Abstract
Polyphenols are important secondary products of plants with the potential to inhibit carbonic anhydrases. The aim of this study was to investigate the inhibition effects of various phenolic standards, honey, propolis, and pollen species on human carbonic anhydrase I and II. The inhibition values (IC50) of the phenolics (gallic acid, protocatechuic acid, quercetin, catechin, tannic acid, and chrysin) ranged from 0.009 to 0.32 μg/mL, tannic acid emerging as the best inhibitor. The inhibition values of three different types of honey, heather, rhododendron, and chestnut ranged between 2.32 and 25.10 μg/mL, the chestnut honeys exhibiting the best inhibition. The ethanolic extracts of pollen and propolis exhibited good inhibitory properties, with IC50 values between 0.486 and 3.320 μg/mL. In order to evaluate the phenolic composition of bee products, phenolic profiles and total phenolic contents (TFC) were also measured. The inhibition ranking among the natural products studied was phenolic standards > propolis > pollen > honeys, and inhibition was related to TFC.
Introduction
Carbonic anhydrases (CA, carbonate hydroliyase, E.C.4.2.1.1) are one of the most studied enzymes. They are present in all species and constitute a family involved in regulating pH and water, electrolyte, and ion transport. Zinc metalloenzymes play an important role in respiration, in the transport of carbon dioxide and bicarbonate, in the pH and CO2 balance in the lungs and tissues, in the release of secretions from various tissues and organs, and especially in biosynthetic metabolic processes such as lipogenesis, glycogenesis, and ureagenesis in mammals. Carbonic anhydrases are also necessary for CO2 fixation in plants, algae, and prokaryotesCitation1,Citation2. The carbonic anhydrase enzyme is common in organisms but has different isoenzymes that vary according to environmental conditions and requirements. There are currently 16 known isozymesCitation3. The most widely available and most studied of these are CA I and CA II. The enzyme exhibits esterase activity in addition to hydrate activity. However, hydrate activity is more important from a physiological standpoint. The enzyme thus plays an important role in the regulation of organisms’ acid–base balance. Intervention in carbonic anhydrase enzyme activity is a treatment modality widely applied in cases where this balance is disrupted, such as increased intraocular pressure. From this perspective, carbonic anhydrase inhibitors are clinically highly important compoundsCitation4.
Acetazolamide, methazolamide, sulfanilamide, dichlorphenamide, and dorzolamide derivatives have been used for many years to reduce intraocular pressure in glaucomaCitation2,Citation5. Systemic carbonic anhydrase inhibitors are one of the most powerful agents used to lower intraocular pressure. However, their use is generally accompanied by many undesirable side-effects. These side-effects of drugs used as CA II inhibitors in glaucoma have resulted in a search for natural inhibitors.
Plant-derived natural products have increased in popularity in recent years. Polyphenols are important secondary metabolites of plants with numerous biological active properties, such as antioxidant, antimicrobial, anticarcinogenic, and antiinflammatory effectsCitation6–8. Plants also have an unlimited ability to synthesize aromatic compounds, as well as phenolicsCitation9. Honey, propolis, and pollen are important plant-derived bee products containing numerous kinds of phenolic substancesCitation10. The composition of bee products is reported to vary depending on climatic conditions, flora, environmental conditions, and the state of the colony, and the quality of honey depends on its chemical composition and floral originCitation11,Citation12. There is known to be a positive relationship between the biological active properties and phenolic content of bee products, such as honey, pollen, and propolisCitation13,Citation14.
The purpose of this study was therefore to investigate various pure phenolic compounds and extracts of bee products from Turkey, including unifloral honeys, pollen, and propolis, in terms of the human CA I and II (hCA I and hCA II) isoforms involved in crucial physiological and pathological processes.
Materials and methods
Reagents
Analytical grade solvents (methanol and ethanol) were obtained from Merck Co. (Merck, Darmstadt, Germany). Buffer and other reagents were the highest purity grade and were obtained from Sigma-Aldrich (Milan, Italy). Carbonic anhydrase isozymes were isolated from human blood cell using affinity chromatography. All standard phenolic compounds, acetazolamide, and sulfanilamide were obtained from Sigma-Aldrich (Milan, Italy). Human CAI and CAII were supplied by Sigma-Aldrich (Germany).
Samples and extractions
Unifloral honeys, pollen, propolis were obtained from experienced bee-keepers in Turkey in 2014. Heather honey I and II samples were obtained from the Mugla region, pollen I samples from the Anzer region, pollen II from Erzurum, propolis I from Ankara, and propolis II from Giresun. Both of the rhododendron honey samples were obtained from Trabzon, and the chestnut honeys were obtained from Sinop and Rize. Unifloral properties of the honeys were tested using melissopalynological characterization. The levels of the major botanical pollen components were between 52% and 84%, since a dominant pollen content more of 45% of total pollen is regarded as constituting unifloral honeyCitation13,Citation15. The crude samples were stored in a refrigerator at +4 °C. The honey samples were diluted in bidistilled water and then filtered before use. Once the raw propolis samples had been powdered, ethanolic extraction was performed. Approximately 5 g power propolis was extracted with 70% ethanol. Following stirring for 24 h at room temperatures, ultrasonic dissolving was performed (Heidolph Promax 2020, Schwabach, Germany) for 30 min. The suspension was then filtered and centrifuged at 10 000 g for 15 min. Finally, the supernatant was evaporated and lyophilized. The residue was resolved in 70% alcohol. Pollen samples were extracted with 70% ethanol. Following stirring for 24 h, ultrasonic dissolving was performed, as with the propolis samples. All phenolic acid and flavonoid standards were dissolved in 70% ethanol. Acetazolamide and sulfanilamide were dissolved in bidistilled water.
In order to prepare the samples for HPLC analysis, once the extracts had been filtrated, pH was adjusted to 1.0 with HCl. Solid phase extraction was then performed using Supelclean TM LC-18 SPE tubes (Bellefonte, PA) to separate phenolic compounds. The solvents of the ethanolic fractions were evaporated to dryness under reduced pressure in a rotary evaporator at 40 °C. The residue was finally dissolved in ethanol for HPLC analysisCitation16.
Determination of total phenolic content
Total polyphenol content was estimated following the Folin–Ciocalteu method using gallic acid as reference standardCitation17. For analysis, ethanolic extracts of the samples were prepared, and the amount of total phenolic compound was determined based on the gallic acid standard. The absorbance values read at 760 nm were placed on the y-axis, and the concentration values were placed on the x-axis to prepare a standard work graph. The results were calculated as mg gallic acid equivalent(GAE)/100 g sample using the standard work graph prepared.
Determination of phenolic profile
Hitachi HPLC (Elite LaChrom, Hitachi, Japonya) analysis of phenolic compounds was performed on a reverse phase Zorbax Eclipse XDB-C18 column (4.6 X 150 mm, 5 μm particle size), using a gradient program with two solvent systems (A, 0.5% acetic acid in acetonitrile: water (1:1); B, 2% acetic acid in water at a constant solvent flow rate of 1.2 mL/min). An aliquot of 10 μL of sample was injected, and signal detection was carried out at 280 nm and 315 by ultraviolet detectionCitation13.
CA esterase activity and inhibition
The carbonic anhydrase enzyme hydrolyzes the p-nitrophenyl acetate substrate to p-nitrophenol or p-nitrophenolate. The activity is determined by the decrease in absorbance at 348 nmCitation18,Citation19. The p-nitro phenyl acetate (p-NFA) used as the substrate in determination was prepared fresh on a daily basis. The enzyme-free part was used as a control during activity measurement. During pipetting, a mixture solution of 0.470 μL 0.05 M, pH 7.4 Tris-SO4 buffer, 3 mM 0.350 μL of p-NPA, and 180 μL of pure water was used as the control. Instead of reducing the amount of water in the control sample, a control sample was prepared using 50 μL of enzyme solution. Absorbances at minutes 0 and 3 were read at 348 nm, and the difference between them was calculated. The difference between the control value obtained with or without enzyme was calculated, and this value was regarded as corresponding to 100% activity. This differential value needs to be higher than the value measured with the addition of the inhibitor. The inhibitions were expressed as IC50 values, representing the concentration of compound producing 50% inhibition of the enzymes. The IC50 value was calculated for each inhibitor by taking an inhibitor at a specific concentration, diluting it with five different concentrations, and then drawing a concentration versus a percentage activity graph.
Statistical analysis
The results are presented as mean values plus standard deviations of triplicate measurements. Data were tested using analysis of variance (ANOVA) on SPSS software (version 9.0 for Windows 98, SPSS Inc., Chicago, Il).
Results and discussion
This study evaluated the inhibition effects of various pure phenolic standards and bee products on hCA-I and hCA-II isoenzymes. First, the total phenolic contents (TPC) and phenolic profiles of the bee products were determined. The results are given in and . Three different unifloral honey species of heather, rhododendron, and chestnut honeys were used. TPC values for the honeys ranged between 32 and 105 mgGAE/100 g samples, with chestnut honeys containing the highest level of TPC. In agreement with this finding, one comparative study of unifloral honeys produced in Turkey also reported that chestnut honeys were contained the highest level of TPCCitation13,Citation20,Citation21. Chestnut honey is a dark-blossom honey reported to contain higher antioxidant and antimicrobial properties than light-color blossom honeysCitation13,Citation14. Total phenolic contents of the propolis samples ranged between 788 and 850 mg GAE/100 g, values nearly 10-fold higher than those of the honeys. Propolis is not obtained in pure form from a single source, and the collection areas are rich in chestnut, poplar, hazelnuts, and cherry trees. TPC values of the pollen samples ranged between 247 and 255 mg/100 g, lower than the propolis samples, but higher than the honeys. Ranking the bee products according to their TPC levels gave the order propolis > pollen > honey.
Table 1. RP-HPLC-UV validation parameters of the studied phenolic compounds.
Table 2. Phenolic composition of the studied honey-bee products (μg/g).
In order to determine phenolic compositions of the honeys, pollens, and propolis, 13 phenolic standards were analyzed qualitatively and quantitatively using RP-UV-HPLC. The results are given in . All phenolic compounds were detected in different varieties and quantities in the bee product samples. Propolis samples contained the four highest phenolic substances, with quercetin, rutin, CAPE, ferulic acid, and coumaric acid being identified as the major components of the samples. Various previous propolis studies have reported CAPE, quercetin, and rutin as the major phenolic componentsCitation22–24. Benzoic acid, coumaric acid, and catechin were determined at higher levels in the chestnut honeys than the other honeys. Our findings are compatible with those of previous studiesCitation13,Citation20,Citation25. Heather honey is a dark honey, and both the heather honeys in this study were rich in flavonoids of quercetin, catechin, and rutinCitation13,Citation26. The lowest TPC and phenolic composition in this study were observed in the rhododendron honeys, light-colored blossom honeysCitation13,Citation14. Rhododendron honey is also known as ‘mad honey’ since it contains the toxic compounds andromedotoxin, acetylandromedol, rhodotoxin species, polyhydroxylated cyclic diterpenes, and grayanotoxin, a diterpene and polyhydroxylated cyclic hydrocarbon that does not contain nitrogenCitation21,Citation28.
Natural compounds and natural extracts exhibit many biological activities, such as inhibition and activation of some clinically important enzymes, such as hCA, urease, acetylcholine esterase, xanthine oxidase, monoamine oxidase, and hyaluronidaseCitation10,27,29–31. In this study, we measured the inhibition effect on hCAI and hCAII of various bee products and phenolic standards and compared the results with acetazolamide and sulfonamide inhibitors (). In additional to having positive effects on glaucoma, CAs inhibitors are reported to exhibit numerous other potential activities, such as anti-cancer, anti-obesity, and anti-infective properties and are used in the treatment of Alzheimer’s diseaseCitation32–34.
Table 3. Inhibitory effects of some bee products and polyphenols against hCA I and hCA II.
Of the six different phenolic compounds in the present study (), catechin was identified as the best potential inhibitor of hCAI, and tannic acid as the best inhibitor of hCAII. The ranking of the inhibitory effects on hCAI of the phenolic standards was in the order catechin > quercetin = gallic acid > chrysin > tannic acid > protocatechuic acid, while for hCAII the ranking was tannic acid > quercetin > gallic acid = chrysin = catechin = protocatechuic acid. No correlation was determined between either enzyme in terms of phenolic inhibitions. Although tannic acid exhibited the highest inhibition of hCAII, catechin was the most effective against hCAII. A naturally occurring plant polyphenol, tannic acid is composed of a central glucose molecule derivatized at its hydroxyl groups with one or more galloyl residues, and exhibits good antioxidant activityCitation34. Apart from the phenolic compounds investigated in this study, a wide series of polyphenolic, including resveratrol, dobutamine, curcumin, catechin, and silymarin, have been investigated in terms of the inhibition of all the catalytically active mammalian isozymes of carbonic anhydrase, and these have been reported to exhibit different inhibition effects on the enzymesCitation2. Catechin has been observed to exhibit effective inhibition of all 13 investigated CA isoforms, including hCA-VII, hCA-II, and hCA-I.
Table 4. Phenolic standards found in the studied samples.
This study investigated the inhibition properties of three different unifloral honeys, heather, rhododendron, and chestnut. Chestnut honey exhibited the highest level of inhibition of both hCA isoforms. The honey inhibition values (IC50) were statistically divided into three categories. The inhibition ranking was chestnut > heather > rhododendron, and inhibition was also correlated with honeys’ TPC.
Of the three different bee products investigated, propolis exhibited the best inhibition concentrations, with values ranging from 0.480 to 1.374 μg/mL. It also exhibited higher hCA-II inhibition. The pollen samples also exhibited inhibition of both hCAs. In contrast to propolis, the inhibition concentrations were also effective against the hCAs. In agreement with our results, other studies have also reported that propolis exhibits the highest inhibition of hCAs among propolis, pollen, and honey.
In conclusion, we investigated the potential inhibition of hCA-I and hCA-II using a group of polyphenolic derivatives and bee products. All the polyphenols effectively inhibited these enzymes, with IC50 values in the range of 0.002 μg/mL–0.350 μg/mL. Tannic acid exhibited good inhibition potential against hCA-II and catechin against hCA-I. We think that these data may assist with the design of more effective forms of hCA-I and hCA-II.
Declaration of interest
The authors declare no conflict of interest with the data from this paper.
Acknowledgements
We would like to thank the Muğla Beekeeping Association for providing the authentic heather honey samples.
Funding
This research was supported by the TUBİTAK under grant number 114O208.
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