Inhibitory effects of some phenolic compounds on the activities of carbonic anhydrase: from in vivo to ex vivo

Abstract

Carbonic anhydrase (CA) inhibitors have been used for more than 60 years for therapeutic purposes in many diseases table such as in medications against antiglaucoma and as diuretics. Phenolic compounds are a new class of CA inhibitor. In our study, we tested the effects of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid on esterase and the CO₂-hydratase activities of CA I and II isozymes purified from in vivo to ex vivo. The K_i values of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid were 203.80, 1170.00 and 910.00 μM, respectively for hCA I and 75.25, 354.00 and 1510.00 μM, respectively for hCA II. Additionally, IC₅₀ values from in vivo studies were found to be in the range of 173.25–1360.0 μM for CA I and II, respectively, using CO₂-hydratase activity methods. These results demonstrated that phenolic compounds used in in vivo studies could be used in different biomedical applications to inhibit approximately 30% of the CO₂-hydratase activity of the total CA enzyme of rat erythrocytes.

• Arachidonoyl dopamine
• carbonic anhydrase
• enzyme inhibition
• ex vivo
• in vivo
• phenolic compounds

Introduction

Carbonic anhydrases (EC 4.2.1.1) as a metalloenzyme family are determined to be encoded from six different independent gene families (α-, β-, γ-, ɛ-, ζ- and n-CA) and are found in archaea, eukaryotic and prokaryotic cells1,2. Mammals, including humans, generally contain α-CAs. Zn²⁺is located in the active site of the enzyme2,3. Until now, 16 different α-CA isoenzymes have been identified in various tissues and organs with different expression levels, kinetic and molecular properties, and oligomeric rearrangements as well as various abilities to respond to different inhibitory classes4,5.

There are 13 catalytic and three acatalytic isoforms of CA-related proteins (CARPs)6. According to the known cellular localisation, these isoenzymes are cytosolic (CA I, II, III, VII V and XIII), membrane-bound (CA IV, IX, XII, XIV and XV), mitochondrial (CA VA and VB) and salivary (CA VI)7. CA XV is not synthesised in humans and other primates and is abundantly found in rodents and other vertebrates as an isoform8. Acatalytic CARP VIII and X appear to be cytosolic proteins9.

CAs play an important role in the respiratory system and transport of CO₂/HCO₃⁻ between the lungs and other tissues. The enzyme also maintains the pH and CO₂ balance by the catalytic reversible hydration10 of CO₂ to produce H⁺and HCO₃⁻:

In addition, they are associated with biosynthetic reactions such as ureagenesis, glycogenesis and lipogenesis, and with other physiological and pathological processes such as tumuorigenicity, bone resorption and calcification in various tissue and organs11,12. Therefore, inhibitors of CAs may be able to be used as therapeutic agents in the treatment and prevention of various diseases13. For instance, sulphonamides have been used as agents in pharmacological application and diagnostics in many areas such as antiglaucoma, anti-obesity, anticonvulsants and anticancer for more than 60 years in clinical settings14.

Recently, many studies have shown that phenolic compounds and their derivatives might have a beneficial inhibitory effect on CAs that is versatile and promising. Thus, phenolic compounds have the potential to become a new class of CA inhibitor and may be an alternative for patients who are allergic to sulpha drugs and derivatives15. The zinc ion (Zn²⁺) in CAs is critical for the inhibition profile of these enzymes. Thus far, some inhibition mechanisms have been proposed for CA isoenzymes. One involves anchoring, the inhibitor to a Zn²⁺ bound solvent molecule, such as water or hydroxide ion. Phenols and polyamines bind CA isoenzymes in this way. Phenolic compounds are very important in our daily lives. They are added to the content of many drugs and foods that are widely used. Natural and non-natural phenolic compounds have been reported to have anticancer, antimutagenic, antiviral, antibacterial, anti-inflammatory and anticarcinogenic effects in addition to their antioxidant properties16.

Arachidonoyl dopamine, which is used in this study, an endogenous lipid of central nervous system that provides each bidirectional transition ionotropic of the vanilloid type 1 receptor17. At the same time, it is one of the best activators of cannabinoid and transient receptor potential vanilloid channel receptors18. Studies have recently demonstrated that hyperalgesia was primarily induced by endovanilloid N-arachidonoyl dopamine (NADA). In addition, environmental endovanilloid causes the contraction of smooth muscle in the guinea-pig bronchus and urinary bladder. Because of the molecular structure, NADA may also indicate the potential properties of phenolic compounds (Figure 1)19.

Figure 1. The chemical structure of phenolic compounds, including arachidonoyl dopamine, 3,4-dihydroxy-5-methoxybenzoic acid and 2,4,6-trihydroxybenzaldehyde as inhibitors for hCA I and II isozymes.

In recent years, a series of phenolic compounds and a phenolic acid were investigated for their in vitro inhibitory effects on CA I and II isoenzymes isolated from human erythrocytes. The significant inhibitory properties of phenolic substances such as phenol20, antioxidant phenols21, phenolic acids22, natural product polyphenols and phenolic acids23, natural phenolic compounds24, dimethoxybromophenol25, bromophenols26,27, catechol, resorcinol, hydroquinone28, taxifolin29 pyrogallol, quercetin, guaiacol and its phenolic derivatives30, vanillin, caffeic acid phenethyl ester31, curcumin, silymarin, resveratrol, morphine, catechin, dobutamine15 and rosmarinic acid32 of CA I and II isoenzymes16 have been reported.

In our study, we also investigated the in vitro inhibitory effects of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid on CA I and II isoenzymes from human erythrocytes. In vivo studies were performed for the phenolic compounds (Figure 1). The results obtained from the study provide important new information concerning phenolic compounds.

Experimental procedures

General information

Ethics committee permission was received for in vitro and in vivo analysis from Experimental Animals Local Ethics Committee and Non-Drug Clinical Applications Local Ethics Committee of Yuzuncu Yıl University.

Chemicals

Arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid were commercially purchased from Sigma-Aldrich (St. Louis, MO). Cyanogen bromide (CNBr)-activated Sepharose 4B, l-tyrosine, sulphanilamide for affinity purification and other chemicals for electrophoresis and all chemicals for enzyme activity measurement were obtained from Merck (Kenilworth, NJ) or Sigma-Aldrich.

Purification of CA isoenzymes from human erythrocytes

Fresh human blood was received from volunteers at the blood centre of Yuzuncu Yıl University, Faculty of Medicine Research Hospital. Blood samples placed into centrifuge tubes with heparin were centrifuged at 1500× g for 15 min and the overlying plasma and leukocyte layers were removed carefully using a dropper. There after, the samples were washed three times with a 0.9% isotonic NaCl solution. Erythrocytes were haemolysed with ice water until five times of their volume33. The haemolysate was centrifuged at +4 °C and 10 000× g for 30 min to remove cell membranes and other impurities. The supernatant was carefully removed using a dropper. A Sepharose-4B-L-tyrosine-sulphanilamide affinity column was equilibrated with a solution of 25 mM Tris-HCl/0.1 M Na₂SO₄ (pH 8.7). The haemolysate was adjusted to pH 8.7 with solid Tris and then they were applied to the column (1.36 × 30 cm, Sigma-Aldrich). The affinity column was then washed with a solution of 25 μM Tris-HCl/22 μM Na₂SO₄ (pH 8.7). Thus, the CA enzyme was bound to the column, and other impurities were removed. There after, the hCA I isoenzyme was eluted by applying a solution of 1 M NaCl/25 mM Na₂HPO₄ (pH 6.3). The hCA II isoenzyme was obtained using a solution of NaCH₃COO 0.1/0.5 M NaClO₄ (pH 5.6) from the column (flow rate: 20 mL h⁻¹, fraction volume: 4 mL). All of the procedures were performed at +4 °C34. The qualitative protein assay was performed at 280 nm in every purification step, spectrophotometrically. CO₂ hydratase activity was determined for all of the eluates and the active fractions were collected (Figure 2)35.

Figure 2. Elution graph of the hCA I and II isoenzymes purified by using Sepharose-4B-L-Tyrosine affinity chromatography.

Determination of quantitative proteins

The protein quantity in all of the samples was determined using bovine serum albumin as a standard according to the Bradford method36 by measuring absorbance at 595 nm as explained previously37.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

The purities of the isozymes were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli’s method34. Thus, samples treated with 1% SDS and 10% 2-mercaptoethanol were incubated in a boiling water bath for 5 min for denaturation. The hCA I and II isoenzyme samples were next applied to B, C, E and F wells in the electrophoresis medium (Figure 3). The gel was dyed by incubating it for 1.5 h in medium containing 0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% acetic acid. The resulting electrophoresis bands were visualised (Figure 3)38,39.

Figure 3. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified CA I and II isozymes from human erythrocytes. Lane A and D: standard proteins (kD): β-galactosidase from E. coli (116), phosphorylase B from rabbit muscle (97), bovine serum albumin (66), chicken ovalbumin (45) and carbonic anhydrase (29). Lanes B and F: human carbonic anhydrase I. Lanes C and E: human carbonic anhydrase II.

CO₂-hydratase activity

The activity studies were performed according to the Wilbur and Anderson method as modified by Rickly et al.40 CA activity was assayed with changes in the pH as non-enzymatic (t_o) and enzymatic (t_c) reactions. An enzyme unit (EU) was calculated using the equation (t_o−t_c/t_c) in this method.

Esterase activity

Esterase activity was measured at 348 nm spectrophotometrically (Shimadzu UV mini-1240, Kyoto, Japan). The measurement was performed for 3 min at room temperature. The substrate was 4-nitrophenylacetate (4-NPA) according to the method defined by Verpoorte et al41. The reaction mix contained 1.4 mL of 0.05 M (pH 7.4) Tris-SO₄ buffer, 1 mL of 4-NPA (3 mM), 560 μL of distilled water and 40 μL of the enzyme solution (final volume 3 ml). Other processes are described in our previous study42,43.

In vitro inhibition studies

Inhibition studies were performed on the esterase activities and CO₂-hydratase activities of hCA I and II of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid. The arachidonoyl dopamine concentrations were changed from 113.7 to 568.75 μM for hCA I and from 113 to 455 μM for hCA II in in vitro inhibitory studies on esterase activity. The concentrations for 2,4,6-trihydroxybenzaldehyde were from 649 to 1947 μM for hCA I and from 32 to 162 μM for hCA II. They were from 815 to 1629 μM for hCA I and from 272 to 1629 μM for hCA II of 3,4-dihydroxy-5-methoxybenzoic acid. The mathematical relationship between the CA activity and inhibitor concentrations was determined using conventional polynomial regression software (Microsoft Office 2007, Excel, Redmond, WA). The IC₅₀ values (the inhibitor concentration that causes up to 50% inhibition) were calculated from graphs. The K_i values and inhibition types of the compounds were determined by Lineweaver–Burk graphics44.

In vivo inhibition studies

Permission from the ethics committee for the in vivo studies was obtained from Yuzuncu Yıl University Experimental Animals Local Ethics Committee. Forty-eight albino rats test animals of (250 ± 20 g) were selected and separated into eight equal groups (n = 6). 2,4,6-Trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid were applied at a dose of 10 mg/kg, and arachidonoyl dopamine was also applied at a dose of 5 mg/kg, intraperitoneally. The blood samples were drawn into the anticoagulant-containing tubes at 1st and 4th hours for each group from the heart under anaesthesia (10 mg/kg ketamine) according to the Gülçin et al.33 Additionally, there were control groups for the 1st and 4th hours. Blood samples were centrifuged at +4 °C and 2500× g for 15 min. Plasma and leucocytes were removed carefully and erythrocytes were haemolysed with ice-water43. Next, CO₂-hydratase activities were assayed colorimetrically according to the Wilbur and Anderson method40 as described above.

Measurement of haemoglobin (Hb)

The determination of haemoglobin was performed using the cyanmethamoglobin method in haemolysates. This method is based on a spectrophotometric measurement at 540 nm of the cyanomethemoglobin concentration formed after oxidation to Fe³⁺from Fe²⁺ions. The experimental procedure was conducted according to that in our previous studies43. The results of the CA activities were given as the EU/gHb for in vivo studies. The time to decrease to half of the enzyme activity (t₅₀) was calculated using % Activity-Time charts.

Statistical analysis

The obtained results were expressed as the mean and standard deviation. Tukey’s range test to compare between groups was used for post-hoc analysis. Student’s t-test was performed for intra-group comparisons with respect to time within the same group. Significance was taken as 5% (p < 0.05) in statistical calculations, and SPSS 15 statistical software package (SPSS Inc., Chicago, IL) was used for calculations.

Results and discussion

Sulphonamides and their derivatives are classic inhibitors of CAs45. Sulphamates and sulphonamides are used in medications associated with different clinical applications such as in anticonvulsant, anti-obesity, anticancer and anti-infective46. However, many of these compounds inhibit 16 different isoforms of the CAs at different levels in mammals. Researchers have recently identified new sulphamides and sulphonamides compounds as inhibitors. Presently, variety inhibitors have been discovered, such as coumarins, polyamines and phenols. They have different features from sulphonamides, including interesting inhibition mechanism47. For instance, it has been demonstrated that mammalian isoforms I–XIV of CA are inhibited at low concentrations by ferulic acid, p-hydroxybenzoic acid, p-coumaric acid, gallic acid, quercetin, ellagic acid and syringic acid23. In addition, Innocenti and co-workers have reported that a series of phenolic compounds such as resveratrol, silymarin, dobutamine, curcumin and catechins inhibited the esterase activity of mammalian CA isoenzymes. They state that these molecules have different molecular structures from other CA inhibitors and exhibited different inhibitory profiles with various CA isozymes. The obtained data can lead to drug design studies with different inhibition mechanism48.

In the present study, we purified the hCA I and II isozymes in one simple step by Sepharose-4B-L-tyrosine-sulphanilamide affinity chromatography, one of the best protein purification methods. To prepare the affinity gel, Sepharose 4B was used with CNBr-activated as a matrix. L-tyrosine was inserted covalently into the matrix as a long arm and sulphanilamide was used as a ligand in the affinity gel49. Enzyme purity was checked by SDS-PAGE (Figure 3). The hCA I and II isoenzymes was purified with yields of 56 and 14%, specific activities of 4709.5 and 11057.1 and fold purifications of at 365 and 856, respectively (Table 1). The activities of the isozymes were measured using the esterase and CO₂-hydratase methods as in our previous studies43,50. Arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid were used as inhibitors for the hCA I and II isozymes. The IC₅₀ values, the concentration with a 50% decrease of the enzyme activity, were determined for each compound. For this purpose, the CA activities were measured at five different concentrations of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid (Tables 2 and 3). The IC₅₀ values were calculated graphically from measurements at five different inhibitor concentrations for the esterase activity of the hCA I and II isoenzymes (Table 2). Next, the average K_i values were also calculated from Lineweaver–Burk plots. Arachidonoyl dopamine has the best inhibition effect on esterase activity of the hCA I and II isoenzymes. The IC₅₀ and K_i values were determined to be 346.50–203.80 and 231.00–75.25 μM for arachidonoyl dopamine in vitro, respectively (Table 2). In addition, 2,4,6-trihydroxybenzaldehyde for the hCA I isoenzyme and 3,4-dihydroxy-5-methoxybenzoic acid for the hCA II isoenzyme showed the best inhibitory effect on the CO₂-hydaratase activities according to arachidonoyl dopamine. The IC₅₀ values were in the range of 115.50–542.88 μM and 1360.00–693.00 μM on the hCA I and II isoenzymes for the CO₂ hydratase activities, respectively (Table 3). Indeed, three compounds also demonstrated the inhibitory effect at low concentrations under in vitro conditions (Table 3).

Table 1. Summary scheme of hCA I and II isoenzymes purified using Sepharose-4B-L-tyrosine affinity chromatography.

Sample type	Activity (EU/mL)	Total volume (mL)	Protein (mg/mL)	Total protein (mg)	Total activity	Specific activity (EU/mg)	Yield (%)	Purification fold
Haemolysate	1020	77.5	78.98	6121.00	79 050	12.9	100	1
Sepharose-4B-L-tyrosine-sulphanilamide affinity column chromatography
CA I	5934	7.5	1.26	19.65	44 505	4709.5	56	365
C CA II	3096	3.5	0.28	0.98	10 836	11057.1	14	856

Table 2. The IC₅₀, K_i and inhibition type of phenolic compounds for the hCA I and II isozymes as ex vivo using the esterase activity method.

	hCA I			hCA II
Phenolic compounds	IC50 (μM)	Ki (μM)	Inhibition type	IC50 (μM)	Ki (μM)	Inhibition type
Arachidonoyl dopamine	346.50	203.80	Non-competitive	231.00	75.25	Uncompetitive
2,4,6-Trihydroxybenzaldehyde	1380.00	1170.00	Competitive	77.00	354.00	Non-competitive
3,4-Dihydroxy-5-methoxybenzoic acid	1550.00	910.00	Competitive	820.00	1510.00	Non-competitive

Table 3. The IC₅₀ values of substances for the hCA I and II isozymes ex vivo using the CO₂-hydratase activity method.

	hCA I		hCA II
Phenolic compounds	IC50 (μM)	r^2	IC50 (μM)	r^2
Arachidonoyl dopamine	173.25	0.9971	1070.00	0.9948
2,4,6-Trihydroxybenzaldehyde	115.50	0.9468	1360.00	0.9635
3,4-Dihydroxy-5-methoxybenzoic acid	542.88	0.9767	693.00	0.9991

In recent years, inhibition studies of many phenolic compounds have been performed extensively. These studies were conducted on various enzymes, including CA isozymes glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and lactoperoxidase51–54. Particularly, it was observed that the inhibition effects of phenolic compounds on CA isozymes are variable, and the inhibition can be in the millimolar to the submicromolar range. Researchers have determined that phenolic compounds are generally bound to the enzyme active site by K_i studies on CA isozymes. CA is a Zn²⁺-related enzyme, and this Zn²⁺-bound water molecule interacts with Thr199 in the active site55. This structure is a ligand for inhibitors such as phenolic compounds56,57. On the other hand, some inhibitors may interact with the active site of the CA.

The in vivo CA activity was assayed using the CO₂-hydratase activity method and represented the physiological activity of the CA enzyme. In vivo results from the enzyme activity studies are crucial to recognise the physiological role of the enzyme. Particularly, drug-enzyme or any chemical compound-enzyme interaction studies are important to understand the toxicological mechanisms. Thus, in vivo studies concerning various enzymes are available in the literature. For instance, Çoban and co-workers reported the in vivo effects of morphine on rat CA enzyme. They provided a single dose of 5 mg/kg to the rats intraperitoneally and found an in vivo inhibitory effect with 4.39 ± 0.63% activity values after 3 h on the CO₂-hydratase activity of rat erythrocyte CA58. In addition, it was demonstrated that the inhibitory levels were approximately 52% with 42% activity values after 1 h in rats treated with dantrolene (10 mg/kg)33. Additionally, Beydemir and colleagues revealed that gentamicin sulphate (3.2 mg/kg) inhibits the rat CA enzyme to approximately at 65% of the level at 1 h under in vivo conditions59. In the present study, we studied 48 albino rats (250 ± 20 g) separated into eight equal groups (n = 6). The dose of 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid was 10 mg/kg, intraperitoneally. The dose of arachidonoyl dopamine was 5 mg/kg, intraperitoneally. We observed approximately 30% inhibition levels as 73, 71 and 68% after 4 h for arachidonoyl dopamine, 3,4-dihydroxy-5-methoxybenzoic acid and 2-4-6-trihydroxybenzaldehyde, respectively. The inhibition effects were almost similar among the three chemicals at 4 h (Table 4). The half-life (t₅₀) values of the substances were calculated by activity%-time graphs in in vivo studies (Figure 4 and Table 5). The half-life (t₅₀) values were 8.34, 7.70 and 6.79 h for arachidonoyl dopamine, 3,4-dihydroxy-5-methoxybenzoic acid and 2,4,6-trihydroxybenzaldehyde, respectively. According to the results obtained from this study, 2,4,6-trihydroxybenzaldehyde in vivo inhibited the CA enzyme to a greater amount in a shorter time in vivo. The inhibition order was determined as 2,4,6-trihydroxybenzaldehyde > 3,4-dihydroxy-5-methoxybenzoic acid > arachidonoyl dopamine according to the activity and t₅₀ values. These results indicate that there is a significant correlation in between the half-life and CO₂-hydratase activity under in vivo conditions.

Figure 4. The half-life (t₅₀) values of the substances were calculated by activity%-elapsed time graphs in in vivo studies. The half-life (t₅₀) values were found for arachidonoyl dopamine, 3,4-dihydroxy-5-methoxybenzoic acid and 2,4,6-trihydroxybenzaldehyde. These experiments were performed using CO₂-hydratase activity assays.

Table 4. The activity values of substances for in vivo rat erythrocyte CA enzyme as IU/gHb using the CO₂-hydratase activity method.

Groups*
Time (hour)	Control	Arachidonoyl dopamine	3,4-Dihydroxy- 5-methoxybenzoic acid	2,4,6-Trihydroxybenzaldeyde
First	54 927.1 ± 1258.8^Ψ^,^a	46 953.0 ± 730.0^ϕ^,^a	47 067.0 ± 1246.9^ϕ^,^a	45 698.8 ± 641.2^ϕ^,^a
Fourth	55 589.5 ± 1262.5^Ψ^,^a	40 661.6 ± 1750.7^ϕ^,^b	39 357.5 ± 930.3^ϕ^,^b	37 781.4 ± 1182.4^Ω^,^b

*CA activity as IU/gHb.

^Ψ,ϕ,Ωp < 0.05. The difference between the groups with different uppercase letters on the same line was significant.

^a,bp < 0.05. The difference between the time values with having different lowercase letters in the same column was significant (first and fourth hours).

Table 5. The half-life (t₅₀) values of substances for rat erythrocytes CA enzyme using the CO₂-hydratase activity method.

Phenolic compounds	Half-life (hour)
Arachidonoyl dopamine	8.34
3,4-Dihydroxy-5-methoxybenzoic acid	7.70
2,4,6-Trihydroxybenzaldeyde	6.79

It is understood from the results that phenolic compounds may generally show inhibition effects with their OH group on CA isoenzymes. Particularly, the CA enzyme has a Zn²⁺in its active site. Zn²⁺may be a ligand for the OH group of the phenolic compound. On the other hand, some groups of the structural parts of the enzyme may interact with the OH group of the phenolic compound. Thus, many phenolic compounds and their synthesised derivatives can act as novel inhibitors for CA isoenzymes.

Consequently, we reported that arachidonoyl dopamine, 3,4-dihydroxy-5-methoxybenzoic acid and 2,4,6-trihydroxybenzaldehyde were effective CA inhibitors both in vitro and also in vivo. CA inhibitors are crucial for different biomedical applications, such as antiglaucoma, metabolic acidosis, epilepsy, acute mountain sickness, anti-obesity and anti-inflammation. However, the use of phenolic compounds in patients with metabolic acidosis may also generate adverse effects. To minimise of the side effects of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid should be used carefully and their doses should be adjusted correctly.

Declaration of interest

There is no conflict among the authors concerning the article. The authors are solely accountable for the content and writing of this article.