Abstract
Different natural products and secondary metabolites from mushrooms, teas, honeys, mosses, plants and seaweeds were investigated for their in vitro inhibitory effects on human carbonic anhydrase (hCA, E.C.4.2.1.1) isoforms I and II. Inhibition data were correlated with the total phenol content in the extract and investigated with the pure compounds believed to be responsible for this activity. Methanolic extracts were prepared for 17 such pure chemicals present in the natural products and for 41 diverse natural products. The IC50 values were in the range of 0.11–66.50 μg/mL against hCA I and of 0.09–54.54 μg/mL against hCA II, respectively. The total phenol content was in the range of 0.02–1318.96 (as milligrams of gallic acid equivalents) per gram of sample. These data offer new insights on possible novel classes of CA inhibitors based on natural products, possessing a range of chemical structures not present in the classical inhibitors with pharmacological applications, such as the sulfonamides and sulfamates.
Introduction
Carbonic anhydrase (CA, E.C.4.2.1.1), an enzyme purified from erythrocytes for the first time in 1993Citation1, plays an important role in mammals, in processes such as pH control, gas balance, ion transport, calcification, secretion of electrolytes, and tumourigenesis among othersCitation2. CAs, of which many diverse isoforms are currently known, effectively catalyze a slow, but fundamental physiological reaction, the conversion of carbon dioxide to bicarbonate and protonsCitation3,Citation4. CAs are classified in five distinct classes, the α, β, γ, δ and ζ familiesCitation5. These types of CAs are present in different organisms, but the α-CAs are the only such enzymes found in mammals. The β-class is mainly found in plants, fungi and prokaryotes; γ-CAs were identified in Archaea and some bacteriaCitation6. The ϵ-CA class was reclassified as a different type of β-CA based on crystallographic dataCitation7,Citation8, which showed a nearly identical fold to those of the canonical, archaeal (cab-type) and plant-type (Pisum sativum) β-CAsCitation9–11. In ζ-CA, the geometry of the active site is nearly identical to that of β-CAs, and there is also some similarity in the protein fold, but these enzymes contain Cd(II) at their active site, not Zn (II) as the other genetic CA familiesCitation6, although they function also with Zn(II) or Co(II) replacing the cadmium ion.
In mammals, 16 different CA isoenzymes have been described so far2. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII and CA XIII), others are membrane bound (CA IV, CA IX, CA XII and CA XIV), two are mitochondrial (CA VA and CA VB) and one is secreted in saliva and milk (CA VICitation12). Human and most mammalian red blood cells comprise two CA isozymes, CA I (slow enzyme of low catalytic efficiency) and human CA (hCA II; rapid, highly effective catalysts for the CO2 hydration reactionCitation13). hCA I and hCA II are two of the most abundant protein (after hemoglobin) in human erythrocytes.
The catalytic mechanism of CAs is understood in detail. In all enzyme classes, a metal hydroxide species of the enzyme is the catalytically active species, acting as a strong nucleophile on the CO2 molecule bound in a hydrophobic pocket nearbyCitation2. This metal hydroxide species is generated from water coordinated to the metal ion, which is found at the bottom of the active site cavity. The active centre normally comprises M (II) ions in tetrahedral geometry, with three protein ligands (L) in addition to the water molecule/hydroxide ion, but Zn(II) and Co(II) were also observed in trigonal bipyramidal or octahedral coordination geometriesCitation2,Citation14–18.
The inhibition of CA is a well understood process, with most (but not all) classes of inhibitors binding to the metal centreCitation19–29. Inhibition can be achieved by four different mechanisms. First, coordination of the inhibitor to the Zn(II) ion by replacing the zinc-bound water/hydroxide ion, leading to a tetrahedral geometry of Zn(II). Second mechanism is addition of the inhibitor to the metal coordination sphere, when the Zn(II) ion is in a trigonal bipyramidal geometryCitation12,Citation30. Third type of inhibition consists of the anchoring of the inhibitor molecule to the Zn (II)-bound solvent molecule, a water or hydroxide ionCitation31. The fourth type of inhibition is achieved by occlusion of the entrance to the active site cavity, when the inhibitors bind in the activator binding regionCitation32–36. Many CA inhibitors were synthesized and evaluated in the last decades, whereas natural product compounds started to be investigated only recentlyCitation37. For this reason, the primary objective of this study was the screening of in vitro inhibitory effects on the cytosolic, widely spread isoforms hCA I and hCA II of some natural product extracts and the corresponding pure chemicals species present in them. Most of these compounds are known to possess antioxidant effects.
Materials and methods
Reagents
Analytical grade methanol was obtained from Merck Co. (Merck, Darmstadt, Germany). Buffers and other reagents were of the highest purity grade, from Sigma-Aldrich (Milan, Italy). CA isozymes were recombinant ones obtained as reported earlier. Folin-Ciocalteu’s phenol reagent was from Fluka Chemie GmbH (Switzerland). Polytetrafluoroethylene membranes (porosity 0.2 μm) for the filtration of the extracts were obtained from Sartorius (Goettingen, Germany).
Samples and preparation of extracts
All samples were prepared in methanol. Because chemicals can be dissolved completely in methanol, an extraction process was not used. The natural products were continuously stirred with a shaker at 60°C for 24 h. The suspension was removed by filtration then centrifuged at 10,000g for 15 min. The supernatant was concentrated in a rotary evaporator under reduced pressure, and the residue resolved in a minimal volume of the same solvent and kept in 4°C until use.
CA catalytic activity and inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as an indicator, working at the absorbance maximum of 557 nm, with 20 mM HEPES (pH 7.5) as buffers and 20 mM Na2SO4 (for maintaining constant ionic strength), following the initial rates of the CA-catalysed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each sample, at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Solutions of extracts (10 mM) were prepared in distilled, deionized water and diluted up to 0.01 nM thereafter with distilled, deionized water. Inhibitory sample and enzyme solutions were preincubated together for 15 min at room temperature before assay to allow for the formation of the enzyme–inhibitor complex. The IC50 represents the concentration of inhibitor producing a 50% decrease of the catalytic rate and was obtained by nonlinear least-squares methods using PRISM 3 and represent the mean from at least three different determinations.
Determination of total phenolics
Total phenolic (TP) content was determined by the Folin-Ciocalteu procedureCitation38 using gallic acid as standard. In brief, 20 μL of various concentrations of gallic acid and samples (20 μL), 400 μL of 0.5 N Folin-Ciocalteu reagent and 680 μL of distilled water was added, and the contents were vortexed. After 3 min incubation, 400 μL of Na2CO3 (10%) solution was added, and after vortexing, the mixture was incubated for 2 h at 25°C with intermittent shaking. The absorbance was measured at 760 nm at the end of the incubation period. The concentration of TP compounds was calculated as milligrams of gallic acid equivalents (GAE) per gram of 100 g FW, by using a standard graph for gallic acid in the concentration range between 0.015 and 0.5 mg/mL (r2 = 0.9997).
Statistic analysis
Results ate presented as mean values of two replicates. Data and regression analyses were tested using SPSS for Windows Release 10 (SPSS Inc.).
Results and discussion
Many natural products are rich in phenolic compounds and possess antioxidant, antibacterial, anti-inflammatory, antiallergic and antithrombotic activitiesCitation37,Citation39,Citation40. As far as we know, such products have never been tested for their inhibitory activities against the CA family of enzymes. Indeed, CA inhibitors, especially aromatic and heterocyclic sulfonamides, have been and are used clinically for more than 50 years in the treatment of a variety of diseases such as glaucoma, epilepsy, obesity, mountain sickness, gastric and duodenal ulcers, osteoporosis and acid–base disequilibriaCitation2. Recently, some sulfonamide CA inhibitors were demonstrated to possess relevant antitumour and antimetastatic effectsCitation41,Citation42.
We have prepared some pure chemical standards and natural compounds, most of which are aromatic derivatives based on the flavone core structure, various substituted phenols/polyphenols and phenolic aromatic acids as methanolic extracts and measured their TP content ( and ). Total phenols were expressed in milligrams of gallic acid per gram of sample. Especially, rhamnetin, protocatechuic acid and catechin extracts showed higher phenolic content than other preparations. It may be observed that in the chemical samples, the total polyphenol contents were between 0.76 ± 0.37 and 1318.96 ± 1.00 mgGAE/g of sample, whereas for the mushrooms 0.57 ± 0.05 and 3.49 ± 0.35, teas 59.71 ± 1.27 and 602.92 ± 1.09, honeys 0.20 ± 0.01 and 0.90 ± 0.05, natural compounds 0.05 ± 0.01 and 37.58 ± 0.01 mgGAE/g sample ().
Table 1. The name of samples and their codes.
Table 2. Total phenolic content (TPs) and inhibitory effects of samples against hCA I and hCA II (as IC50-s) from three different determinations.
Table
Table
We measured thereafter the inhibitory effects of these samples against the purified cytosolic CA isoforms hCA I and II, which are among the physiologically most relevant such enzymesCitation2. The inhibition results are expressed as IC50 and were found to be in the range of 0.11–66.50 μg/mL for hCA I and 0.09–54.54 μg/mL for hCA II (). The meaning of IC50 is the concentration of compound (molarity or in this specific case, expressed in mg/mL) that reduces the CA activity by 50%. Among all the investigated samples, it may be observed that the extraction method highly influenced the CA inhibitory activity against both isoforms. Because the components are pure chemicals belonging to the polyphenol class, they are expected to show some inhibition against hCA I and II, and phenols are highly investigated as CA inhibitorsCitation37. Rhamnetin showed the highest value of total polyphenol content, according to the gallic acid standard, and it was also a potent inhibitor of hCA I and II. trans-Cinnamic acid (C17) was the best inhibitor, with IC50 values of 0.11–0.09 μg/mL (). It should be mentioned that 2-hydroxy-trans-cinnamic acid was recently discovered to be a CA inhibitor, being formed from coumarin (a prodrug) by active-site–mediated hydrolysisCitation33,Citation34. This compound binds in a particular manner to the enzyme, at the entrance of the active site cavity, as shown by X-ray crystallography. Coumarins also led to highly isoform-selective CA inhibitorsCitation33,Citation34, and one such compound has notable antitumour and antimetastatic properties, being in preclinical evaluation as an anticancer drugCitation41.
White tea is prepared from young tea leaves or buds covered with tiny, silvery hairs, which are harvested only once a year in the early spring. It is richer in phenolic compounds, especially catechin and its derivatives, compared with mature teaCitation43. Pleurotus eryngii contains various phenolic components especially catechin, similar to white teaCitation44. Because of the high catechin content, white tea (T3), among the tea species and P. eryngii (M3) among the mushrooms, showed the lowest IC50 and highest total polyphenol value among the investigated extracts ().
Different honey types contain diverse components that have antioxidant, antimicrobial, antiviral, and antifungal effects, by mechanisms not well elucidated yetCitation45–47. Chestnut honey is one type of honey known for its excellent properties. Küçük et al.Citation45 reported that chestnut honey contains superior amounts of the total polyphenols (which are responsible for the antioxidant effects) compared with rhododendron and flower honeys. Chestnut honey (H5) investigated here showed the best value on hCA I and II inhibition among the various honeys.
Phenolic compounds were investigated in Mentha piperita by Areias et al.Citation48 According to this study, M. piperita contains a high percentage of phenolic compounds such as eriodictyol, luteolin, hesperetin, apigenin, and rosmarinic acid, etc.Citation48 These compounds might be responsible for the inhibition of hCA I and II observed in the current study, but this hypothesis must be verified.
Marine algae are potentially prolific sources of highly bioactive secondary metabolites that might represent useful leads in the development of new pharmaceutical agentsCitation49. According to Chewa et al.Citation50, Padina pavonica contains a high amount of polyphenolsCitation50. However, our results show that the value of TP compounds is similar to each other among all investigated seaweeds (). This also may explain the CA inhibition effects of the algal extracts, which were similar to each other ().
In conclusion, we report that a CA I and II inhibition study with natural product compounds that contain polyphenols and flavones, extracted from various plants, mushrooms and honey. Many of these rather effective inhibitors detected here can be used as lead compounds for developing novel classes of CA inhibitors, presumably with a new mechanism of inhibition, which may be similar to that of the coumarins, newly discovered class of inhibitors of these enzymes.
Acknowledgments
We also thank Assoc. Prof. Dr. Turan ÖZDEMIR for providing and identifying the moss sample Biology Department, Karadeniz Technical University, and also thanks to Assoc. Prof. Dr. Ömer ERTÜRK for providing and identifying the seaweeds sample Biology Department, Ordu University. Work from Supuran lab was financed by two FP7 EU projects (Metoxia and Gums and Joints).
Declaration of interest
This study was supported by Research Fund of Karadeniz Technical University (Project No: 2009.111.002.5). One of the authors (H. Sahin) would like to thank TUBİTAK, BİDEB, TURKEY, for the financial support given to him.
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