Inhibition properties of propolis extracts to some clinically important enzymes

Abstract

The present study was conducted to envisage inhibition effects of propolis on the crucial enzymes, urease, xanthine oxidase (XO) and acetylcholinesterase (AChE). Some of the antioxidant properties of the propolis samples were determined using the total phenolic content (TPE) and total flavonoids in the eight different ethanolic propolis extracts (EPE) samples. Inhibition values of the enzymes were expressed as inhibition concentration (IC₅₀; mg/mL or μg/mL) causing 50% inhibition of the enzymes with donepezil, acetohydroxamic acid and allopurinol as reference inhibitors. All the propolis extracts exhibited variable inhibition effects on these enzymes, but the higher the phenolic contents the lower the inhibitions values (IC₅₀ = 0.074 to 1.560 mg/mL). IC₅₀ values of the P5 propolis sample having the highest TPE, obtained from Zonguldak, for AChE, urease and XO were 0.081 ± 0.009, 0.080 ± 0.006 and 0.074 ± 0.011 μg/mL, respectively. The EPE proved to be a good source of inhibitor agents that can be used as natural inhibitors to serve human health.

Keywords:

• Acetylcholinesterase
• enzyme inhibition
• propolis
• urease
• xanthine oxidase

Introduction

Bee products like honey, pollen and propolis have been used in traditional and complementary medicine from ancient times. Scientific studies reveal that the natural products are effective in treating and protecting human health. The treatment with bee products is called apitherapy1–4. Propolis is a resinous mixture used to protect hives from many diseases and threats. The composition of propolis depends mainly on floral sources, it contains nearly 40–50% resins, 30% wax, 5–10% essential oils and 3–10% phenolic substances, such as phenolic acids, flavonoids, tannins, etc.5,6 Propolis is a good pharmaceutical mixture that includes many biological active compounds and has many biological active features such as antioxidants, antimicrobial, anti-inflammatory, anti-tumoral, hepatoprotective and anti-neurodegenerative activities6–11.

As planned in this study, inhibitory effects of propolis samples were investigated on acetylcholinesterase (AChE), urease and xanthine oxidase (XO) enzymes which are crucial for human health. The inhibition of the catalytic functions of these enzymes is important for the treatment of many diseases. For example, AChE inhibition is related to many neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s12, XO inhibition obstructs accumulation of uric acid and stops the growth of gout diseases3, and the inhibition of urease prevents gastric disorders caused by Helicobacter pylori3,13,14.

Additionally to the literature, this work covers the investigations of urease, AChE and XO enzymes inhibition properties of the different propolis extracts and the relation between their phenolic contents and inhibition values. The aim of these observations was to evaluate inhibitory potentials of the bee product and that it can be used for clinical purposes as pharmaceutical agents.

Materials and methods

Chemicals

Folin-Ciocalteu’s phenol reagent, Trolox® (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), gallic acid and quercetin were purchased from Sigma Chemical Co. (St Louis, MO). Enyzmes of AChE (from Electric Eel), urease (Jack Bean Urease) and XO (bovine milk xanthine oxidase), standard inhibitors and substrates such as acetohydroxamic acid, allopurinol, donepezil, xanthine, acetylthiocholine chloride (ATC) and 5,5-dithio-bis(2-nitrobenzoic) acid (DTNB) were obtained from Sigma-Aldrich, St. Louis, MO.

Samples

Raw propolis samples were obtained from experienced beekeepers in 2015 in different areas of Turkey. Only one raw propolis sample was purchased from bee product markets in Brazil. Raw propolis samples were frozen at −20 °C than grinded to powder. For extraction, 5 g of the powdered propolis was placed with 50 mL 70% ethanol in a glass flask and stirred on a shaker (Heidolph Promax 2020, Schwabach, Germany) at room temperature for 24 h. The suspension was centrifuged at 10 000 g for 15 min, then supernatants were evaporated. The residue was resolved in minimal volumes of 70% ethanol.

Total phenolic contents

The total phenolic compounds of the ethanolic extracts of propolis samples were determined using the Folin-Ciocalteu method15. Briefly, 680 μL distilled water, 20 μL propolis extract, 400 μL of 0.2 N Folin reagent and 400 μL Na₂CO₃ (7.5%) were added in a test tube. After 2 h of incubation at room temperature, the absorbance was measured at 740 nm. The result was expressed as mg of gallic acid equivalents per g samples.

Total flavanoid contents

The total flavonoid concentration was measured using a spectrometric assay. Briefly, 0.5 mL samples, 0.1 mL of 10% Al(NO₃)₃ and 0.1 mL of 1 M NH₄(CH₃COO) were added to a test tube and incubated at room temperature for 40 min. The absorbance was measured against a blank at 415 nm. Quercetin was used for the standard calibration curve. The total flavonoid concentration was expressed as mg of quercetin equivalents per 100 g sample16.

Urease inhibition assay

Urease catalyzes the hydrolysis of urea into carbon dioxide and ammonia. The production of the ammonia was measured using the indophenol method17. Reaction mixtures including 200 μL of Jack Bean Urease, 500 μL of buffer (100 mM urea, 0.01 M K₂HPO₄, 1 mM EDTA and 0.01 M LiCl, pH 8.2) and 100 μL of the propolis extract were incubated at room temperature for 20 min. The phenol reagent (550 μL, 1% w/v phenol and 0.005% w/v sodium nitroprusside) and alkali reagent (650 μL, 0.5% w/v sodium hydroxide and 0.1% v/v NaOCl) were added to each tube and an increasing absorbance at 625 nm was measured after 50 min, using a UV/vis spectrophotometer (1601UV-Shimadzu, Australia). Acetohydroxamic acid (AA) was used as standard inhibitor. In order to calculate IC₅₀ values, different concentrations of each extracts and standards were assayed at the same reaction conditions. The inhibition concentrations of the extracts (IC₅₀) were calculated from the dose response curve, which reduced absorbance by 50%.

In vitro anti-xanthine oxidase assay

The inhibition of XO was measured with the UV spectroscopy technique at 295 nm which attributes to released uric acid from xanthine. The inhibitory activity of each extract was determined using a slight modification of the reference methods18,19. Briefly, the reaction mixture consisted of 500 μL of the propolis extract, 770 μL of phosphate buffer (pH 7.8) and 70 μL of bovine milk XO (Sigma-Aldrich, St. Louis, MO), which was prepared immediately before usage. After preincubation at 25 °C for 15 min the reaction was initiated by the addition 660 μL of substrate solution into the mixture. The assay mixture was incubated at 25 °C for 15 min. The reaction was stopped by adding 200 μL of 0.5 N HCl and the absorbance was measured at 295 nm using UV/VIS spectrophotometer (1601UV-Shimadzu, Australia). A well-known XO inhibitor (XOI), allopurinol (Sigma-Aldrich, St. Louis, MO) was used as a positive control for the inhibition test. The assay was done in triplicate for calculating standard deviation. The IC₅₀ values of extracts were determined as the concentration of extract that give 50% inhibition of maximal absorbance.

Acetylcholinesterase inhibition assay

Propolis extracts were subjected to the method of Ellman’s test20 in order to evaluate their potency to inhibit the AChE from Electric Eel (Sigma-Aldrich, St. Louis, MO). This method is based on the reaction of released thiocholine to give color to a product with a chromogenic reagent DTNB. Enzyme solution was prepared in gelatin solution (1%), at a concentration of 2.5 units/mL. AChE (50 μL) and propolis samples (50 μL) were added to 3.0 mL phosphate buffer (pH 8.0, 0.1 M) and incubated at 25 °C for 5 min. The reaction was started by adding DTNB (100 μL) and ATC (20 μL) to the enzyme–inhibitor mixture. The production of the yellow anion was recorded after 10 min at 412 nm, using a UV/VIS spectrophotometer (1601UV-Shimadzu, Australia). As a control, an identical solution of the enzyme without the inhibitor was processed the same protocol. Donepezil hydrochloride was used as a positive control. All processes were assayed in triplicate.

Statistical analysis

The data were calculated in the form of arithmetical mean values and standard deviations. The SPSS 13.00 for Windows software package was used for the statistical analysis of the gathered data (SPSS Inc., Chicago, IL). Significance of the results was based on the Kruskal–Wallis test and Pearson correlations, significant differences were p < 0.05.

Results and discussion

Total phenolic contents and enzyme inhibition studies

The results of the phenolic contents and total flavonoids of the ethanolic propolis extracts (EPE) were summarized in Table 1. The total phenolic contents (TPC) of the EPE samples were changed between 88.675 and 261.055 mg GAE/g (Table 1). Eight different propolis samples have also shown different amounts of the TPE and total flavonoids. It was interesting that all propolis samples had significantly different TPC, and the highest phenolic contents were found in the P5 and P8 samples which were the Zonguldak propolis and the Brazil red propolis, respectively.

Table 1. IC₅₀ values of each enzymes and antioxidant properties and location of the ethanolic propolis extracts*.

Samples	Location	Total phenolic contents mg GAE/g	Total flavonoids mg Q/100g	Inhibition of acetylcholinesterase IC50 (mg/mL)	Inhibition of xanthine oxidase IC50 (mg/mL)	Inhibition of urease IC50 (mg/mL)
P1	Ankara, Turkey	138.812 ± 3.831^abc	65.406 ± 7.803^bc	1.340 ± 0.020^d	0.188 ± 0.010^b	0.144 ± 0.009^ab
P2	Kars, Turkey	170.461 ± 8.090^c	72.215 ± 4.605^c	0.340 ± 0.045^b	0.195 ± 0.002^b	0.170 ± 0.027^ab
P3	Nahcıvan, Azerbaycan	88.675 ± 4.562^a	37.526 ± 7.810^a	1.353 ± 0.062^d	0.763 ± 0.011^d	1.560 ± 0.045^f
P4	Erzurum	150.013 ± 10.643^abc	65.580 ± 3.250^bc	0.315 ± 0.014^ab	0.165 ± 0.004^b	0.411 ± 0.398^d
P5	Zonguldak, Turkey	261.055 ± 2.500^e	150.412 ± 12.505^e	0.081 ± 0.009^a	0.074 ± 0.011^a	0.080 ± 0.006^a
P6	Düzce, Turkey	138.321 ± 6.043^abc	52.243 ± 1.452^abc	0.882 ± 0.022^c	0.356 ± 0.031^b	0.301 ± 0.027^cd
P7	Giresun, Turkey	114.207 ± 7.207^abc	45.086 ± 3.004^abc	1.053 ± 0.016^c	0.387 ± 0.017^b	0.240 ± 0.017^bc
P8	Red, propolis, Brazil	220.205 ± 5.101^d	98.303 ± 1.702^d	0.221 ± 0.013^ab	0.317 ± 0.023^b	0.680 ± 0.051^e
AA				–	–	25.110 ± 0.120
Allopurinol				–	0.520 ± 0.010	–
Donepezil				16.800 ± 0.110	–	–

*Means ± standard deviations; different letters in the same lines are significantly different at the 5% level (p < 0.05). IC₅₀ values of acetohydroxamic acid (AA), donepezil and allopurinol were given in terms of μg/mL.

It was reported that the TPE was found in Chinese propolis ranged from 43 to 302 mg GAE/g21. The TPE was reported to be between 115–210 mg GAE/g in different origin of Turkish propolis22. A slightly narrow range of TPE was reported in the Poland propolis samples between 150 and 190 mg GAE/g7. Therefore, the Zonguldak and the Brazilian red propolis were distinguished from the other propolis samples. The total flavonoids of the propolis samples were ranged from 37.526 to 150.412 mg Q/100 g (Table 1). The highest total flavonoids was in the P5 (Zonguldak) propolis, followed by the Brazilian red propolis. Much smaller differentiations in the amount of total flavonoids in the Poland propolis samples, between 35–62 mgQ/100 g, were reported7. Comparing the studied propolis to other studies, the range of the TPE and flavonoids was wider which may be explained by the different origins of the samples7. A strong significant correlation between the TPE and total flavonoids in the samples (R²: 0.950, p < 0.01, Table 2) was found. Many studies have reported that propolis samples contained both high total phenolics and high total flavonoids4,7.

Table 2. Correlations between studied parameters.

		Total phenolic contents	Total flavonoids	Inhibition of acetylcholinesterase	Inhibition of xanthine oxidase	Inhibition of urease
Total phenolic contents	Pearson Correlation	1	0.950**	−0.679**	−0.542**	−0.371
	p, Sig. (two-tailed)		0.000	0.000	0.006	0.074
	N	24	24	24	24	24
Total flavonoids	Pearson Correlation	0.950**	1	−0.640**	−0.563**	−0.364
	p, Sig. (two-tailed)	0.000		0.001	0.004	0.080
	N	24	24	24	24	24
Inhibition of acetylcholinesterase	Pearson Correlation	−0.679**	−0.640**	1	0.646**	0.372
	p, Sig. (two-tailed)	0.000	0.001		0.001	0.073
	N	24	24	24	24	24
Inhibition of xanthine oxidase	Pearson Correlation	−0.542**	−0.563**	0.646**	1	0.882**
	p, Sig. (two-tailed)	0.006	0.004	0.001		0.000
	N	24	24	24	24	24
Inhibition of urease	Pearson Correlation	−0.371	−0.364	0.372	0.882**	1
	p, Sig. (two-tailed)	0.074	0.080	0.073	0.000
	N	24	24	24	24	24

**Correlation is significant at the 0.01 level (two-tailed).

All propolis extracts showed variable inhibition values, ranging from 0.081 to 1.353 mg/mL for AChE (Table 1). While the P3 sample showed the highest IC₅₀ value (1.353 mg/mL), the lowest IC₅₀ value (0.081 mg/mL) was found in the P5 propolis sample (Zonguldak propolis). Water-soluble propolis extract was reported to be used as food supplements which improved memorial functions in mice by inhibited AChE23. A strong significant negative correlation between the TPE and AChE activity of the EPE extracts (R²: −0.679, p < 0.01, Table 2) has been found. In recent years, some in vivo and in vitro investigations indicated that polyphenol-rich extracts have substantial inhibitory activity of AChE and improve memory in experimental animals23,24.

Previous studies as well as our results have shown that the relationship between propolis phenolic contents and the results of AChE inhibition gives us important information that the enzyme was probably inhibited by phenolic substances. We also compared our inhibition values to donepezil that is frequently used as AChE inhibitor in Alzheimer’s disease25. The IC₅₀ value of donepezil was 0.0168 mg/mL, smaller than our inhibition results of the propolis samples. However, propolis is a complex extract, specific phenolics in this mixture may exhibit higher AChE inhibitions at smaller concentrations.

In this study, the second enzyme used to examine inhibition effects of EPE extracts was XO which catalyzes the formation of uric acid from purine degradation. XOI block the production of uric acid. The enzyme XO is responsible for oxidative damage that causes many pathological diseases, such as gout, hyperuricemia, atherosclerosis, hepatitis, carcinogenesis, vascular endothelium damage and ageing26–29. Allopurinol has been the sole XOI under the clinical application for the past three decades30. However, this drug gives inevitably rise to severe adverse effects such as hepatitis, nephropathy, allergic reactions and 6-mercaptopurine toxicity31.

All of the EPE inhibited XO enzyme at a wide inhibition degree, ranging from 0.074 to 0.387 mg/mL (Table 1). The P5 sample from the studied EPE was distinguished from the others, showing the highest inhibition effect. When comparing the results to their TPE, there was a strong relation between TPC and XO inhibition results (R²: −0.542, p < 0.01, Table 2). Similarly, a number of natural compounds, rich in polyphenolic agents, such as caffeic acid32,33, rutin34, and chestnut and oak honeys3, have been reported to inhibit XO, and foods rich in phenolic compounds are recommended to reduce blood concentrations of uric acid in gout. Also, correlation coefficients and significant values (Table 2) show positive strong correlations between inhibition of XO and inhibition of urease (R²: 0.882; p: 0.000); inhibition of AChE and inhibition of inhibition of XO (R²: 0.646; p: 0.001).

H. pylori is an anaerobic microorganism that survives in an acidic environment with the help of the enzyme urease, a Nichel-metallo enzyme, causing numerous gastric disorders. The main survival way of this bacterium is being prevented from adhering in the gastric system by urease inhibition, urease inhibitors are particularly important in the treatment of gastric ulcers. Propolis is accepted as a natural antibiotic, and inhibits many infection bacteria as well as H. pylori35,36. All EPE inhibited urease with different extract concentration, changing from 0.080 to 0.301 mg/mL (Table 1). The P5 sample showed the highest activity (the lowest IC₅₀ value) in lower concentration compared to others propolis samples (Table 1).

For the results of this study, the P5 propolis sample is distinguished from other propolis, having the highest total phenolics as well as total flavonoids and it showed the highest inhibition effect (the lowest IC₅₀ value) against AChE, urease and XO enzymes. The P5 propolis sample was obtained from Zonguldak, in the east of the Black Sea Region, where the flora is richer in chestnut trees and flowers37. However, chestnut honey is also a valuable product having high phenolic contents and antioxidant capacity38.

Our study indicated that propolis has many pharmacokinetic and pharmaceutical properties, contains many inhibitor agents, its inhibition effect is related to the total phenolic substances, which is effected by floral sources. Consequently, the propolis extracts exhibited good potentials towards the inhibition of urease, XO and AchE. Consequently, the propolis extracts exhibited a good potential towards the inhibition of urease, XO and AchE. Propolis is a mixture containing many bioactive compounds and which compounds inhibited these enzymes are not clear. But, phenolic compounds (phenolic acids and flavonoids) present in the propolis extracts to be appear responsible for these inhibitions. For this reason, further studies are necessary in order to account the inhibition mechanism. Considering that some commercially urease, XO and AchE inhibitors such as acetohydroxamic, allopurinol and donepezil could not be developed into therapeutic agents because of their toxicity and rapid metabolic degradation. In conclusion, we think that regular consumption of these natural products rich in phenolic compounds can contribute to a reduction in several forms of H. pylori-associated diseases, neurodegenerative and gout disease.

Declaration of interest

This study was supported by the project of Tubitak (114Z370).