Antioxidant, antiradical, and anticholinergic properties of cynarin purified from the Illyrian thistle (Onopordum illyricum L.)

Abstract

Cynarin is a derivative of hydroxycinnamic acid and it has biologically active functional groups constituent of some plants and food. We elucidated the antioxidant activity of cynarin by using different in vitro condition bioanalytical antioxidant assays like DMPD^•+, ABTS^•+, , DPPH^• and H₂O₂ scavenging effects, the total antioxidant influence, reducing capabilities, Fe²⁺ chelating and anticholinergic activities. Cynarin demonstrated 87.72% inhibition of linoleic acid lipid peroxidation at 30 µg/mL concentration. Conversely, some standard antioxidants like trolox, α-tocopherol, butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA) exhibited inhibitions of 90.32, 75.26, 97.61, 87.30%, and opponent peroxidation of linoleic acid emulsion at the identical concentration, seriatim. Also, cynarin exhibited effective DMPD^•+, ABTS^•+, , DPPH^•, and H₂O₂ scavenging effects, reducing capabilities and Fe²⁺ chelating effects. On the contrary, IC₅₀ and K_i parameters of cynarin for acetylcholinesterase enzyme inhibition were determined as 243.67 nM (r²: 0.9444) and 39.34 ± 13.88 nM, respectively. This study clearly showed that cynarin had marked antioxidant, anticholinergic, reducing ability, radical-scavenging, and metal-binding activities.

• Antiacetylcholinesterase
• anticholinergic
• antioxidant activity
• cynarin
• Onopordum illyricum
• radical scavenging

Introduction

Oxidation is the transfer of electrons between two atoms and stands for a required part of aerobic life and our metabolism. In many living organisms, it is essential for the production of energy to fuel biological processes. However, problems may occur when the electron flow becomes disconnected, generating free radicals and reactive oxygen species (ROS)1^,2. A free radical has one or more uncoupled electron situated outermost orbital of molecular or atomic orbitals. ROS includes non-free radical kinds like ozone (O₃), singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), and free radical varieties like superoxide anion radicals (), peroxyl (ROO·), hydroxyl radicals (OH·), and hydroperoxyl radicals (HOO^.)3^,4. Free radicals may be occurring by living cells during pathophysiological and biochemical processes as well as due to environmental pollutants, radiation, chemicals, and toxins5^,6. They are produced naturally in mammalian systems as a result of oxidative metabolism2.

A normal cell has convenient pro-oxidant-antioxidant equilibrium. Yet, this equilibrium can be changed towards the pro-oxidants when the production of ROS increases enormously or when ranges of antioxidants are decreased. This stage is entitled as oxidative stress1. Antioxidants are compounds that prevent or retard the oxidation of substrates even if the compounds are available in substantially lower concentrations than the oxidized substrates. Also, they have features that safeguard the person from free radicals and effects of ROS. In recent years, interest has remarkably increased in identifying alternate safe and innate source materials of food antioxidants, and the search for natural antioxidants, particularly of plant basis7^,8. Also, restricting the use of synthetic antioxidants has led to an incremental interest of natural antioxidant sources. Therefore, there is an increasing way in consumer priorities based on native antioxidants, all of them, which has given driving force to the initiatives to detect native source material of antioxidants9^,10.

Cynarin, 1,3-dicaffeoylquinic acid, is formed from esterification of two units of caffeic acid and one unit of quinic acid. It inhibits taste receptors, making water to be sweet. It has been shown to have some pharmacological properties including hypocholesterolemic11, hepatoprotective12, antiviral, antibacterial, and antihistamic effects13. Obtaining pure cynarin from the mixture of mono- and dicaffeoylquinic acids in the crude extract of the plant material is problematic. The isolation of cynarin from natural sources has been reported, but yields are low and the purity is poor14, even following extensive chromatographic isolation. While a synthetic method for cynarin has been described15, the synthesis is uneconomical because of the high cost of the starting material and the low overall yield expected from the multistep reaction sequence. For these reasons, we have isolated cynarin from Illyrian thistle and its structure has been identified by NMR and Mass data.

Alzheimer’s disease (AD) is characterized by memory loss, dementia, and cognitive impairment. This disease is one of the most common diseases in elderly people16^,17. Acetylcholinesterase (AChE) is considered to be a drug target. AChE inhibitors (AChEIs) are used in the treatment of neuromuscular disorders such as myasthenia gravis. On the other hand, a variety of compounds are known to inhibit AChE and to act via different mechanisms. Based on these considerations, the design and identification of new AChEIs are pursued by several research groups. Currently, the treatment of AD focuses on acetylcholinesterase (AChE, E.C. 3.1.1.7) inhibitors, such as tacrine, donepezil, rivastigmine, and galantamine. AChEIs are used in the treatment of several neuromuscular diseases and were studied for the treatment of AD18^,19. However, the potential effectiveness of these inhibitors in clinical use is often complicated by their associated side effects. For example, clinical studies have shown that tacrine, which is putative AChEI, causes hepatotoxicity20. Since AD is a multi-pathogenic illness, a current drug-discovery strategy is to develop novel anti-acetylcholinesterase agents with multiple potencies such as inhibition of AChE21. AChE is found in high concentrations in the brain and in red blood cells22. This enzyme is involved in the hydrolysis and regulation of acetylcholine (ACh). The use of agents with enhanced selectivity for AChE indicated potential therapeutic benefit of inhibiting AChE in Alzheimer’s disease and related dementias23. Therefore, AChE might be considered as an important target for novel drug development to treat AD.

In this study, we determined the antioxidant, antiradical, and anticholinergic activities of cynarin used by different bioanalytical methods. Also, another intention of the current study was to explain the antioxidant, anticholinergic, antiradical, and chelating ability with metal of cynarin.

Methods

Chemicals

Ellman’s reagent [5,5′-dithio-bis(2-nitro- benzoic acid), DTNB], acetylthiocholine iodide (AChI), N,N-dimethyl-p-phenylenediamine, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 2,9-dimethyl-1,10-phenanthroline, butylated hydroxyanisole, nitrobluetetrazolium, butylated hydroxytoluene, 1,1-diphenyl-2-picryl-hydrazyl, 3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine, methionine, riboflavin, linoleic acid, trichloroacetic acid, and α-tocopherol were purchased commercially from Sigma-Aldrich GmbH, Sternheim, Germany. Ammonium thiocyanate was obtained from Merck, Darmstadt, Germany. All other chemicals used were of analytical grade and attained from either Sigma-Aldrich (Sternheim, Germany) or Merck (Darmstadt, Germany).

Isolation of cynarin

The compound of cynarin was isolated from the Onopordum illyricum L. The plant species was collected from Saribeyler Dam area, Savaştepe, Balıkkesir (altitude: 245 m) in July 2010. A voucher specimen of species was deposited in Balıkesir University, Education Faculty, Department of Biology, Herbarium no. TD3735. Eight gram of methanol extract of species was partitioned in 60% aqueous methanol and hexane (7:3). The aqueous methanol layer was re-extracted by dichloromethane (6:4) and after then vacuum evaporation of solvent 4.8 g of residual was acquired. The residual was fractionated on a silica gel column (3 × 60 cm) with a gradient of hexane, CH₂Cl_2, acetone, and methanol. TLC runs were done during the elution and the similar fractions were combined. LC-MS/MS analysis was carried out to determine the cynarin-rich fractions and the cynarin was mostly found in the fractions of C and D. These fractions of cynarin were further purified by preparative HPLC with a gradient of acetonitrile: 0.1% TFA and H₂O: 0.1% TFA on XTerra Prep OBD MSC18 column (5.0 µm, 19 mm × 50 mm column) with several runs. About 42 mg of cynarin was isolated and the structure of compound was identified by NMR and Mass data.

Total antioxidant activity detection by ferric thiocyanate method

For the evaluation of total antioxidant activity of cynarin and reference antioxidants, ferric thiocyanate method was used. This method, in other words inhibition of linoleic acid emulsion (LAE), was described previously24^,25. Cell membranes contain phospholipid bilayers with peripheral proteins and are the direct destination of lipid peroxidation. Preparation of stock solution and LAE was defined in our previous studies26. The peroxides obtained through peroxidation of linoleic acid will oxidize Fe²⁺ to Fe³⁺, which generates a complex with thiocyanate which has a maximum absorbance at 500 nm. The assay steps were renewed every 12 h increasing to a high scale. In linoleic acid emulsion, the percent inhibition of lipid peroxidation (ILP) was computed by the following equation: where A_C is the absorbance value of the control reaction, which includes only sodium phosphate buffer (NaH₂PO₄/Na₂HPO₄) and linoleic acid emulsion. In the existing solution, the absorbance value of cynarin and other tested samples is called A_S22.

Fe³⁺ reducing antioxidant power assay

For the determination of Fe³⁺ reducing ability of cynarin, Fe³⁺(CN⁻)₆ to Fe²⁺(CN⁻)₆ reduction method was used24^,26. In brief, various concentrations of cynarin (10–30 µg/mL) in 0.75 mL of deionized H₂O were added with 1.25 mL of sodium phosphate (NaH₂PO₄/Na₂HPO₄) buffer (0.2 M, pH 6.6) and 1.25 mL of potassium ferricyanide [K₃Fe(CN)₆] (1%). Then, the solution was incubated at 50 °C during 20 min. After the incubation period, trichloroacetic acid (TCA) was added (1.25 mL, 10%). Finally, a portion of FeCl₃ (0.5 mL, 0.1%) was transferred to this mixture and the absorbance value was enrolled at 700 nm in a spectrophotometer. According to the obtained results, when reduction capability increases, absorbance indicates greater value27.

Cu²⁺ reducing power-CUPRAC assay

Cupric ions (Cu²⁺) reducing power was used as a second method for the reducing ability of cynarin. Cu²⁺ reducing capability was performed according to the method of Apak et al.28 with slight modification. For this purpose, aliquots of CuCl₂ solution (0.25 mL, 0.01 M), ethanolic neocuproine solution (0.25 mL, 7.5 × 10⁻³M), and NH₄Ac buffer solution (0.25 mL, 1 m) were transferred to a test tube, which contains cynarin at different concentrations (10–30 µg/mL). Total volume was completed with distilled H₂O to 2 mL and shaken vigorously. The absorbance of samples was recorded at 450 nm after 30 min29.

Fe²⁺ chelating activity

Fe²⁺ binding ability of cynarin was predicted according to Dinis et al.30 with minor modification1. Fe²⁺ chelating capacity of cynarin was spectrophotometrically recorded by the absorbance value of the ferrous iron–bipyrdyl complex at 522 nm. In brief, to a mixture of FeSO₄ (0.1 mL, 0.6 mM), cynarin was added at three different concentrations (10–20 µg/mL) in the presence of methanol (0.4 mL). The reaction was started by the addition of bipyrdyl solution (0.1 mL, 5 mM). After that, the solution was mixed and incubated at room temperature for 10 min. Finally, the absorbance value of the mixture was quantified spectrophotometrically at 522 nm versus blank sample.

H₂O₂-scavenging activity

It was decided to use the methodology of Ruch et al.31 for H₂O₂₋scavenging activity. According to the method, a decrease in the absorbance value of H₂O₂ upon oxidation of H₂O₂ is measured spectrophotometrically. A stock mixture of H₂O₂ (40 mM) was prepared in phosphate (NaH₂PO₄/Na₂HPO₄) buffer (0.1 M, pH 7.4). Cynarin (at a concentration of 30 µg/mL) in phosphate buffer (3.4 mL) was mixed to an aliquot of H₂O₂ mixture (0.6 mL, 40 mM) and the absorbance value of solution was enrolled at 230 nm. An empty solution included sodium phosphate (NaH₂PO₄/Na₂HPO₄) buffer without H₂O₂.

Superoxide anion radical scavenging activity

Superoxide radicals () have one electron more than an oxygen molecule. In other words, it is reduced to the shape of molecular oxygen. scavenging activity of cynarin was done in accordance with the methodology of Zhishen et al.32 with slight adjustment33. is produced in methionine and riboflavin enlightens system and determined by decreasing NBT to generate blue formazan (NBT²⁺). All mixtures were prepared in phosphate buffer (0.05 M, pH 7.8). Photo-induced reactions were realized by using fluorescent lamps (20 W). The mixture was enlightened at 25 °C for 40 min. Photochemically reduced riboflavin produced , which reduced NBT to form NBT²⁺. The absorbance was measured spectrophotometrically at 560 nm34.

DPPH·scavenging activity

The radical scavenging activity of cynarin was gauged in the same way of hydrogen-donating capability, using the steady 1,1-diphenyl-2-picrylhydrazyl (DPPH^•) reagent, formerly described by Göçer and Gülçin35. When an electron or hydrogen atom was moved to the unpaired electron in DPPH^•, the absorbance value at 517 nm decreased proportionately to the increases of DPPH₂36. The solution of DPPH^• was freshly prepared daily, stored in a flask coated with aluminum foil, and kept in the dark at 4 °C between the measurements. In brief, fresh solution of DPPH^• (0.1 mM) was prepared in ethanol. Then, 1.5 mL of cynarin in ethanol was added to an aliquot of this solution (0.5 mL) (10–30 µg/mL). These mixtures were mixed vigorously and incubated in the dark for 30 min. Finally, the absorbance value was recorded at 517 nm in a spectrophotometer7.

ABTS^•+ scavenging activity

ABTS^•+ scavenging activity of cynarin was gauged using the methodology of Re et al.37. The ABTS radical cation (ABTS^•+) was acquired by reacting 7 mM solution of ABTS with 2.45 mM potassium persulfate. The radical cation was steady in this form for more than 2 d which was stored in the dark at room temperature. Prior to assay, the ABTS radical cation solution was diluted with ethanol to an absorbance value of 0.750 ± 0.05 at 734 nm. This solution was equilibrated at 30 °C. Then, 1 mL of ABTS^•+ solution was supplemented with 3 mL of cynarin or positive control solutions in ethanol at different concentrations (10–30 µg/mL). The extent of decolorization is calculated as percentage reduction of absorbance6.

DMPD^•+ scavenging activity

DMPD radical scavenging capability of cynarin was found as defined by Fogliano et al.38. DMPD (0.1 M) was prepared by dissolving 0.2 g of DMPD in 10 mL of deionized H₂O. Then an aliquot of this solution (1 mL) was supplemented to acetate buffer (100 mL, 0.1 M and pH 5.3). Colored radical cation (DMPD^•+) was attained by adding 0.2 mL of FeCl₃ solution (0.05 M). Diverse concentrations of positive controls or cynarin (10–30 μg/mL) were transferred in test tubes. Distilled water was added up to complete 0.5 mL of volume and the absorbance value was gauged at 505 nm after 10 min. One milliliter of DMPD^•+ mixture was added to the solution and its absorbance value was recorded at 505 nm.

Percentage of Fe²⁺ chelating, H₂O₂ scavenging, superoxide anion scavenging, ABTS^•+ scavenging, DPPH^• radical scavenging and DMPD^•+ radical was computed using the following equation: where A_C is the absorbance value of control and A_S is the absorbance value of sample39.

Determination of anticholinesterase activity

Inhibitory effects of cynarin on AChE activities were measured by slightly modifying the spectrophotometric method of Ellman et al.40. AChI was used as a substrate of the reaction. DTNB (Product no: D8130-1 G, Sigma-Aldrich, Sternheim, Germany) was used for the determination of the AChE activity. Namely, 100 mL of Tris/HCl buffer (1 M, pH 8.0), 10 mL of sample solution dissolved in ultra pure water at different concentrations, and 50 mL AChE (5.3210⁻³ EU) solution were mixed, and incubated for 10 min at 25 °C. Then a portion of DTNB (50 mL, 0.5 mM) was added. The reaction was then started by the addition of 50 mL of AChI (10 mM, Product no: 01480-1 G, Sigma-Aldrich, Sternheim, Germany). The hydrolysis of these substrates was observed spectrophotometrically by the formation of yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with thiocholine, determined by the enzymatic hydrolysis of AChI, at a wavelength of 412 nm. In order to determine the effect of cynarin on AChE, different cynarin concentrations were added to the reaction mixture. Then, AChE activity was measured18. IC₅₀ values were obtained from activity (%) versus compounds plots41–44.

Statistical analysis

Each experiment was repeated three times. The acquired data were enrolled as mean ± standard deflection and analyzed by SPSS (version 11.5 for Windows 2000, SPSS Inc., Chicago, IL). One-way analysis of type ANOVA was implemented by procedures. Considerable distinctions between means were identified by Duncan’s Multiple Range tests, and p < 0.05 was seen as significant and p < 0.01 as very significant.

Results and discussion

Antioxidant compounds from natural sources are the only alternative to synthetic antioxidants counteracting the ROS-associated diseases. For this purpose, majority of naturally taking place substances have been noticed to own antioxidant abilities. Also, various in vitro methods have been used to assess antioxidant activity1. In the present study, several different antioxidant activity assays based on different reaction mechanisms are used to detect the potent antioxidant activity of cynarin. The first of these methods is FTM. These methods determine the quantity of peroxides produced throughout the first steps of peroxidation of lipid. Lipid oxidation occurs in food and pharmaceutical applications and is one of the major anxieties for both food and pharmaceutical technologies. It is important for nutritional and pharmaceutical qualities and safeties which are caused by the formation of secondary, potentially toxic compounds. This is very important to human health protection and it is also economically important45. Cynarin demonstrated effective inhibition of lipid peroxidation in the linoleic acid emulsion. The impression of 30 µg/mL concentration of cynarin on the lipid peroxidation of linoleic acid emulsion is demonstrated in Figure 1 and was identified to be 87.72%. Otherwise, the positive controls like trolox, α-tocopherol, BHT, and BHA demonstrated 90.32%, 75.26%, 97.61%, and 87.30% peroxidation of linoleic acid emulsion at the identical concentration (30 µg/mL), respectively. Without cynarin or positive controls, the auto-oxidation of linoleic acid emulsion was accompanied by a rapid increase in peroxides. These outcomes expressly showed that cynarin had the greatest antioxidant activity compared with BHA and α-tocopherol but lower than that of BHA and trolox.

Figure 1. Total antioxidant activities of cynarin and standard antioxidant compounds like trolox, α-tocopherol, BHT, and BHA at the same concentration (30 µg/mL) assayed by the ferric thiocyanate method. The control value reached a maximum 50 h (BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene).

Generally, reducing properties depend on the presence of reductones, which have been shown to exert antioxidant activity and radical scavenging ability by donating a hydrogen atom46. The Fe³⁺(CN⁻)₆ reduction method detects the antioxidant effect of any molecule as reducing capability in the reaction. Cynarin had the most influential reducing capacity using Fe³⁺(CN⁻)₆ reduction and Cu²⁺ ions reducing ability when classed with the standards (trolox, α-tocopherol, BHT, and BHA). As shown in Figure 2(A), cynarin (r²: 0.9082) demonstrated potent Fe³⁺ reducing capability and these diversities were statistically seen to be considerably important (p < 0.01). Reducing capacity of cynarin (r²: 0.9947), trolox (r²: 0.9943), α-tocopherol (r²: 0.9987), BHT (r²: 0.9967), and BHA (r²: 0.9976) increased constantly when the concentration of sample was increased. Reducing capacity of cynarin and standard chemical agents were given in the following order: cynarin (3.613) > BHA (3.086) > BHT (1.918) > α-tocopherol (1.335) > trolox (1.184) at the same concentration (30 µg/mL). Results proved that cynarin had marked ferric ions (Fe³⁺) reducing capability and also had electron-releasing features to neutralize free radicals by creating steady products. In vivo, as a result of reduction reactions, radical chain reactions are complete and it may be very destructive.

Figure 2. Reducing power of cynarin. (A) Fe³⁺→ Fe²⁺ reductive potential of different concentrations (10–30 µg/mL) of cynarin (r²: 0.983) and reference antioxidants. (B) Cu²⁺ reducing ability of different concentrations (10–30 µg/mL) of cynarin (r²: 0.840) and reference antioxidants (BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene).

On one hand, Cu²⁺ reducing power of cynarin and positive controls is shown in Figure 2(B). A positive relationship was found between Cu²⁺ reducing power and different concentrations of cynarin (r²: 0.9572). It was detected that Cu²⁺ reducing capacity of cynarin was addicted to the concentration (10–30 μg/mL). Cu²⁺ reducing capability of cynarin and positive controls at the same concentration (30 μg/mL) showed the following order: BHA (r²: 0.9752) > BHT (r²: 0.9955) > cynarin (r²: 0.9572) > α-tocopherol (r²: 0.9999) > trolox (r²: 0.9703).

On the other hand, cynarin had also effective Fe²⁺ ions chelating effect. The distinction between different concentrations of cynarin (10–30 µg/mL) and the control value was fixed to be statistically important (p < 0.01). Furthermore, it is found that cynarin exhibited 58.90% chelating of ferrous ion at 20 µg/mL concentration (r²: 0.966). As shown in Figure 3, chelating capacity of cynarin with ferrous was classed with that of trolox (r²: 0.960), α-tocopherol (r²: 0.962), BHT (r²: 0.931), and BHA (r²: 0.961). Whereas, at the same concentration, Fe²⁺ ions chelating capacity of positive controls like trolox, α-tocopherol, BHT, and BHA was found to be 63.72, 52.96, 55.13, and 52.96%, respectively. These results clearly introduce that Fe²⁺ ions chelating effect of cynarin was parallel to trolox, α-tocopherol, BHA, and BHT (p > 0.05).

Figure 3. Comparison of Fe²⁺ chelating activity of cynarin (r²: 0.898) and standard antioxidant compounds like trolox, α-tocopherol, BHT, and BHA at the concentrations of 10–20 µg/mL (BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene).

Based on the superoxide anion radical scavenging activity results of cynarin, it was asserted that the activity of α-tocopherol and trolox is lower than cynarin whereas the activity of BHA and BHT is higher than cynarin. As shown in Figure 4, the IC₅₀ value of superoxide anion radical scavenging of cynarin was found to be 11.76 µg/mL (r²: 0.9699). Conversely, IC₅₀ values of trolox, α-tocopherol, BHT, and BHA were found as 36.44 µg/mL (r²: 0.9833), 56.42 µg/mL (r²: 0.9931), 53.57 µg/mL (r²: 0.9980), and 38.88 µg/mL (r²: 0.9986), respectively. When the EC₅₀ value is lower, superoxide anion radical scavenging activity increases. These results demonstrated that cynarin had the highest activity when compared with superoxide anion radical scavenging activity in general chemical agents, and these distinctions were found to be statistically significant.

Figure 4. Comparison of superoxide anion radical () scavenging activities of cynarin and standard antioxidant compounds like trolox, α-tocopherol, BHT, and BHA at the concentration of 30 µg/mL (BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene).

DPPH test is generally used as the substrate to gauge free radical scavenging effectiveness of antioxidants47. This methodology is based on the reduction of a DPPH solution in alcohol in the source of a hydrogen donating antioxidant, owing to the formation of non-radical form DPPH-H by the reaction48^,49. Cynarin has the ability to reduce steady radical DPPH to yellow-colored DPPH₂. Figure 5(A) defines a crucial decrement (p < 0.01) in the concentration of DPPH radical owing to the scavenging capability of cynarin and reference chemical agents like trolox, α-tocopherol, BHT, and BHA. IC₅₀ values were found to be 3.98 µg/mL (0.9425) for cynarin, 14.16 µg/mL (0.9992) for trolox, 12.84 µg/mL (0.9991) for α-tocopherol, 38.13 µg/mL (0.9768) for BHT, and 13.43 µg/mL (0.9925) for BHA. DPPH radical scavenging of samples increased in the order of cynarin > α-tocopherol ≈ BHA ≈ trolox > BHT. A lower IC₅₀ value demonstrates a higher DPPH radical scavenging activity.

Figure 5. Radical-scavenging activities of cynarin. (A) DPPH free radical scavenging activity of different concentrations (10–30 µg/mL) of cynarin (r²: 0.950) and reference antioxidants. (B) ABTS radical scavenging activity of different concentrations (10–20 µg/mL) of cynarin (r²: 0.956) and reference antioxidants. (C) DMPD radical scavenging activity of different concentrations (10–30 µg/mL) of cynarin (r²: 0.982) and reference antioxidants (BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; DPPH•, 1,1-diphenyl-2-picryl-hydrazyl free radical; ABTS^•+, 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid; DMPD^•+, N,N-dimethyl-p-phenylenediamine radical).

Efficient radical cation scavenging activity was seen on tested compounds. As shown in Figure 5(B), cynarin is an efficient ABTS^•+ radical scavenger in a concentration-dependent manner (10–30 µg/mL, r²: 0.9377). The EC₅₀ value for cynarin in this analysis was 10.60 µg/mL (r²: 0.937). It is seen that the concentration of ABTS^•+ (p < 0.01) declines substantially owing to the scavenging capability at all cynarin concentrations. Moreover, EC₅₀ values for trolox, α-tocopherol, BHT, and BHA were found to be 15.98 µg/mL (r²: 0.985), 15.98 µg/mL (r²: 0.938), 15.67 µg/mL (r²: 0.996), and 7.32 µg/mL (r²: 0.993), respectively. The scavenging efficacy of cynarin and standards on the ABTS^•+ increased in the following order: cynarin ≥ BHA > BHT > α-tocopherol ≈ trolox. As well as in DPPH free radical scavenging activity, a lower EC₅₀ value indicates a higher ABTS^•+ scavenging activity.

Previously, as in both radical scavenging methods, cynarin was an efficient DMPD^•+ radical scavenger in a concentration-dependent manner (10–30 µg/mL, r²: 0.935). The value EC₅₀ for cynarin was 48.68 µg/mL (r²: 0.935). This value was found to be 47.55 µg/mL (r²: 0.986) for trolox, 45.44 µg/mL (r²: 0.943) for α-tocopherol, 41.65 µg/mL (r²: 0.959) for BHT, and 40.21 µg/mL (r²: 0.953) for BHA (Figure 5C). There was an significant decline (p < 0.05) in the DMPD^•+ concentration owing to the scavenging ability at all cynarin concentrations. Within different concentrations of cynarin, crucial differences could not be determined in ABTS^•+ scavenging potential.

Lipid peroxidation is a free-radical chain reaction, and ROS can quicken this process. Mechanistic approaches of spectrophotometric analysis of lipid hydroperoxides based on the oxidation of Fe²⁺ to Fe³⁺ ions and following chelation of the latter by thiocyanate (SCN⁻) are thought. Ferric thiocyanate method measures the amount of peroxide generated along the first steps of lipid peroxidation. In this test, hydroperoxides produced from linoleic acid supplement to the reaction solution, which has oxidized in air through the experimental period, were immediately evaluated. Thiocyanate and FeCl₂ react with one another to generate Fe(SCN⁻)₂ through the agency of hydroperoxides1^,50.

Reducing capability of a bioactive compound can be calculated by means of direct reduction of Fe[(CN)₆]₃ to Fe[(CN)₆]₂. In this technique, the presence of reductants like cynarin would result in the reduction of Fe³⁺ to Fe²⁺. Addition of free Fe³⁺ to the reduced product brings about the formation of intensive Perl’s Prussian blue complex, Fe₄[Fe(CN⁻)₆]₃, which has a strong absorbance at 700 nm. Fe³⁺ reducing assay gets advantage of an electron chain reaction where a ferric salt is utilized as an oxidant51. In addition, the yellow color of the tested mixture changes into diverse tons of green and blue by ability of cynarin.

The CUPRAC method is a simple, rapid, selective, cost-effective, steady, and versatile antioxidant assay useful for a wide variety of polyphenols, as well as for thiols, synthetic antioxidants, and vitamins C and E. Also, this chromogenic redox reaction is conducted at a pH (7.0) and the method allows measuring antioxidants including thiol like glutathione and non-protein thiols. CUPRAC reactions are essentially complete within 30 min28.

Fe²⁺ ions are the most efficient pro-oxidants in pharmacology systems and food52. Ferrozine can create complexes with Fe²⁺. In the presence of Fe²⁺ chelating compounds, ferrozine–Fe²⁺ complex formation is a broken down, resulting in a decrease in the red color of ferrozine–Fe²⁺ complex1^,53. Data shown in Figure 3 display that cynarin has a strong capability to bind Fe²⁺. It shows that its main action as a peroxidation inhibitor may be involved to its Fe²⁺ linking capacity. Cynarin prohibited the formation of ferrous–ferrozine complex. Cynarin is able to catch ferrous ion before ferrozine. It can convert Fe²⁺ ions into insoluble metal complexes or generate sterically hindrance, which can prevent the interactions between metals and lipid intermediates54^,55. The chemical structure of cynarin and its metal binding sites is given in Figure 6(B). It may chelate two ferrous ions through the agency of its two-hydroxyl groups. It was known that biological active molecules with structures including functional groups like C–OH and C=O can bind Fe²⁺ ions. Additionally, the compounds with molecules contain two or more of the following functional groups: −S−, −O−, −OH, −SH, C=O, −NR₂, −COOH, and −H₂PO₃ in favor of structure–function configuration. In this way, the compounds can chelate Fe²⁺ ions1^,56^,57. In our previous study, it was demonstrated that l-carnitine, which is required for the transport of fatty acids from the cytosol into the mitochondria during the breakdown of lipids, chelated ferrous ions (Fe²⁺) thanks to the hydroxyl and carbonyl groups3. Similarly, it was specified that curcumins, natural phenols, are responsible for the yellow color of turmeric-bounded Fe²⁺ ions thanks to hydroxyl and carbonyl groups36. In a similar vein, l-adrenaline, which is used for the treatment of a number of conditions including anaphylaxis, cardiac arrest, and superficial bleeding, bounded with Fe²⁺ ions with hydroxyl and amine groups10. The purposed Fe²⁺ ions chelating of cynarin are presented in Figure 6(B). In this study, we demonstrated that two cynarin compounds linked ferrous ions (Fe²⁺) by the agency of hydroxyl groups. Recently, Fiorucci et al.58 have proved that quercetin complexioned metal ions in the same way.

Figure 6. (A) The reaction scheme between DPPH free radicals and cynarin. (B) The proposed reaction for chelating of ferrous ions by cynarin.

Figure 7. Stabilization of radicals by the phenol group of cynarin.

H₂O₂ is a biologically relevant, non-radical reactive oxygen species and is ineluctably generated as a by-product of normal aerobic metabolism10. H₂O₂ is not very reactive; whereas it may have hazardous effects on the cells because it may cause to produce OH^• within the cells. When the concentration increases under oxidative stress or stress conditions, H₂O₂ could be detrimental to cells. Accessing of H₂O₂ to cells in culture can cause transition metal ion-dependent OH^• mediating oxidative DNA damage59. Therefore, removing superoxide anion and H₂O₂ is very crucial for the protection of pharmaceutical and food systems7. It is well known that biologically active phenolic compounds play an important role in the protection of mammalian or bacterial cells from cytotoxicity induced by H₂O₂.

DPPH exposed an absorbance at 517 nm, which vanished after acceptation of an electron or a hydrogen radical from an antioxidant compound to become a steadier diamagnetic molecule45^,60. DPPH radicals scavenging of cynarin are summed up in Figure 6(A). It is well known that a radical can be stabilizing through the agency of resonance structure of phenolic compounds. Chemically, it is an ester formed from quinic acid and two units of caffeic acid. Cynarin has monophenol and diphenol phenolic rings. Separation of a hydrogen atom from monophenolic and diphenolic hydroxyl group may be occurring readily. Also, cynarin scavenged a third DPPH radical on the carboxyl group of quinic acid. The reasons of powerful radical scavenging activity of cynarin molecule are its conjugation structures and resonance stability (Figure 6A). Stabilization of radicals by the phenol group of cynarin is given in Figure 7.

Another improved technique for the determination of radical scavenging is ABTS^•+ scavenging activity. Generation of ABTS^•+ defined here includes direct production of blue/green ABTS^•+ chromophore thanks to the reaction between ABTS and K₂S₂O₈. ABTS^•−, the oxidant, was produced by the agency of K₂S₂O₈ oxidation of ABTS²⁻ and radical cation is calculated spectrophotometrically. This is a direct production of a steady form of radical to create a blue-green ABTS^•+ chromophore before the reaction with antioxidants61. ABTS^•+ cation can be prepared by running distinct oxidants like permanganate (), chromate (), and perchlorate (). In this sense, the oxidizing agent can be called an oxygenation reagent or oxygen-atom transfer agent. Results acquired using K₂S₂O₈ as an oxidant show that the occurrence of K₂S₂O₈ increases the ratio of ABTS^•+. These radicals were generated in ABTS/K₂S₂O₈ system1.

The basic guideline of DMPD^•+ scavenging assay is that when it is used at acidic pH and in convenient oxidant solution, DMPD can form a steady and colored radical cation (DMPD^•+). The UV–visible spectrum of DMPD^•+ shows a maximum absorbance at 505 nm. Cynarin, which is able to transfer a hydrogen atom to DMPD^•+, quenches the color and produces discoloration of the solution. This reaction is rapid and the end point, which is steady, is taken as a measure of antioxidative competence. Also, this assay reflects the talent of radical hydrogen-donors to scavenge the single electron from DMPD^•+. Actually, radical cation forms slowly and, therefore, the absorbance value increases continuously28. Contrary to the ABTS^•+ scavenging method, DMPD^•+ scavenging procedure guarantees a very steady end point. This is quite significant when a large-scale screening is necessary. It was shown that the main drawback of DMPD^•+ scavenging procedure is the fact that its sensitivity and renewability significantly decreased when hydrophobic antioxidants like BHT or α-tocopherol were used. Therefore, these positive controls were not suitable for use in DMPD^•+ scavenging assay1^,62.

In living systems, plays a crucial role in the formation of other ROS like H₂O₂,·OH or ¹O₂ (A). can react with NO· and form peroxynitrite (ONOO^–), which can produce toxic compounds like ·OH and nitric dioxide (B)63^,64. (A) (B)

In this study, derived from dissolved oxygen through the riboflavin–methionine–illuminate system will reduce NBT65. The reactants were illuminated at 25 °C for 40 min. In this way, the reactants reduced riboflavin generated , which reduced NBT to form blue formazan66^,67. In our methodology, reduces yellow dye (NBT²⁺) to generate blue formazan, which has an absorbance value at 560 nm in the spectrophotometer. Cynarin as an antioxidant is able to block blue NBT formation. Reduction of absorbance at 560 nm with antioxidants shows superoxide anion68. Figure 4 shows the inhibition of generation by 30 µg/mL concentration of cynarin and positive controls like trolox, α-tocopherol, BHT, and BHA.

Phenolic structures comprise a diverse group of molecules classified as secondary metabolites in plants that have a large range of structures and functions69^,70. Cynarin is effective phenolic compounds and sold as food and nutritional supplements. Cynarin is a phenolic compound, which are very important molecules in terms of biological activities71^,72. A greater number of useful health effects, like hepatoprotective, antiviral, antibacterial, and antihistamic and inhibition effects on HIV-1 replication in MT-2 cell culture, have been reported1^,73.

Different types of AChEIs have been studied for the treatment of AD. AChE inhibitor rivastigmine and galanthamine are used as drugs for the treatment of AD. Besides, rivastigmine was shown to be effective of opposite from the scopolamine of consciousness disorders74. Rivastigmine rapidly causes inhibition on AChE and BChE alone75. Results obtained from these studies showed that an investigation into the mechanism of action of AChE might cause the design of inhibitors, which can be used as a therapeutic in the future. Additionally some studies showed that AChEIs have different properties in terms of action mechanism, metabolism, and brain selectivity.

In the current study, we focused on the influence of cynarin on the inhibition kinetics of AChE. In our study, cynarin was investigated for their ability to inhibit AChE in detail. According to our data, inhibitory effect of cynarin revealed significant elevation in the case of AChE. Generally, these compounds showed higher inhibition property76–78. Considering the results, cynarin expressed significantly higher inhibition activity. Cynarin had significantly higher AChE inhibitory activity than that of putative standard acetylcholinesterase inhibitors such as rivastigmine and galanthamine87. Also, as can be seen in Figure 8(A), the IC₅₀ values of cynarin are 243.67 nM (r²: 0.9444). On the contrary, it was reported that both standard compounds demonstrated IC₅₀ values of 501.0 and 4.0 µM, respectively88. As can be seen in the results obtained from Figure 8(B), AChE was effectively inhibited by cynarin, with a K_i value of 39.34 ± 13.88 nM.

Figure 8. Determination of IC₅₀ (A) and K_i (B) values of cynarin for acetylcholinesterase enzyme by the Lineweaver–Burk graph.

Conclusion

On one hand, cynarin was found to be a powerful antioxidant, antiradical, and anticholinergic in different in vitro bioassays when compared with standard antioxidant and anticholinergic compounds. As discussed above, cynarin can be used for minimizing or preventing lipid oxidation in food or pharmaceutical products, delaying the formation of toxic oxidation products, maintaining nutritional quality, and prolonging the shelf life of food or pharmaceutical materials. On the other hand, acetylcholine is one of the key neurotransmitters for the peripheral nervous system and, therefore, inhibition of AChE has been proposed as a drug for neurotoxicity. Cynarin had effective AChE inhibition properties and this compound can be used for the treatment of mild-to-moderate Alzheimer’s disease and various other memory diseases. Also, cynarin should be further evaluated as potent AChE inhibitor for the treatment of Alzheimer's disease.

Declaration of interest

The authors report that they have no conflicts of interest. I. G. and S. E. would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research, RGP-VPP-254.