Acethylcholinesterase inhibitory potential and antioxidant properties of pyrogallol

Abstract

Pyrogallol is found naturally in crops and fruits of many plants. It is also an active ingredient of many pharmaceuticals. For this reason, we employed different in vitro antioxidant assays such as cupric ion Cu²⁺ reducing power, Fe³⁺ reducing power, total antioxidant activity by ferric thiocyanate method, ABTS radical scavenging, DMPD radical scavenging, DPPH • scavenging, Fe²⁺ chelating, scavenging and H₂O₂ scavenging activities of pyrogallol. Pyrogallol inhibited 77.95% lipid peroxidation of linoleic acid emulsion at 30 μg/mL concentration. BHA, BHT, α-tocopherol and trolox exhibited inhibitions of 89.88, 89.97, 83.82 and 91.85% against peroxidation of linoleic acid emulsion at the same concentration, respectively. In addition, pyrogallol was an effective of all the scavenging and reducing power results. In this study, pyrogallol was also evaluated as potential inhibitor for acethycholinesterse enzyme. The results showed that pyrogallol exhibited potent acetylcholinesteras inhibitory activity with IC₅₀ and K_i values 10.2 and 8.6 μM, respectively.

• Acetylcholinesterase
• anticholinergic
• antioxidant activity
• pyrogallol

Introduction

For maintaining the structural integrity of cells and tissues, and in fulfilling the normal functions, it is very remarkable that an important balance is always needed between oxidant and antioxidants. By scavenging free radicals, antioxidants reduce agents and limit oxidative damage in biological structures1. Being related to the formation of lipid peroxidation, antioxidants play an important role in preventing many diseases and aging2.

Polyphenols are naturally occurring plant metabolites and also called phenolic compounds. They are widely available in vegetables, fruits, seeds, nuts and flowers. They can easily react with reactive oxygen species (ROS), resulting in powerful antioxidant activity. So it could be considered that – the more the intake of polyphenols, less the risk for cancer and various diseases. Polyphenols are a structural class of natural, but also synthetic chemicals characterized by the presence of large multiples of phenol structural units3^,4. They, integral part of both human and animal diets, constitute one of the most ubiquitous groups of plant metabolites. Because of owing to their antioxidant capacity and their possible beneficial implications in human health, such as in the treatment and prevention of cancer and cardiovascular disease, recent interest in food phenolics has increased greatly5. Recently, the importance of phenolic compounds is described extensively in the Gulcin’s review1.

Pyrogallol (1,2,3-trihydroxybenzene) is a polyphenol found various of fruits and vegetables such as avocado and apricot. It is used as an active reducer for gold, silver and mercury salts; a chemical reagent for antimony and bismuth; and a developer in photography and holography. Additionally, pyrogallol is used in the manufacture of pharmaceuticals and pesticides and has been used for medicinal purposes as a topical antipsoriatic. Owing to its antioxidant properties, pyrogallol is used as a corrosion inhibitor (i.e. oxygen scavenger) in boilers.

On the other hand, Alzheimer’s disease (AD) is characterized by dementia, cognitive impairment and memory loss, and is one of the most common diseases in elderly people6^,7. Recently, treatment of AD focuses on AChE inhibitors (AChEI), such as tacrine, donepezil, rivastigmine and galantamine. However, the potential effectiveness of such inhibitors in clinical use is often complicated by their associated side effects. It was reported that clinical studies have shown that the AChEI tacrine causes hepatotoxicity8. Since AD is a multi-pathogenic illness, a current drug-discovery strategy is needed to develop novel anti-Alzheimer’s agents with multiple potencies such as inhibition of AChE. In the future, the development of specific and new AChEI may lead to improved clinical outcomes.

In this study, the ferric ion (Fe³⁺) reducing power, cupric ion (Cu²⁺) reducing power, total antioxidant activity by ferric thiocyanate method, ABTS radical scavenging, DPPH radical scavenging, DMPD radical scavenging, ferrous ion (Fe²⁺) chelating, superoxide () scavenging and hydrogen peroxide (H₂O₂) scavenging activity of pyrogallol were investigated.

Materials and methods

Chemicals

Pyrogallol, butylated hydroxyanisole (BHA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), butylated hydroxytoluene (BHT), α-tocopherol, N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD), 2,9-dimethyl-1,10-phenanthroline (neocuproine), 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), trichloroacetic acid (TCA), acetylcholinesterase enzyme and other chemicals were purchased from Sigma-Aldrich GmbH (Sternheim, Germany).

Fe³⁺ reducing power

For measuring power of pyrogallol, Oyaizu’s the ferric reducing antioxidant powder method, with slight modification, was used. According to this method, Fe³⁺(CN⁻)₆ is reduced to Fe²⁺(CN⁻)₆ and absorbance is measured by using spectrophotometer9. The absorbance was measured at 700 nm in a spectrophotometer10.

Cu²⁺ reducing method

We measured reducing power of pyrogallol with a slight modification of Cuprac method11. Zero-point 25 mL CuCl₂ (0.01 M), 0.25 mL ethanol neocuproine (7.5 mM), 0.25 mL CH₃COONH₄ buffer solution (1.0 M) and standard antioxidants (10, 20 and 30 μg/mL) were added to each test tube. Total volume was adjusted to 2 mL and kept at room temperature during 30 min. Absorbance was measured at 450 nm in spectrophotometer12.

Ferric thiocyanate method (total antioxidant activity)

The ferric thiocyanate method was used to evaluate the effects of pyrogallol. With this method, peroxide formation occurred during the oxidation of linoleic acid oxidation13^,14. These compounds oxidized Fe²⁺–Fe³⁺ and ions form a complex with thiocyanate. Reaction mixture has a maximum absorbance at 500 nm. The percent inhibition of lipid peroxidation was calculated by the following equation:

AS is the absorbance of the sample in the presence of pyrogallol or other test compounds. AC is the absorbance of the control reaction contains only sodium phosphate buffer and linoleic acid emulsion15^,16.

ABTS^•+ scavenging activity

ABTS^•+ scavenging activity assay was improved by Re et al.17 with minor modification. After an antioxidant is added to a pre-formed ABTS^•+ radical solution, the remaining ABTS^•+ is determined at 734 nm in a spectrophotometer. The ABTS radical solution was prepared by using 2 mM ABTS in H₂O with potassium persulfate (2.45 mM). Then, ABTS^•+ solution stored in the dark at room temperature until used. Finally, 1 mL of ABTS^•+ solution was added to 3 mL of pyrogallol solution in ethanol at different concentrations (10–30 μg/mL). The mixture was incubated in the dark for 30 min and measured at 734 nm18.

DPPH^• scavenging activity

This activity was measured by the method of Blois described previously by Gulcin19. DPPH solution has deep purple color and if the sample contains an antioxidant this color disappears16^,20. One microliter of ethanolic DPPH solution (0.1 mM) was added to the different concentrations (10–30 μg/mL) of pyrogallol. These samples were vortexed and incubated in the dark at 30 °C for 30 min. The absorbance was measured at 517 nm against blank samples.

DMPD^•+ scavenging activity

DMPD^•+ scavenging activity was determined with minor modifications by Gülcin15 according to the method of Fogliano et al. Firstly, 105 mg DMPD was dissolved in 5 L of distilled water and 1 mL of this solution was added to 100 ml of acetate buffer (0.1 M, pH 5.3). DMPD^•+ was obtained by adding 0.3 ml ferric chloride (0.05 M) to this solution. After the addition of different concentrations (10–30 μg/mL) of pyrogallol standard antioxidants and 1 μL of DMPD^•+ solution was added to the reaction mixture, they incubated in dark for 15 min. Absorbance of mixture was measured at 505 nm.

Fe²⁺ ions chelating activity

Pyrogallol’s chelating activity was measured using the method of Dinis et al.21 and the absorbance was measured at 562 nm spectrophotometrically. This method is based on the absorbance measurement of Fe²⁺–ferrozine complex. In the experiment, 0.1 mL of FeCl₂ (0.6 mM) solution was added to pyrogallol (10–30 μg/mL) in 0.4 mL methanol. The reaction was initiated by adding 5 mM ferrozine. Then, it was incubated for 10 min and the mixture was measured spectrophotometrically at 562 nm.

scavenging activity

scavenging activity was measured according to the method of Zhishen et al. with slight modification22. In this method, the total volume of the reaction mixture was 3 mL. The reaction mixture comprising riboflavin, methionine and NBT were illuminated at 25 °C for 40 min. The absorbance was measured at 560 nm. Different concentrations (10–30 μg/mL) of pyrogallol were added to the reaction mixture, in which was scavenged, thereby inhibiting the NBT reduction23.

H₂O₂ scavenging activity

The H₂O₂ scavenging was described following the procedure of Ruch et al.24. According to this method, a solution of 40 mM H₂O₂ was prepared in 0.1 M phosphate buffer (pH 7.4). Pyrogallol (10 μg/mL) in 3.4 mL phosphate buffer was added to 0.6 mL of H₂O₂ solution (40 mM) and of the reaction mixture’s absorbance was measured at 230 nm.

Acetylcholinesterase activity method

Acetylcholinesterase enzyme catalyzes the breakdown of acetylcholine to acetate and thiokols. Pyrogallol was assayed for cholinesterase by the method of Ellman et al.25 using 3 mM acetylthiocholine iodide as substrate. According to the Ellman method thiocholine, produced by AChE, reacts with 5,5′-dithiobis(2-nitrobenzoic acid) to form a colorimetric product. The absorbance at 412 nm was measured immediately. In this method, 100 μL Tris–HCl (pH 8), 50 μL DTNB and 40 μL enzyme were added into the test tubes and total volume was adjusted with distilled water to 950 μL. Then, 50 μL of acetylthiocholine iodide was added to this mixture and absorbance was measured at 412 nm. Distilled water was used instead of enzyme for blank sample.

Results

In the linoleic acid emulsion system, pyrogallol showed effective antioxidant activity. The effect of 30 μg/mL concentration of pyrogallol on the lipid peroxidation of linoleic acid emulsion was found to be 77.95% (Figure 1). On the other hand, the same concentration of standard antioxidants (BHA, BHT, α-tocopherol and trolox) exhibited 89.88, 89.97, 83.82 and 91.85% peroxidation of linoleic acid emulsion, respectively. The autoxidation of linoleic acid emulsion without pyrogallol or standard compounds was accompanied by a rapid increase in peroxides. According to results of this experiment, pyragallol was found to have potent activity. On account of the deleterious role of free radicals in biological systems and foods, radical scavenging activity is very significant. Chemical assays relate to the ability of scavenge synthetic free radicals, using a variety of radical generating methods and systems for detection of the oxidation endpoint. Antioxidant capacities of components are determined with commom spectrophotometric procedures such as DMPD^•+, DPPH^• and ABTS^•+ radical scavenging methods26. All of the tested compounds showed remarkable radical scavenging activity. As shown in Supplementary Figure 2A, there was a significant decrease in the concentration of ABTS^•+ owing to the scavenging capacity at all pyrogallol concentrations (10–30 μg/mL). EC₅₀ values for standard antioxidants (BHA, BHT, α-tocopherol and trolox) were found to be 9.26, 8.02, 12.98 and 11.49 μg/mL, respectively. Pyrogallol and standards’ EC₅₀ values on the ABTS^•+ scavenging decreased in the following order: pyrogallol (7.08 μg/mL) > BHT (8.02 μg/mL) > BHA (9.26 μg/mL) > troloks (11.49 μg/mL) > α-tokoferol (12.98 μg/mL). Lower EC₅₀ values point out high ABTS^•+ scavenging ability.

Figure 1. Total antioxidant activities of pyrogallol and standard antioxidant compounds such as BHA, BHT, α-tocopherol and trolox at the same concentration (30 μg/mL) assayed by ferric thiocyanate method13^,14.

In Supplementary Figure 2B, pyrogallol was an efficient DMPD^•+ radical scavenger in a concentration-dependent manner (10–30 μg/mL). Pyrogallol and standards’ EC₅₀ values on the DMPD^•+ decreased in the following order: pyrogallol (10.44 μg/mL) > BHA (44.75 μg/mL) > troloks (59.38 μg/mL). Owing to the scavenging capacity at all the phenolic compounds concentrations, there was a notable decrease in the concentration of DMPD^•+. Reportedly, when hydrophobic antioxidants such as BHT or α-tocopherol were used, the main drawback of the DMPD^•+ method is that its sensitivity and reproducibility dramatically reduced2.

In Supplementary Figure 2C, owing to the scavenging ability of pyrogallol and the reference compounds, there was a significant reduction in the concentration of DPPH radical. Standard antioxidants (BHA, BHT, α-tokoferol and trolox) were used as references for radical scavenger activity. Pyrogallol and standard antioxidants’ (BHA, BHT, α-tokoferol and trolox) EC₅₀ values on the DPPH radical were found to be 10.71, 15.07, 31.45, 11.48 and 8.63 μg/mL and decreased in the order of troloks > pyrogallol > α-tokoferol > BHA > BHT. A lower EC₅₀ values point higher DPPH radical scavenging activity.

Due to the fact that antioxidant compounds are able to donate electrons to reactive radicals, reducing them into more stable and unreactive species, the reduction capacity of a compound may serve as a significant indicator of its potential antioxidant activity27. In this article, Fe³⁺ reducing power and CUPRAC assays were used for reducing power of pyrogallol. When pyrogallol was compared to the standards, both methods were found to be effective. As seen in Supplementary Figure 3A, pyrogallol demonstrated powerful Fe³⁺ reducing activity. So these differences were statistically found to be major. The reducing power of pyrogallol and standards (BHA, BHT, α-tocopherol and trolox) increased with increasing concentration of samples. The Fe³⁺–Fe²⁺ reducing powers of the phenolic and standard compounds at the same concentration (20 μg/mL) were as follows: pyrogallol > BHA > BHT > troloks ≥ α-tokoferol.

Cu²⁺ reducing capability of pyrogallol was measured by CUPRAC method was found to be concentration dependent (10–30 μg/mL). As shown in Supplementary Figure 3B, Cu²⁺ ions reducing power of pyrogallol and standard compounds at the same concentration (20 μg/mL) were as follows: pyrogallol > BHA > BHT > α-tokoferol > troloks. Similar results were observed for pyrogallol between Cu²⁺ ions reducing and Fe³⁺ ions reducing ability methods.

Among the various species of metal ions, Fe²⁺ ion is the most strong pro-oxidant. Ferrozine and Fe²⁺ can quantitatively form complexes. The complex formation is disrupted, resulting in a decrease in the red color of the complex in the presence of chelating agents. For this reason, measurement of color reduction provides estimating the metal chelating activity of the coexisting chelator. If absorbance is lower this means that metal chelating activity is higher. Fe²⁺ chelating activities of pyrogallol and standard antioxidants (BHA, BHT, α-tocopherol and trolox) are shown in Supplementary Figure 4. EC₅₀ values for pyrogallol, standard antioxidants (BHA, BHT, α-tokoferol and trolox) on the Fe²⁺ chelating activity were found to be 44.08, 19.08, 17.65, 15.76 and 17.27 μg/mL, respectively, and decreased in the order of α-tokoferol > troloks > BHT > BHA > pyrogallol. A lower EC₅₀ value exhibits a higher Fe²⁺ chelating activity.

Oxidative stress can generate included in free radical species. This radical is a factor that can destruct cell membranes and induce aging. In Supplementary Figure 5A, EC₅₀ values for pyrogallol and standards on the scavenging were as follows: BHA (17.92 μg/mL) > pyrogallol (18.93 μg/mL) > BHT (28.53 μg/mL). EC₅₀ value was not determined for α-tocopherol and its water-soluble analog, trolox.

Scavenge H₂O₂ ability of pyrogallol is shown in Supplementary Figure 5B and compared with standard antioxidants (BHA, BHT, α-tocopherol and trolox). BHA, BHT, α-tocopherol and trolox exhibited 32.9, 57.5, 53.2 and 56.6% H₂O₂ scavenging activity, respectively, at 10 μg/mL. On the other side, H₂O₂ scavenging activity of pyrogallol was found to be 20.9% at the same concentration. Then, EC₅₀ values were calculated. EC₅₀ values for pyrogallol, BHA, BHT, α-tokoferol and trolox were found to be 23.92, 15.19, 8.69, 9.30 and 8.80 μg/mL. These results indicate that pyrogallol has a powerful O₂ and H₂O₂ anion radical scavenging activity.

The mechanism of action of AChE might lead to the design of the mechanism-based inhibitors, which could be of future therapeutic use. Different types of AChEIs have been studied for the treatment of AD. Some of the AChEI differ in their mechanism of action, metabolism and brain selectivity. AChE was very effectively inhibited by pyrogallol, with K_i value of 8.6 μM (IC₅₀: 10.22, r²: 0.9952). On the other hand, comparing to inhibitor effect, tacrine was used. Tacrine, which is used for the treatment of mild, inhibited AChE with K_i value of 0.19 μM (IC₅₀: 0.48, r²: 0.9933). IC₅₀ and K_i values for pyrogallol and tacrine are shown in Table 3.

Discussion

Polyphenols, or phenolic compounds, is a common term that refers to a large number of compounds (>8000). They have at least one aromatic ring attached with one or more hydroxyl group and they are produced in plants as secondary metabolites via the Shikimic acid pathway4. As a result of studies, many new plant component parts, among these are the polyphenols, on antioxidant activity have been identified. Antioxidant activity of phenolic compounds is mainly owing to their redox properties, which permit them to act as reducing agents, singlet oxygen quenchers or hydrogen donors. In addition to their roles as antioxidants, they exhibit a wide spectrum of medicinal properties, such as anti-inflammatory, cardioprotective, antimicrobial, antithrombotic and anti-allergic effects28.

In this study, we have used different in vitro bioanalytical methodologies (Tables 1 and 2) and studied the antioxidant and radical scavenging activity of pyrogallol. Its antioxidant and radical scavenging activities were compared to standard antioxidants (BHA, BHT, α-tocopherol and its water-soluble analog, trolox). These collations were made using a series of in vitro tests. For this purpose, ABTS^•+ scavenging, DPPH^• free radical scavenging, DMPD^•+ scavenging, scavenging, Fe³⁺–Fe²⁺ transformation and CUPRAC methods were used.

Table 1. Reducing ability of pyrogallol by Fe³⁺–Fe²⁺ transformation and CUPRAC method and standards such as BHA, BHT, α-tocopherol and trolox at the same concentration (20 μg/mL).

Compound	Fe^3+ ions reducing ability (700 nm)	Cu^2+ ions reducing ability (450 nm)
Pyrogallol	1.945	2.750
BHA	1.735	2.251
BHT	1.429	2.092
Trolox	1.161	0.808
α-Tocopherol	1.156	1.144

Table 2. Concentration required for 50% scavenging (IC₅₀) of DPPH^• scavenging activity, ABTS^•+ scavenging activity, DMPD^•+ scavenging activity, scavenging activity, Fe²⁺ chelating activity and H₂O₂ scavenging activity of pyrogallol and standard compounds such as BHA, BHT, α-tocopherol and trolox.

Compound	H2O2 scavenging	Fe^2+ chelating	scavenging	DPPH^• scavenging	ABTS^•+ scavenging	DMPD^•+ scavenging
BHA	15.19	19.08	17.92	15.07	9.26	44.75
BHT	8.69	17.65	28.53	31.45	8.02	–
α-Tocopherol	9.30	15.76	–	11.48	12.98	–
Trolox	8.80	17.27	–	8.63	11.49	59.38
Pyrogallol	23.92	44.08	18.93	10.71	7.08	10.44

Lower IC₅₀ values indicate higher scavenging activity.

Table 3. IC₅₀ and K_i values, and inhibition types of tacrine and pyrogallol.

Inhibitor	IC50 (μM)	[I] (μM)	Ki values	Mean Ki (μM)	r^2	Inhibition type
Tacrine	0.48	0.21	0.194	0.195 ± 0.004	0.9933	Non-competitive
		0.42	0.192
		0.63	0.201
Pyrogallol	10.22	7.9	9.626	8.680 ± 1.227	0.9952	Non-competitive
		9.9	9.119
		11.8	7.294

Varied methods such as DPPH^•, ABTS^•+ and DMPD^•+ are currently used to assess the antioxidant activity of plant phenolic compounds and these methods are rapid; no sophisticated instrumentation is required, a sample analysis takes 15 min in total, or no expensive reagents17^,26.

DPPH^• and ABTS^•+ are the most common methods used to show significant differences in their response to antioxidants. The ABTS^•+ must be generated by enzymes or chemical reactions, conversely the DPPH^• does not require any special preparation. Furthermore, ABTS^•+ can be dissolved in aqueous and organic media due to the hydrophilic and lipophilic nature of the compounds. However, DPPH can only be dissolved in organic media, especially in ethanol. When interpreting the roles of hydrophilic antioxidants, this is a very important limitation29.

ABTS free radicals occur by oxidation of ABTS and potassium persulfate. This assay is based on the inhibition of the absorbance of ABTS^•+, which has a characteristic long-wave length absorption spectrum showing absorption at 734 nm. In this method, reaction with ABTS^•+ is both faster and is completed in a short time (0.25–0.5 min)30.

To evaluate the free radical scavenging effectiveness of different antioxidant substances, DPPH method is used. In this method, DPPH decreases in the presence of a hydrogen-donating antioxidant owing to the formation of the non-radical form DPPH-H in the reaction. DPPH accepts an electron or hydrogen radical and is a stable free radical to become a stable diamagnetic molecule31.

DMPD radical scavenging method is more suitable for hydrophilic antioxidants, but is less sensitive to hydrophobic bioactive compounds. In spite of the ABTS procedure, the DMPD^•+ method guarantees a very stable endpoint. When a large-scale screening is required, this is especially important. According to the studies, the main drawback of the DMPD method when using the hydrophobic antioxidants such as α-tocopherol or BHT, its reproducibility and sensitivity decrease. It was reported that organic acids might cause interference. Therefore, these standard antioxidants were not used in the antiradical assay.

Antioxidant compounds are able to donate electrons to reactive radicals, reducing them into more stable and unreactive species27. Fe³⁺ and CUPRAC methods were used for reducing power of pyrogallol.

Antioxidant compounds decrease Fe³⁺–ferricyanide complexes to the ferrous (Fe²⁺) form. The Prussian blue-colored complex is formed by adding FeCl₃ to the ferrous (Fe²⁺) ions. In this method, depending on the reducing power of the antioxidant, the yellow color of the test solution changes to blue or green. The higher the absorbance, the higher the ferric reducing power.

Cu²⁺ is reduced to Cu⁺ by antioxidants according to the CUPRAC method. This method is suitable for a variety of antioxidants regardless of chemical type or hydrophilicity; furthermore, it is cost-effective, rapid and stable. Studies also indicate that in vitro Cu²⁺-reducing measurement results might be more efficiently extended to the possible in vivo reactions of antioxidants. This method can be used for both hydrophilic and lipophilic antioxidants.

Metal chelating capacity is important, on account of the fact that it reduces the concentration of the catalyzing transition metal in lipid peroxidation1. According to this method, free Fe²⁺ and ferrozine forms a complex, but not with Fe²⁺ bound to other chelators. Therefore, if there is a presence of antioxidant chelators, this means that there is a decrease in the amount of ferrozine–Fe²⁺ complex formed after treatment. This complex is measured spectrophotometrically at 562 nm. A significant drawback of this complexation in measuring the presence of antioxidant chelator is – the reaction is affected by both the antioxidant–Fe²⁺ and ferrozine–Fe²⁺ complex formation constants. Therefore, a weak antioxidant iron chelator could be underestimated in quantitative determination.

When one electron transfers to molecular oxygen, are formed. In this study, we used riboflavin/methionine/illuminate system for generation of O₂ anion radicals. According to the method, producing the blue formazan, O₂ anion reduces the yellow dye (NBT²⁺), which is measured at 560 nm. Antioxidants inhibit the blue NBT formation.

Riboflavin activating photochemically reacts with NBT to generate NBTH, eventually it leads to formazan according to reaction. Radical species are controlled by a quasi-equilibrium in the presence of oxygen. For this reason, when the assay is performed under aerobic conditions, appear indirectly32.

H₂O₂ can be produced by biological systems and can be formed in vivo by several oxidizing enzymes such as O₂ dismutase. When H₂O₂ is added to cells in culture, it can cause transition of metal dependent on OH radicals and lead to DNA damage33. For this reason, removing H₂O₂ as well as , is very significant for protection of food systems and pharmaceuticals15.

For food and medicinal bioactive components, total antioxidant capacity is used as a parameter. In this method, thiocyanate method, a compound inhibits oxidative degradation like lipid peroxidation and is used to measure the peroxide level along the initial stage of lipid oxidation34. During the linoleic acid oxidation, peroxides are formed and react with Fe²⁺ to form Fe³⁺. Then, ions and thiocyanate (SCN⁻) form a complex, which has a maximum absorbance at 500 nm. In the presence of antioxidants, oxidation of linoleic acid will be slow. Moreover, the color development will be slow as a result of formation of thiocyanate35.

Acetylcholine-mediated neurotransmission is fundamental for nervous system function. While the physiological role of the AChE in neural transmission has been well-known, it is still the focus of pharmaceutical research, targeting in treatments of some diseases such as gravis, glaucoma and AD. It has been elucidated that cholinergic deficiency is associated with AD. Therefore, one of the major therapeutic strategies is to inhibit the biological activity of AChE and hence, to increase the acetylcholine level in the brain. Recently, most of the drugs used for the treatment of AD are AChEI such as tacrine, donepezil and rivastigmine6. In our study, the inhibition study made for tacrine and pyrogallol, and IC₅₀ and K_i values were calculated (Table 3). It has been observed pyrogallol was effective inhibitor for AChE with non-competitive inhibition.

Conclusion

According to these results, pyrogallol was powerful antioxidant in different in vitro assays, including the ferric thiocyanate method, DPPH^• scavenging, ABTS^•+ scavenging, DMPD^•+ scavenging, Fe²⁺ chelating, scavenging and H₂O₂ scavenging activities, Fe³⁺–Fe²⁺ and Cu²⁺–Cu⁺ reducing power assays when compared to the standard antioxidant compounds such as BHA, BHT, α-tocopherol as a natural antioxidant and trolox. An anticholinergic agent is a substance that blocks the neurotransmitter acetylcholine in the central and the peripheral nervous system. It has been observed pyrogallol was effective inhibitor for AChE. For this reason, pyrogallol can show effect acting as an anticholinergic substance. Also, it is known that drugs which inhibit acetylcholinesterase are used for the treatment of Alzheimer’s disease. In conclusion, pyrogallol can be used anticholinergic compound or nutritional product as a source of natural antioxidant for pharmaceutical industry.

Supplementary material available online

Supplementary Figures 2–5.

Declaration of interest

The author reports no conflicts of interest.