Inhibitory Effects and Kinetic-Docking Studies of Xanthohumol From Humulus lupulus Cones Against Carbonic Anhydrase,Acetylcholinesterase,and Butyrylcholinesterase

Abstract

Xanthohumol is an essential prenyl flavonoid of Humulus lupulus L. cones, and the taste of beer is due to this compound. Lately, xanthohumol has earned significant interest due to its potential anticancer, antigenotoxic, and adipogenesis effects. In this paper, the inhibitory effects of xanthohumol on human carbonic anhydrase isozymes (hCAI and hCAII), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) were studied. Also, molecular docking studies were used to investigate ligand interaction diagrams of xanthohumol at the binding cavities of hCAI and II. Xanthohumol was isolated from hop cones by silica gel column chromatography. Carbonic anhydrase enzyme activities were determined spectrophotometrically. In addition, molecular modeling approaches were used for the hCAI and hCAII isoenzymes. Ellman’s method was used for the inhibitor activities of AChE and BChE. The K _I values of xanthohumol were detected as 0.085 µM for hCAI, 0.049 µM for hCAII, 95.5 nM for AChE, and 124.9 nM for BChE. In conclusion, xanthohumol can pleiotropically exert health promoting effects. It has antiglaucoma, anticonvulsant, antiepileptic, and anticancer activities due to its potent inhibitory effects on hCAI and hCAII. These findings may open new avenues for the design and development of novel hCAI, hCAII, AChE, and BChE inhibitors compared with sulfonamide/sulfamate.

Humulus lupulus L. (hop) is used as a raw material in the beer industry. Female hop cones are a bitter tasting agent in beer, and thus, hops have considerable economic value. Cultivation of hops is climatically and ecologically appropriate in temperate regions of the world. ^1,2 The major prenylated chalcone of hop cones is xanthohumol, which has important pharmacological activities, such as antiproliferative, anti-inflammatory, antioxidant, pro-apoptotic, antibacterial, and anti-adhesive effects. ³ Especially, the anticancer properties of this compound were found in recent studies. Xanthohumol is reported to play a central role in the induction of apoptosis in A549 lung cancer cells. ⁴ The compound has also been tested for efficacy against different cancer cell lines and experimentally verified data substantiated its role as an effective anticancer agent. ^{5 -7}

Flavonoids have attracted considerable attention because of their wide ranging pharmacological properties. These compounds have been shown to control strategically a myriad of cellular mechanisms to safeguard the proper functionality of cells and organs. ^{8 -10} Previously, we studied the interactions between natural phenolic compounds with 2 cytosolic isoforms CAI and II. ¹¹ Carbonic anhydrases (CAs) are zinc-containing metalloenzymes with a ubiquitous distribution. These enzymes play a leading role in the transportation of CO₂ and protons. Carbonic anhydrase isoenzymes are heterogeneously distributed within tissues, organs, and cells. Carbonic anhydrases can be therapeutically targeted for the treatments of epilepsy, glaucoma, and cancers. ^12,13 Carbonic anhydrase isoenzymes are reportedly involved in the regulation of pH homeostasis, transportation of ions, electrolyte balance, bone resorptions, and tumorigenesis. Carbonic anhydrases catalyze the reversible hydration of CO₂ to form HCO₃ ⁻. They are involved in the modulation of biosynthetic reactions, including biosynthesis of amino acids, gluconeogenesis, lipogenesis, and biosynthesis of pyrimidine nucleotides. ¹³ Phenol binds to CA in a diverse manner as compared with most CA inhibitors. Interestingly, most of the clinically used CA inhibitors belong to the sulfonamide class, as they possess primary sulfonamide (sulfamate and sulfamide) moieties as the zinc-binding functions. ¹⁴ Nair et al reported the X-ray crystal structure for the adduct of hCAII with phenol. Mechanistically, it was shown that phenol physically interacted with hCAII and anchored its OH moiety to the zinc-bound H₂O/hydroxide ion of the enzyme through a hydrogen bond, as well as to the NH amide of threonine-199. ¹⁴ In our recent work, we explored an interaction potential of different chemical compounds with 2 catalytically active isoforms of CAs. ^{15 -19} Inhibitory effects of different chemicals have previously been tested against many mammalian, fish, fungal, and bacterial CAs. ^{11,14 -21} The use of enzyme inhibitors has a great effect in the treatment of various diseases. Acetylcholinesterase (AChE) is responsible for the termination of signal transduction in the cholinergic system due to its superior hydrolytic potential. Acetylcholinesterase substrate, acetylcholine, is a neurotransmitter of the cholinergic system and has a dominant effect on motor neurons that play a role in memory formation. ²² Acetylcholinesterase, located in the postsynaptic membrane, hydrolyzes acetylcholine and ends neuronal signal transduction. Butyrylcholinesterase (BChE) is produced in the liver and is found mainly in blood plasma, and the central nervous and peripheral nervous systems. ^23,24 In clinical trials, AChE inhibitors have been reported to increase the amount of acetylcholine in cholinergic synapses and to increase cholinergic function. ²⁵ Under normal conditions, acetylcholine is hydrolyzed more dominantly by AChE in comparison with BChE. Although the BChE enzyme is thought to play a minor role in regulating brain acetylcholine levels, it has been reported that BChE levels are directly related to drug metabolism and detoxification. ^{26 -28} Specific inhibitors can be used in the treatment of certain motor neuron diseases, such as myasthenia gravis, dementia, and Alzheimer’s, by reducing the activities of AChE and BChE. ²²

In the current work, we have isolated, xanthohumol, the principal component of H. lupulus cones and evaluated its ability to inhibit hCAI and II isoenzymes and AChE and BChE. Also, molecular docking studies were used to investigate ligand interaction diagrams of xanthohumol at the binding cavities of hCAI and hCAII (Figures 1 and 2).

Figure 1

3D (top) and 2D (bottom) ligand interaction diagrams for xanthohumol at the binding pocket of hCAI.

Figure 2

3D (top) and 2D (bottom) ligand interaction diagrams for xanthohumol at the binding pocket of hCAII.

K _I values calculated to compare the inhibitor effects of xanthohumol and positive controls are summarized in Supplemental Table S1. According to the results of the study, hCAI and hCAII were strongly inhibited by xanthohumol. K _I values were determined as 0.085 and 0.049 µM for hCAI and hCAII isoenzymes, respectively. Also, catechin, quercetin, p-coumaric acid, and phenol were used as positive controls for inhibitors. Their K _I values were reported as 6.8, 3.6, 1.07, and 17.3 µM, respectively. ^29,30

When compared with positive controls, the highest inhibitory effects were exhibited by xanthohumol on the CA isoenzymes. Between all the tested compounds, xanthohumol was the strongest inhibitor. More importantly, its inhibitory effect on hCAII was also found to be effective with a value of 0.049 µM K _I. According to our literature survey, the inhibition effects of xanthohumol on hCAI and hCAII were evaluated for the first time in the current study. It is relevant to mention that the presence of a ring between the phenol rings of catechin and quercetin makes these molecules structurally rigid. However, there is no ring between the phenol rings of xanthohumol, so it is structurally flexible and can act as p-coumaric acid. As indicated in Supplemental Table S1, they showed weaker inhibitory effects than xanthohumol. Among all the compounds in Supplemental Table S1, phenol has a single basic ring. Because it had a smaller chemical structure, it showed inhibition at higher concentrations, since it could not chemically interact with other compounds.

K _I values were determined as 95.5 and 124.9 nM for AChE and BChE, respectively (Supplemental Table S1). When compared with xanthohumol and tacrine, which is a specific inhibitor, the values are very close to each other and, accordingly, xanthohumol showed an effective inhibition of AChE and BChE. ³¹ In accordance with our results, xanthohumol, which has a prenyl skeleton, showed a strong inhibitory effect, such as the prenylated flavonoids isolated from Sophora flavescens. ³² Induced Fit Docking (IFD) scores of xanthohumol in binding to the catalytic domains of hCAI and hCAII are calculated in Figures 1 and 2. Figure 1 shows 2D and 3D ligand interaction diagrams for the top-docking pose of xanthohumol at the active site of hCAI. While Trp5, His67, and Gln92 construct H-bonds with the ligand, Phe91 forms π-π stacking interactions. Figure 2 represents the top-docking pose of xanthohumol at the binding pocket of hCAII. The observed crucial amino acids were His94, Gln92, and Thr199 for the ligand interaction.

Hop cones are natural products used in the production of beer, giving it a bitter taste. Xanthohumol, an essential bioactive molecule isolated from hop cones, has considerable pharmacological importance. It was found to be effective against hepatic steatosis and fibrosis. ³³ In this work, we have investigated a new biological activity of xanthohumol. We have shown that it strongly inhibits hCAI, hCAII, AChE, and BChE in vitro. Until today, many studies have been carried out on CA isoenzymes, which have characteristically unique features and an extraordinary ability to modulate different biological mechanisms. In this study, catechin, quercetin, p-coumaric acid, and phenol were used as standards against hCAI and hCAII. Xanthohumol exhibited the strongest inhibitory activity among the tested compounds. Furthermore, it also showed an inhibitory activity comparably closer to tacrine. Butyrylcholinesterase enzymes are key players in the regulation of brain AChE levels. Acetylcholinesterase inhibitors are currently being tested as possible new agents in the treatment of Alzheimer’s disease. ³¹ Hence, xanthohumol may be suggested as a potential new natural agent in the treatment of this disease. In accordance with our results, in a recent study, xanthohumol was demonstrated to exert a moderate inhibitory activity toward AChE and BChE (IC₅₀ = 71.34 ± 2.09 and 32.67 ± 2.82 µM, respectively) in comparison with that of galanthamine (IC₅₀ = 2.52 ± 0.15 and 46.58 ± 0.91 µM, respectively). ³⁴ In conclusion, xanthohumol was noted to exert potent inhibitory effects at μM levels on the metabolic enzymes hCAI and hCAII and AChE and BChE. In particular, xanthohumol was found to be a potent inhibitor of hCAII isoenzyme for first time in the present study. Our data clearly suggest that xanthohumol may prove to be a strong candidate for the treatment of some diseases such as glaucoma, mountain sickness, epilepsy, Alzheimer’s, and ulcers. However, further studies are needed to determine the safety, toxicity, and economical value of xanthohumol as a new drug agent.

Experimental

General

Hop cones were obtained from the Pazaryeri District Directorate of Agriculture in August 2014, in Turkey. Plant samples were carefully stored in the Central Research and Application Laboratory, Agri Ibrahim Cecen University. Chemicals were purchased from Merck, Fluka, Alfa, and Aldrich. Thin layer chromatography (TLC) and prep. TLC: silica gel 60 F-254 (Merck, precoated plates); visualization by UV₂₅₄ and UV₃₆₅, and by spraying with 1% vanillin-H₂SO₄, followed by heating (105°C). Column chromatography: silica gel 60 (Merck, 70-230 and 200-400 mesh). UV/VIS: Jasco V-530 spectrophotometer; λ_max in nm. 1D- and 2D-NMR spectra: Bruker 400 MHz (¹H: 400 MHz and ¹³C: 100 MHz) spectrometer; CDCl₃, soln.; δ in ppm rel. to Me₄Si as an internal standard, J in Hz.

Extraction of Hop Cones and Isolation of Xanthohumol

Xanthohumol was isolated from hop cones and characterized as in our previous study. ³⁵

Inhibition of hCAI and hCAII Isozymes

Activity of enzymes was determined spectrophotometrically. Absorbance was recorded at 348 nm upon conversion of 4-nitrophenylacetate to 4-nitrophenylate over a 3-minute duration at 25°C. ²⁰ Quercetin, catechin, resorcinol, catechol, p-coumaric acid, and phenol were used as test compounds. Various inhibitory concentrations were tested and triplicate analysis of all compounds was done. Control cuvette activity was recorded as 100% in the absence of any inhibitor. For each inhibitor, an activity %-[inhibitor] graph was drawn. ^21,36 The curve-fitting algorithm was used to obtain IC₅₀ values, working at the lowest concentration of substrate of 0.15 mM, from which K _I values were calculated. ^{13 -15,19} Catalytic activities of these enzymes were calculated from Lineweaver-Burk plots. ³⁶ The CA isoenzymes were purified from human blood, as previously described. ^21,37,38

Induced Fit Docking Simulations

The IFD module of Maestro molecular modeling package was used for the docking simulations. ^39,40 Induced Fit Docking provides partial flexibility to the active site residues together with the full conformational flexibility of ligand. The IFD procedure was established in 3 consecutive stages: (i) docking of the ligand to the binding pocket of the target structure; (ii) refining of amino acid residues within 5 Å of docked poses; and (iii) re-docking of docked ligands against the refined structure.

Inhibition of AChE and BChE

The inhibitory effects of xanthohumol on AChE and BChE were measured using Ellman’s colorimetric method. ⁴¹

Supplemental Material

Supplemental Table S1 - Supplemental material for Inhibitory Effects and Kinetic-Docking Studies of Xanthohumol From Humulus lupulus Cones Against Carbonic Anhydrase, Acetylcholinesterase, and Butyrylcholinesterase

Supplemental material, Supplemental Table S1, for Inhibitory Effects and Kinetic-Docking Studies of Xanthohumol From Humulus lupulus Cones Against Carbonic Anhydrase, Acetylcholinesterase, and Butyrylcholinesterase by Tuba Aydin, Murat Senturk, Cavit Kazaz and Ahmet Cakir in Natural Product Communications