 3a and 3c). Moreover, their dose- dependent inhibition curves against EcGUS were also depicted. As shown in Figures 3b and 3d, HZ-1 and HZ-2 displayed evident concentration-dependent inhibition of EcGUS mediated DDAOG hydrolysis. The IC₅₀ values of HZ-1 and HZ-2 against EcGUS-mediated DDAOG hydrolysis were evaluated to be 25.88 μM and 2.24 μM, respectively (Table I). These results demonstrated that both HZ-1 and HZ-2 exhibited inhibitory effects towards EcGUS, among which, HZ-2 was a more potent inhibitor of EcGUS. Notably, the IC₅₀ of HZ-2 determined in our study was in agreement with that reported previously (Feng et al., 2018), wherein it was identified to be one of the main constituents of Rheum palmatum L responsible for the strong inhibitions of EcGUS.

FIGURE 3
(a) Inhibitory effects of HZ-1 and HZ-2 at the concentrations of 0, 1, 10, 100 μg/mL on EcGUS-mediated DDAOG hydrolysis, and (b) the concentration dependent inhibition of EcGUS-catalyzed DDAOG hydrolysis by HZ-1 and HZ-2. Each data point corresponds to the average values calculated from triplicate measurements.

TABLE I
IC50 and Ki values of HZ-1 and HZ-2 against GUS

Compound name	IC50 (μM)	Ki (μM)	Inhibition type
HZ-1	25.88	9.95	Mixed
HZ-2	2.24	1.95	Mixed

Inhibition kinetics of the two identified constituents against EcGUS

Inhibition kinetics experiments were further carried out to calculate the K _i values of the identified constituents for EcGUS and to characterize their corresponding inhibition behaviors. As shown in Figure 4a-4f, both Lineweaver-Burk and Dixon plots indicated that HZ-1 and HZ-2 followed the mixed inhibition behavior against EcGUS-mediated DDAOG hydrolysis. The K _i values for HZ-1 and HZ-2 were evaluated as 9.95, and 1.95 μM, respectively. These results further demonstrated that HZ-1 and HZ-2 were potential inhibitors towards EcGUS and responsible for the strong inhibitory effects of P. cuspidatum towards EcGUS. In addition, HZ-2 displayed much stronger inhibition potency towards EcGUS compared with HZ-1.

FIGURE 4
(a) Lineweaver-Burk plot of HZ-1’s inhibition towards DDAOG hydrolysis in EcGUS, (b) Dixon plot of HZ-1’s inhibition towards DDAOG hydrolysis in EcGUS, (c) Second plot with the slopes from Lineweaver-Burk plot towards the concentrations of HZ-1, (d) Lineweaver-Burk plot of the inhibition of HZ-2 towards EcGUS-catalyzed DDAOG hydrolysis, (e) Dixon plot of the inhibition of HZ-2 towards EcGUS-catalyzed DDAOG hydrolysis, and (f) Second plot with the slopes from Lineweaver-Burk plot towards the concentrations of HZ-2. The results shown are the means of duplicate experiments.

P. cuspidatum and its constituents are known for their wide spectrum of pharmacological activities, such as anti-asthmatic, anti-oxidant, anti-inflammatory, and anti-cancer effects (Peng et al., 2013). In this study, HZ-1 and HZ-2 from P. cuspidatum were identified as a new class of naturally occurring EcGUS inhibitors. Inhibitory effects and molecular mechanism of the identified EcGUS inhibitors were systemically characterized using a selective fluorescent probe substrate for EcGUS. Compared with synthetic GUS inhibitors, these two naturally occurring dietary compounds might be readily available and safe owing to their natural, nontoxic and multiple pharmacological effects.

Notably, HZ-1 could also be found in grapes and several traditional medicinal plants, including Rheum tanguticum, R. rhaponticum, and P. multiflorum (Zhao et al., 2019), whereas HZ-2 is abundantly distributed in teas including green, oolong, and black tea (Tao et al., 2016). It is readily conceivable that these food plants and herbal medicines may also display strong inhibitory effects towards EcGUS due to the presence of HZ-1 or HZ-2. Furthermore, it is admitted that whether these inhibitors can selectively inhibit human gut GUS needs to be evaluated both in vitro and in vivo. Therefore, further studies are warranted to determine the inhibitory potency of these two inhibitors against human β-glucuronidases and other bacteria strains.

Molecular docking

Molecular docking was used to evaluate the potential determinants responsible for the inhibitory effects of HZ-1 and HZ-2 toward EcGUS. The crystal structure of EcGUS was downloaded from Protein Data Bank (http://www.rcsb.org). As depicted in Figures 5a and 5c, HZ-1 and HZ-2 could enter the active site of EcGUS and occupy the active pocket to prevent DDAOG hydrolysis. As shown in Figures 5b and 5d, HZ-1 and HZ-2 could form hydrogen bonds and have, van der Waals, π-π stacked, T-shaped, and π-alkyl interactions with the amino acid residues Asp163, Ser360, Leu361, Ile363, Glu413, Val446, Met447, Tyr468, Tyr472, Val473, Glu504, Trp549, Leu561, and Lys568. More importantly, the amino acid residues Asp163, Ser360, Ile363, Glu413, Glu504, and Lys568 could interact with the hydroxy and carbonyl groups of HZ-1 and HZ-2, Glu413 and Glu504, especially, in the active site of GUS were responsible for the hydrolysis of the glucuronide glycoside bond. Notably, same bonds with the enzyme are formed with amentoflavone, demethylbellidifolin, and gentisin, which were reported to be inhibitors of EcGUS in the previous researches (Tian et al., 2021; Sun et al., 2012). These findings explain the molecular mechanism of the inhibition of EcGUS by HZ-1 and HZ-2.

FIGURE 5
A stereo view of the docking conformation of HZ-1 (a) and HZ-2 (c) (stick model) in the active site of EcGUS. The carbon atoms in these two molecules (HZ-1 and HZ-2) were colored in cyan. Residues in EcGUS interacting with HZ-1 (b) and HZ-2 (d) are shown (conventional hydrogen bond, green; π-donor hydrogen bond, light green; π-π stacked, magenta).

CONCLUSIONS

Taken together, our results demonstrated that HZ-1 and HZ-2 from P. cuspidatum were the key components responsible for EcGUS inhibition. Inhibition kinetic analysis demonstrated that both HZ-1 and HZ-2 were mixed-type inhibitors of EcGUS-mediated DDAOG hydrolysis. Molecular docking results elucidated the importance of amino acid residues Asp163, Ser360, Ile 363, Glu413, Glu504, and Lys 568 in EcGUS inhibition by HZ-1 and HZ-2. These findings demonstrated the inhibitory effects of P. cuspidatum and its two components toward EcGUS, thereby supporting their further evaluation and development as novel candidates for alleviating drug-induced intestinal damage.

ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (No 82174064), National Key R&D Program of China (2018YFC1705900), Distinguished professor of Liaoning Province, Open Fund of Key Laboratory of Biotechnology and Bioresources Utilization, Ministry of Education, the Innovative Entrepreneurship Program of High-level Talents in Dalian (2016RQ025 & 2017RQ121), Dalian Young Star of Science and Technology Project (2020RQ068), “1+X ”program for Clinical Competency enhancement- Interdisciplinary Innovation Project, Second Hospital of Dalian Medical University and National Training Program for College Students’ Innovation and Entrepreneurship (S202010161009).

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  These authors contributed equally to this work.

Publication Dates

• Publication in this collection
  16 Jan 2023
• Date of issue
  2022

History

• Received
  05 Aug 2021
• Accepted
  07 Dec 2021