Insight into the biological properties and

Industrial Crops & Products 113 (2018) 422–428

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Insight into the biological properties and phytochemical composition of Ballota macrodonta Boiss. et Balansa, — an endemic medicinal plant from Turkey

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Sengul Uysala, Zaahira Aumeeruddy-Elalfib, Gokhan Zengina, , Abdurrahman Aktumseka, Andrei Mocanc, Luisa Custodiod, Nuno R. Nenge, José M.F. Nogueirae, Ana Ćirićf, Jasmina Glamočlijaf, Marina Sokovićf, M.Fawzi Mahomoodallyb a

Selcuk University, Science Faculty, Department of Biology, Campus, 42250, Konya, Turkey Department of Health Sciences, Faculty of Science, University of Mauritius, Réduit, Mauritius Department of Pharmaceutical Botany, “Iuliu Hațieganu” University of Medicine and Pharmacy, 23, Ghe. Marinescu Street, 400337 Cluj-Napoca, Romania d Centre of Marine Sciences, Faculty of Sciences and Technology, Campus of Gambelas, University of Algarve, Faro, Portugal e Faculty of Sciences of the University of Lisbon, Centre of Chemistry and Biochemistry/Department of Chemistry and Biochemistry,Building C8, Floor 5, Campo Grande, 1749-016, Lisbon, Portugal f Institute for Biological Research “Siniša Stanković” University of Belgrade, Belgrade, Serbia b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Ballotama crodonta Phenolics Antimicrobial Antioxidant Enzyme inhibitor Natural bioactive agents

Ballota macrodonta Boiss. et Balansa., is a traditionally used endemic medicinal plant used to manage a plethora of diseases in Turkey. Nonetheless, few studies have endeavoured to highlight its therapeutic potential and its phytochemical profile. We hypothesized that B. macrodonta would possess multi-pharmacological propensities and bioactive compounds that would justify its use as a folk remedy. Aerial part of B. macrodonta was assessed for its antioxidant, antimicrobial, and enzymatic inhibitory potential using different extraction solvents. The total bioactive components determination and compound identification using High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) method were used to correlate the biological properties. HPLC-DAD profile showed the presence of 19 components (e.g. hydroxybenzoic acid, catechin hydrate, vanillic acid, caffeic acid, syringic acid, epicatechin, coumaric acid, ferulic acid, luteolin-7-O-glucoside, rutin, and quercetin). The water and methanolic extracts showed high total phenolic and flavonoid contents, respectively. The overall antioxidant capacity of the water extract was higher than the one of the methanol extract. The extracts showed inhibitory activity against cholinesterases, tyrosinase, α-amylase and α-glucosidase. The extracts also presented antimicrobial activity against eight Gram positive/negative bacteria (MIC – 0.05–0.15 mg/ mL) and eight fungi (MIC – 0.0125–0.20 mg/mL). The presence of active biomolecules with multi-pharmacological properties makes B. macrodonta a potential source of health-promoting compounds that can be exploited as a novel medicinal herbal product.

1. Introduction Pharmacological properties of medicinal agents from plants center on secondary metabolites, also referred to as phytochemicals (Mahomoodally, 2013). Phytochemicals, such as plant-based phenolics are widely distributed and abundant in the kingdom Plantae. A multitude of bioactive phytochemicals have been probed as invaluable sources of novel antimicrobial agents, anti-tumour drugs, cholesterollowering compounds, immuno-modulatory, and anti-inflammatory agents. In literature, many studies have been dedicated to the

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antioxidant phenolics available in aromatic plants, medicinal plants, and edible plants (Giada, 2013; Rockenbach et al., 2011; Vinson et al., 2001). An array of phytochemicals have been reported to play fundamental roles in the treatment and/or prevention and/or management of a wide panoply of pathologies, many of which were challenging to manage until these products were discovered. In addition, several studies have revealed the role of plant phenolics in the management of communicable and noncommunicable diseases such as cardiovascular and neurodegenerative diseases, diabetes, urinary tract infections, and diseases caused by pathogenic bacteria and

Corresponding author. E-mail address: [email protected] (G. Zengin).

https://doi.org/10.1016/j.indcrop.2018.01.001 Received 24 August 2017; Received in revised form 30 December 2017; Accepted 2 January 2018 Available online 05 February 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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the sample. Rutin was used as a reference standard, and the total flavonoid content was expressed as milligrams of rutin equivalents (mg RE/g extract)

fungi (Maddox et al., 2010; Rodriguez-Mateos et al., 2014). Also, the ability of plant phenolics to inhibit the activity of enzymes have been previously investigated (Ademiluyi et al., 2015; Altunkaya and Gökmen, 2008). The aim of such studies is to bring forward alternative sources of natural products, to minimize substantial side effects associated with conventional drugs, particularly for pathologies linked to the excessive production of specific enzymes with health deterrent actions. In the current study, the biological properties of an endemic folk medicinal plant, Ballota macrodonta were highlighted. The genus Ballota belongs to the Lamiaceae family. This family, consisting of about 236 genera (Kubitzki et al., 2004) is represented worldwide and comprises mostly of flowering plants, many of which are aromatic plants used as culinary herbs. The Turkish flora comprises of 16 species and 12 subspecies of the Ballota genus among which eight are endemic to Turkey. The traditional usages of members of the Ballota genus have been reported by several authors, in Anatolian folk medicine. For instance, these plants have been used to treat gastrointestinal disorders, inflammation, cough, and hemorrhoids (Mericli et al., 1988; Tuzlacı and Tolon, 2000; Yeşilada et al., 1995; Yeşilada et al., 1993). Ballota macrodonta Boiss. et Balansa, is an endemic medicinal plant growing in Turkey and together with several other members of the same genus is traditionally used as an antiulcer, antispasmodic, diuretic, choleretic, antihemorrhoidal, and sedative agent (Citoğlu et al., 2004). Nonetheless, few studies have endeavoured to highlight its therapeutic potential. Indeed, a comprehensive literature survey shows that there is scarcity of studies focused towards validating its medicinal uses and establishing its phytochemical profile. In light of the traditional usages of species from Ballota genus, we hypothesized that B. macrodonta would possess multi-pharmacological potential and bioactive compounds that would justify its use as a folk remedy. To this effect, the present study was designed to highlight the antioxidant, antimicrobial, and enzyme inhibitory potential of B. macrodonta from Turkey.

2.3. HPLC-DAD analysis and identification of the main compounds The extracts at the concentration of 10 mg/mL in ultrapure water were analyzed by an HPLC-DAD (Agilent 1100 Series LC system. Germany), constituted by the following modules: vacuum degasser (G1322A), quaternary pump (G1311A), autosampler (G1313A), thermostatted column compartment (G1316A), and the diode array detector (G1315B). The data acquisition and instrumental control were performed by the software LC3D ChemStation (Agilent Technologies). Analyses were performed on a “Mediterranean sea 18” column, 15 × 0.21 cm, 5 μm particle size (Teknokroma, Spain). The mobile phase consisted on a mixture of MeOH (solvent A) and 2.5% acetic acid aqueous solution with the following gradient: 0–5 min: 10% A, 5–10 min: 10–30% A, 10–40 min: 30–90% A, 40–45 min: 90% A, 45–55 min: 90-10% A, and 55–60 min: 10% A, using a flow of 0.5 mL/ min. The injection volume was 20 μL with a draw speed of 200 μL/min. The detector was set at 210, 280 (use for quantification), 320 and 350 nm. For identification, the retention parameters of each peak were compared with the standard controls and the peak purity with the UV–Vis spectral reference data. The levels of the different compounds were extrapolated from calibration standard curves. Commercial standards were prepared in methanol (1 mg/L) and diluted with ultrapure water in desired concentration as described previously (Rodrigues et al., 2015). 2.4. Antioxidant activity The antioxidant activity of the Ballota species was evaluated using different assays. These assays were free radical scavenging (DPPH and ABTS), reducing power (CUPRAC and FRAP), phosphomolybdenum, and metal chelating. The experimental procedures were as previously described (Zengin et al., 2015), and they are summarized below. For the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay: Sample solution (2 mg/mL; 1 mL) was added to 4 mL of a 0.004% methanol solution of DPPH. The sample absorbance was read at 517 nm after a 30 min incubation at room temperature in the dark. DPPH radical scavenging activity was expressed as milligrams of trolox equivalents (mg TE/g extract). For ABTS (2,2′-azino-bis(3-ethylbenzothiazoline) 6-sulfonic acid) radical scavenging assay: Briefly, ABTS+ was produced directly by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to incubate for 12–16 h in the dark, at room temperature. Prior to beginning the assay, ABTS+ solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. Sample solution (2 mg/mL; 1 mL) was added to ABTS solution (2 mL) and mixed. The sample absorbance was read at 734 nm after a 30 min incubation at room temperature. The ABTS radical scavenging activity was expressed as milligrams of trolox equivalents (mg TE/g extract) For CUPRAC (cupric ion reducing activity) assay: Sample solution (2 mg/mL; 0.5 mL) was added to premixed reaction mixture containing CuCl2 (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH4Ac buffer (1 mL, 1 M, pH 7.0). Similarly, a blank was prepared by adding sample solution (0.5 mL) to premixed reaction mixture (3 mL) without CuCl2. Then, the sample and blank absorbances were read at 450 nm after a 30 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. CUPRAC results were expressed as milligrams of trolox equivalents (mg TE/g extract). For FRAP (ferric reducing antioxidant power) assay: Sample solution (2 mg/mL; 0.1 mL) was added to premixed FRAP reagent (2 mL) containing acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) (10 mM) in 40 mM HCl and ferric chloride (20 mM) in a

2. Materials and methods 2.1. Plant materials and preparation of extracts Aerial parts of Ballota macrodonta were collected from Nigde (around Camardi) during summer of 2014, and allowed to air dry at room temperature. Taxonomic identification was carried out by Dr. Murad Aydın Sanda, senior taxonomist of the Department of Biology, Selcuk University, Turkey. The dried plant samples were ground and the powdered plant material (2 g) was extracted with 20 mL of solvent (methanol and water), for 60 min in a sonication bath at 30 °C. The extracts were filtered and concentrated under vacuum at 40 °C. The extracts were stored at +4 °C in dark until further analysis. 2.2. Total bioactive components Total phenolic content was determined as described previously (Slinkard and Singleton, 1977). Briefly, sample solution (2 mg/mL; 0.25 mL) was mixed with diluted Folin-Ciocalteu reagent (1 mL, 1:9, v/ v) and shaked vigorously. After 3 min, Na2CO3 solution (0.75 mL, 1%) was added and the sample absorbance was read at 760 nm after a 2 h incubation at room temperature. Results were expressed as milligrams of gallic acid equivalents (mg GAE/g extract). The total flavonoids content was determined using AlCl3 method (Zengin et al., 2014). Briefly, sample solution (2 mg/mL; 1 mL) was mixed with the same volume of aluminum trichloride (2%) in methanol. Similarly, a blank was prepared by adding sample solution (1 mL) to methanol (1 mL) without AlCl3. The sample and blank absorbances were read at 415 nm after a 10 min incubation at room temperature. The absorbance of the blank was subtracted from that of 423

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For α-glucosidase inhibitory activity assay: Sample solution (1 mg/ mL; 50 μL) was mixed with glutathione (50 μL), α-glucosidase solution (from Saccharomyces cerevisiae, EC 3.2.1.20, Sigma) (50 μL) in phosphate buffer (pH 6.8) and PNPG (4-N-trophenyl-α-D-glucopyranoside, Sigma) (50 μL) in a 96-well microplate and incubated for 15 min at 37 °C. Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was then stopped with the addition of sodium carbonate (50 μL, 0.2 M). The sample and blank absorbances were read at 400 nm. The absorbance of the blank was subtracted from that of the sample and the α-glucosidase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract).

ratio of 10:1:1 (v/v/v). Then, the sample absorbance was read at 593 nm after a 30 min incubation at room temperature. FRAP activity was expressed as milligrams of trolox equivalents (mg TE/g extract). For phosphomolybdenum method: Sample solution (2 mg/mL; 0.3 mL) was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The sample absorbance was read at 695 nm after a 90 min incubation at 95 °C. The total antioxidant capacity was expressed as millimoles of trolox equivalents (mmol TE/g extract). For metal chelating activity assay: Briefly, sample solution (2 mg/ mL; 2 mL) was added to FeCl2 solution (0.05 mL, 2 mM). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL). Similarly, a blank was prepared by adding sample solution (2 mL) to FeCl2 solution (0.05 mL, 2 mM) and water (0.2 mL) without ferrozine. Then, the sample and blank absorbances were read at 562 nm after 10 min of incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. The metal chelating activity was expressed as milligrams of EDTA (disodium edetate) equivalents (mg EDTAE/g extract).

2.6. Antibacterial activity The antibacterial activity of the Ballota extracts was evaluated using several bacterial strains. Escherichia coli (ATCC 35210), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium (ATCC 13311), Listeria monocytogenes (NCTC 7973), Enterococcus faecalis (human isolate) were used as Gram-negative bacteria. For Gram-positive bacteria, Bacillus cereus (clinical isolate), Micrococcus flavus (ATCC 10240) and Staphylococcus aureus (ATCC 6538) were used. The antibacterial activity was evaluated via the microdilution method as reported previously (Zengin et al., 2017).

2.5. Enzyme inhibitory activity Enzyme inhibitory effects of the Ballota extracts were investigated against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, α-amylase, and α-glucosidase and the experimental procedures of these assays were previously described (Zengin, 2016). These assays were summarized below. For Cholinesterase (ChE) inhibitory activity assay: Sample solution (1 mg/mL; 50 μL) was mixed with DTNB [5,5-dithio-bis(2-nitrobenzoic) acid, Sigma, St. Louis, MO, USA] (125 μL) and AChE [acetylcholinesterase (Electric ell acetylcholinesterase, Type-VI-S, EC 3.1.1.7,Sigma)], or BChE [butyrylcholinesterase (horse serum butyrylcholinesterase, EC 3.1.1.8, Sigma)] solution (25 μL) in Tris–HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of acetylthiocholine iodide (ATCI, Sigma) or butyrylthiocholine chloride (BTCl, Sigma) (25 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after 10 min incubation at 25 °C. The absorbance of the blank was subtracted from that of the sample, and the cholinesterase inhibitory activity was expressed as galantamine equivalents (mgGALAE/g extract) For Tyrosinase inhibitory activity assay: Sample solution (1 mg/mL; 25 μL) was mixed with tyrosinase solution (40 μL, Sigma) and phosphate buffer (100 μL, pH 6.8) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of LDOPA (40 μL, Sigma). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbances were read at 492 nm after a 10 min incubation at 25 °C. The absorbance of the blank was subtracted from that of the sample, and the tyrosinase inhibitory activity was expressed as kojic acid equivalents (mgKAE/g extract) For α-amylase inhibitory activity assay: Sample solution (1 mg/mL; 25 μL) was mixed with α-amylase solution (ex-porcine pancreas, EC 3.2.1.1, Sigma) (50 μL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37 °C. After pre-incubation, the reaction was initiated with the addition of starch solution (50 μL, 0.05%). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (αamylase) solution. The reaction mixture was incubated 10 min at 37 °C. The reaction was then stopped with the addition of HCl (25 μL, 1 M). This was followed by addition of the iodine-potassium iodide solution (100 μL). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from that of the sample, and the α-amylase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract)

2.7. Antifungal activity The antifungal activity of the studied extracts was evaluated using different fungi species. These species include: Aspergillus versicolor (ATCC 11730), Aspergillus fumigatus (plant isolate), Aspergillus ochraceus (ATCC 12066), Aspergillus niger (ATCC 6275), Penicillium ochrochloron (ATCC 9112), Penicillium funiculosum (ATCC 36839), Penicillium verrucosum (food isolate) and Trichoderma viride (IAM 5061). The experimental procedures for microdilution method were given in our previous study (Zengin et al., 2017). 2.8. Statistical analysis All values were expressed as mean ± S.D of three parallel measurements. Statistical evaluation was performed using SPPS v. 14.0 program (one-way ANOVA with Tukey’s assay). 3. Results and discussion 3.1. Total bioactive components and HPLC analysis The qualitative composition of the extracts was assessed through the evaluation of the total phenolic content (TPC) and total flavonoid content (TFC) using standard protocols. It was found a significant higher (Fig. 1) TPC in the water extract (57.77 ± 1.98 mgGAE/g extract) in comparison with the methanol extract (38.38 ± 0.31 mgGAE/ g extract). On the other hand, the TFC was lower in both extracts, and

Fig. 1. Total phenolic and flavonoid contents of B. macrodonta extracts (mean of three parallel measurements ± SD). Different letters indicate significant difference in the studied solvents (p < 0.05).

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higher for the methanolic (21.18 ± 0.46 mgRE/g extract) than for the water extract (5.49 ± 0.59 mgRE/g extract). It has been stated in numerous scientific publications that phenolic compounds possess redox potential allowing them to operate as antioxidants (Lamounier et al., 2012; Schilderman et al., 1995; Vázquez et al., 2008; Vinson et al., 2001; Williams et al., 2004). Such phenolic antioxidants have been identified in most cases as radical scavengers and metal chelators, but they have also exhibited their effects in cell signaling pathways and gene expression. The radical scavenging properties are thought to be due to the presence in their structure of the hydroxyl groups (Lamounier et al., 2012; Schilderman et al., 1995). In addition, flavonoids which are also secondary metabolites present naturally in plants, have been well documented to exhibit in vivo and in vitro antioxidant properties due to the presence of the free OH groups in their structure (Agati et al., 2012; Shimoi et al., 1996). Based on the above, the antioxidant potential of the methanolic and water extracts of B. macrodonta were investigated as well as total and individual phenolics profile was established. Results obtained from the analysis of the extracts using HPLC/DAD showed the presence of 19 components including hydroxybenzoic acid, catechin hydrate, vanillic acid, caffeic acid, syringic acid, epicatechin, coumaric acid, ferulic acid, luteolin-7-O-glucoside, rutin, and quercetin among the abundant components. However, a considerable variation was observed in the amounts of these components in the methanol and water extracts (Table 1). Epicatechin, was the most abundant compound in both methanol and water extracts. Catechin and epicatechin are well known as natural effective antioxidants belonging to the flavonoid group (Brewer, 2011). Moreover, other species from Ballota genus have been documented to contain several secondary metabolites such as diterpenoids, flavonoids, phenylpropanoids, essential oils, tannins, and saponins (Erdogan-Orhan et al., 2010; Sever-Yılmaz and Citoglu-Saltan, 2003). It was also reported that different extracts (ethyl acetate, methanol, and water) from several species of Ballota showed moderate antioxidant activity in the ferric-reducing test compared to chlorogenic acid (Erdogan-Orhan et al., 2010).

Table 2 Antioxidant properties of B. macrodonta extracts*.

Table 1 Phenolic components in B. macrodonta extracts (mg/g extract). MeOH

Water

gallic acid neochlorogenic acid hydroxybenzoic acid gentisic acid catechin hydrate 4-hydroxybenzaldehyde chlorogenic acid vanillic acid 4-O-caffeoylquinic acid caffeic acid epigallocatechin gallate syringic acid epicatechin coumaric acid ferulic acid salicylic acid naringenin-7-O-glucoside luteolin-7-O-glucoside rutin rosmarinic acid ellagic acid quercetin flavone

2.8 6.5 10.3 10.5 10.9 11.9 13.0 13.1 13.3 13.8 14.0 14.5 14.7 17.2 18.1 19.7 20.1 21.1 22.1 22.4 23.1 27.2 33.2

< 0.01 0.2 0.43 nd 0.42 nd nd 0.17 nd 0.86 < 0.01 0.44 1.73 0.50 0.19 0.07 0.12 0.36 0.43 0.09 0.03 0.63 nd

< 0.01 0.15 0.64 nd 0.17 < 0.01 nd 0.09 nd 1.44 < 0.01 0.16 0.85 0.91 0.46 0.13 0.01 0.08 0.57 0.05 < 0.01 0.08 nd

Water extract

DPPH radical scavenging (mgTE/g extract) ABTS radical scavenging (mgTE/g extract) CUPRAC (mgTE/g extract) FRAP (mgTE/g extract) Phosphomolybdenum (mmolTE/g extract) Metal chelating activity (mgEDTAE/g extract)

157.88 ± 1.46b 145.74 ± 2.59b 221.19 ± 5.93b 138.01 ± 3.69b 1.05 ± 0.06b 10.54 ± 0.68a

339.06 ± 5.71a 368.04 ± 7.17a 302.46 ± 3.80a 219.14 ± 1.27a 1.20 ± 0.01a 9.39 ± 0.22b

included: 2,2-diphenyl-1-picryl hydrazyl radical (DPPH•) scavenging activity, and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) radical cation assay. The scavenging potential of the water and methanolic extracts was then further compared to their reducing power through the ferric reducing antioxidant power (FRAP) assay, and cupric ion reducing antioxidant capacity (CUPRAC), as well as with their metal chelating activity, and total antioxidant capacity through phosphomolybdenum assay (Table 2). The methods mentioned above were selected and executed as the assessment of plant extracts through their hydrogen transfer, electron transfer, and metal chelating properties consists as a more reliable approach. Furthermore, these evaluations are the main reported mechanisms through which natural antioxidants are known to exhibit their protective role (Perera et al., 2016). For instance, the ABTS assay monitors the decay of the radical-cation ABTS•+; the CUPRAC method is based on the reduction of Cu2+ to Cu+, resulting in the formation of coloured copper I-neocuproine complex; the FRAP method assesses the reducing capacity of the antioxidants based on ferric ion; the phosphomolybdenum assay is based on the reduction of Mo (VI) to Mo (V) by antioxidants, forming subsequently a green phosphate/Mo (V) complex at acid pH; and in the metal chelating activity, Ferrozine chelates Fe2+ to form a red coloured chromophore, which has an absorption maxima at 562 nm (Zengin et al., 2018). Data obtained through the antioxidant assays revealed that the water extract was significantly more active than the methanolic extract of B. macrodonta. For the metal chelating activity, the result obtained for the water extract is close to the one obtained for the methanol extract. When assessing these results with the profile obtained through HPLC-DAD analysis, it was noticed for example that three phenolic acids, namely ferulic acid, coumaric acid, and caffeic acid were present in higher amounts (0.46, 0.91, and 1.44 mg/g of extract respectively) in the water extract of B. macrodonta compared to the methanol extract 0.19, 0.50, and 0.86 mg/ g of extract respectively). These three phenolic compounds are well known as naturally occurring antioxidant compounds identified in plants. Ferulic acid is the outcome of the Shikimic pathway in plants via metabolism of phenylalanine and tyrosine. Due to its strong membrane antioxidant potential, ferulic acid has been associated to a multitude of therapeutic effects including but not limited to cancer, diabetes, cardiovascular, and neurodegenerative diseases. Ferulic acid has been purported as an effective free radical scavenger and listed as food additive preventing lipid peroxidation (Srinivasan et al., 2007). Lipid peroxidation is a destructive process and it occurs from free radicals attack to unsaturated fatty acids in a lipid membrane. This process can initiate an oxygen-mediated chain reaction that leaves the membrane perforated with lipid hydroperoxides (Girotti, 1998). The hydroxy and phenoxy groups of ferulic acid are thought to donate electrons to quench the free radicals. However, the exact mechanism for the antioxidant property of ferulic acid and its possible role in therapeutic usage are still not well known (Srinivasan et al., 2007; Graf, 1992; Kanski et al., 2002; Ohnishi et al., 2004; Ou et al., 2003).

In vitro antioxidant assays were further selected to assess the biological potential of the extracts to scavenge free radicals which

Retention time (min)

MEOH extract

* Values expressed are means ± S.D. of three parallel measurements. Data marked with different letters within the same row indicate statistically significant differences (p < 0.05). TE, trolox equivalents; EDTAE, EDTA equivalents.

3.2. Antioxidant activity

Phenolic compounds

Antioxidant assays

nd: not detected.

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The dual potential of the methanolic extract of B. macrodonta, that is, rich in phytochemicals and potential inhibitors of AChE and BChE tend to further confirm its potential therapeutic benefits. Moreover, it has been argued that both AChE and BChE may play fundamental roles in the etiology and progression of AD beyond regulation of synaptic AChE levels (Greig et al., 2002). Therefore, the assessment of the chemical composition coupled with enzyme inhibitory properties may therefore lead to a multi-pharmacological profile of B. macrodonta extracts.

In the same way, p-coumaric acid (4-hydroxycinnamic acid), a phenolic acid, and an ubiquitous plant metabolite has been reported in literature as a radical scavenger and a reducing agent (Kiliç and Yeşiloğlu, 2013; Luceri et al., 2007). Zang et al. (2000) also stated that p-coumaric acid effectively scavenged hydroxyl radical in a dose-dependent manner. In addition, both water and methanolic extracts showed significant antioxidant potential, which may be partly explained by the fact that there are other phenolic compounds present in the extracts, such as catechin and epicatechin which are two flavan-3-ols stereoisomers with important antioxidant features. These phytochemicals have been reported to have the capacity to scavenge hydroxyl, peroxyl, and 2,2diphenyl-1-picrylhydrazyl (DPPH) radicals, and chelate the Fe2+ as well as to retard lipid oxidation. Also, catechin has been purported to exhibit antioxidant activity in human plasma by delaying the degradation of endogenous tocopherol and β-carotene, as well as by inhibiting the oxidation of plasma lipids (Nimse and Pal, 2015). In a previous study, Nakao et al. (1998) argued that catechin and epicatechin have peroxyl radical scavenging activity almost 10 times higher than those of L-ascorbate and β-carotene, when evaluated against bacteria.

3.3.2. Tyrosinase inhibitory activity The capability of the methanolic extract of B. macrodonta to inhibit tyrosinase activity (mean value of 10.18 ± 0.63 mgKAE/g extract) (Table 3) can be translated to its potential as a skin whitening agent as well as in the management of hyperpigmentation pathologies. Melanin production is impaired when tyrosinase enzyme is inhibited, resulting in a less pigmented skin. However, the inhibition of tyrosinase activity for the methanol extract of B. macrodonta was found to be relatively low compared to other values reported in literature. For instance, Uysal et al. (2017) reported a high anti-tyrosinase activity with a mean value of 123.36 ± 4.63 mgKAE/g of extract for the methanolic extract of Potentilla reptans. The methanolic extract of B. macrodonta was found to contain several phytochemicals, such as flavonols, flavanols, and phenolic acids, reported to inhibit tyrosinase activity (Lin et al., 2008). Also, quercetin, a flavonol which is well known to inhibit tyrosinase enzyme better than kaempferol (Chang, 2009), was detected in the extract of B. macrodonta by HPLC-DAD analysis with an abundance of 0.63 mg/g in the methanolic extract. The inhibition of tyrosinase has an important role in the hindrance of melanin accumulation. Natural tyrosinase inhibitors such as the extract of B. macrodonta represent a potential target in cosmetics and skin treatment formulations.

3.3. Enzyme inhibitory activity 3.3.1. AChE and BChE inhibitory activity The assessment of the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potential of B. macrodonta extracts hinted the possibility of the methanolic extract as inhibitor of AChE and BChE (mean values of 2.50 ± 0.01 and 1.89 ± 0.06 mgGALAE/g extract respectively) (Table 3). AChE has been acknowledged as the enzyme responsible for regulating the brain neurotransmitter, acetylcholine. On the other hand, BChE is less actively involved in the modulation of acetylcholine. However, BChE has been alleged to progressively increase in patients diagnosed with Alzheimer's disease (AD). To this effect, both enzymes represent valuable therapeutic targets aiming towards the improvement of cholinergic deficit in patients. Cholinergic deficit in patients leads to the declines in cognitive, behavioral and global function, which represent the major decline of AD (Doraiswamy et al., 2002). The exact cause of the neuronal degeneration is usually attributed to cellular and molecular events, including neuro-inflammation, increases in oxidative stress, iron and/or depletion of endogenous antioxidants (Barzilai and Melamed, 2003). In addition, a growing body of evidence also tend to indicate that phenolic compounds might be involved in the management of the development of Alzheimer’s disease and help in counteracting the age-related cognitive declines due to their capacity to modulate brain cellular and molecular architecture involved in memory (Bahadori et al., 2016; Schluesener and Schluesener, 2014; Vauzour, 2014).

3.3.3. α-Amylase and α-glucosidase inhibitory activity The antihyperglycemic activity of the water and methanol extracts of B. macrodonta was evaluated by studying their inhibitory potential on α-amylase and α-glucosidase enzymes. In α-glucosidase inhibitory activity, both extracts exhibited moderately-high inhibitory activities with the values of 15.48 ± 0.96 and 11.03 ± 0.67 mmolACAE/g extract, for the methanol and water extracts, respectively. In a previous study dealing with the inhibitory properties of phenolic-rich extracts of medicinal and edible plants, Savran et al. (2016) reported a lower activity on α-glucosidase (0.7 ± 0.1 and 0.4 ± 0.1 mmol ACAE/g of extract) for the methanol and water extracts of Pseudosempervivum sempervivum. The α-amylase inhibitory activity of the two extracts was significantly lower than that of the α-glucosidase, with mean values of 0.59 ± 0.03 and 0.20 ± 0.01 mmolACAE/g extract for the methanol and water extracts, respectively. These values tend to corroborate with those observed in the study reported by Savran et al. (2016) which reported lower values in terms of α-amylase inhibitory activity compared to α-glucosidase. Nevertheless, it has been reported that several natural compounds such as triterpenoids and phenolics exhibited antidiabetic activities via inhibition of carbohydrate hydrolysis enzymes, which may partly explain the results obtained herein (Etxeberria et al., 2012)

Table 3 Enzyme inhibitory effects of B. macrodonta extracts*. Enzyme inhibitory assays

MEOH extract

Water extract

Acetylcholinesterase inhibition (mgGALAE/g extract) Butrylcholinesterase inhibition (mgGALAE/g extract) Tyrosinase inhibiton (mgKAE/g extract) α-Amylase inhibition (mmolACAE/g extract) α-Glucosidase inhibition (mmolACAE/g extract)

2.50 ± 0.01a

0.39 ± 0.09b

1.89 ± 0.06

na

10.18 ± 0.63 0.59 ± 0.03a 15.48 ± 0.96a

na 0.20 ± 0.01b 11.03 ± 0.67b

3.4. Antibacterial and antifungal activity The methanol and water extracts of B. macrodonta, were further investigated for their possible antimicrobial activities (Tables 4 and 5). For instance, in the HPLC-DAD profile, antimicrobial compounds like hydroxybenzoic acid and vanillic acid were identified. Moreover, it is well known that esters of hydroxybenzoic acid, also called parabens, are widely used as antimicrobial agents in a large variety of food,

* Values expressed are means ± S.D. of three parallel measurements. Data marked with different letters within the same row indicate statistically significant differences (p < 0.05). GALAE, galantamine equivalents; KAE, kojic acid equivalents; ACAEs, acarbose equivalents; na, not active.

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Table 4 Antibacterial activity of B. macrodonta extracts (MIC and MBC in mg/ml)*. Gram Negative Samples B. macrodonta MEOH B. macrodonta Water Streptomycin Ampicillin

MIC MBC MIC MBC MIC MBC MIC MBC

S. typhimurium 0.15 ± 0.009b 0.22 ± 0.008b 0.15 ± 0.02b 0.22 ± 0.03b 0.05 ± 0.003a 0.10 ± 0.030a 0.15 ± 0.030b 0.20 ± 0.030b

Gram Positive E. coli 0.08 ± 0.11 ± 0.08 ± 0.11 ± 0.10 ± 0.50 ± 0.30 ± 1.00 ±

0.000a 0.02a 0.003a 0.02a 0.009a 0.060b 0.000b 0.100c

E. cloacae 0.15 ± 0.02ab 0.22 ± 0.03a 0.11 ± 0.03a 0.22 ± 0.02a 0.10 ± 0.010a 0.50 ± 0.030b 0.20 ± 0.060b 1.00 ± 0.060c

P. aeruginosa 0.08 ± 0.002b 0.11 ± 0.03a 0.08 ± 0.005b 0.11 ± 0.02a 0.05 ± 0.006a 0.10 ± 0.020a 0.10 ± 0.030c 0.20 ± 0.010b

M. flavus 0.11 ± 0.03b 0.22 ± 0.04b 0.08 ± 0.002a 0.11 ± 0.02a 0.125 ± 0.03b 0.25 ± 0.030b 0.30 ± 0.060c 0.50 ± 0.030c

S. aureus 0.11 ± 0.02a 0.22 ± 0.03a 0.15 ± 0.03ab 0.22 ± 0.01a 0.25 ± 0.06b 0.50 ± 0.060b 0.30 ± 0.060c 0.50 ± 0.030b

B. cereus 0.05 ± 0.000a 0.11 ± 0.02a 0.08 ± 0.002ab 0.11 ± 0.03a 0.05 ± 0.003a 0.10 ± 0.010a 0.10 ± 0.030b 0.30 ± 0.060b

L. monocytogenes 0.15 ± 0.02b 0.22 ± 0.02a 0.11 ± 0.01a 0.22 ± 0.03a 0.15 ± 0.030b 0.30 ± 0.030ab 0.15 ± 0.030b 0.50 ± 0.060b

* Values expressed are means ± S.D. of three parallel measurements. Data marked with different letters within the same column indicate statistically significant differences (p < 0.05). Table 5 Antifungal activity of B. macrodonta extracts (MIC and MFC in mg/ml)*. Samples B. macrodonta MEOH B. macrodonta Water Bifonazole Ketoconazole

MIC MFC MIC MFC MIC MFC MIC MFC

A. niger

A. versicolor

A. fumigatus

0.0125 ± 0.003a 0.02 ± 0.002a 0.20 ± 0.03b 0.45 ± 0.02bc 0.20 ± 0.060b 0.30 ± 0.020b 0.20 ± 0.010b 0.50 ± 0.060c

0.025 ± 0.000b 0.05 ± 0.003b 0.0125 ± 0.001a 0.025 ± 0.003a 0.15 ± 0.030c 0.20 ± 0.060c 0.20 ± 0.000d 0.50 ± 0.030d

0.05 0.11 0.08 0.11 0.15 0.20 0.20 0.50

± ± ± ± ± ± ± ±

0.0002a 0.02a 0.000b 0.02a 0.060c 0.030b 0.060d 0.000c

A. ochraceus 0.05 0.11 0.11 0.22 0.15 0.20 0.15 0.20

± ± ± ± ± ± ± ±

0.000a 0.02a 0.03b 0.08b 0.030c 0.060b 0.010c 0.000b

P. funiculosum

P. ochrochloron

P. veruccosum

0.05 ± 0.000b 0.11 ± 0.02b 0.0125 ± 0.003a 0.025 ± 0.002a 0.20 ± 0.020c 0.25 ± 0.030c 2.50 ± 0.060d 3.50 ± 0.300d

0.05 ± 0.000b 0.11 ± 0.02b 0.025 ± 0.003a 0.050 ± 0.002a 0.20 ± 0.000c 0.25 ± 0.010c 0.20 ± 0.030c 0.50 ± 0.060d

0.05 0.11 0.05 0.11 0.20 0.30 1.00 1.00

± ± ± ± ± ± ± ±

0.002a 0.02a 0.003a 0.02a 0.060b 0.030b 0.030c 0.100c

T. viride 0.05 ± 0.000b 0.11 ± 0.03b 0.0125 ± 0.003a 0.025 ± 0.002a 0.10 ± 0.010c 0.20 ± 0.030c 0.20 ± 0.020d 0.30 ± 0.060d

* Values expressed are means ± S.D. of three parallel measurements. Data marked with different letters within the same column indicate statistically significant differences (p < 0.05).

phytochemical profile of B. macrodonta, a folk medicinal plant endemic to Turkey. The HPLC/DAD profile showed the presence of 19 bioactive components including hydroxybenzoic acid, catechin hydrate, vanillic acid, caffeic acid, syringic acid, epicatechin, coumaric acid, ferulic acid, luteolin-7-O-glucoside, rutin, and quercetin among the determined compounds. Both water and methanol extracts revealed high values in terms of total phenolic and flavonoid contents, respectively. According to all assays, the overall antioxidant capacity of the water extract was higher than the values obtained for the methanol extract, except for the metal chelating assay. Furthermore, the extracts were active against a wide range of bacterial and fungal strains. Nonetheless, particularly B. macrodonta methanol extract possesses inhibitory potential against key enzymes involved in hyperpigmentation, diabetes, and neurodegenerative diseases. In conclusion, B. macrodonta deserve due attention as a folk medicinal plant that can be further exploited as a pharmaceutical herbal product.

pharmaceutical, and cosmetic products (Młynarczyk et al., 2008; Rastogi et al., 1995). These compounds are commonly used in these formulations as they provide efficient antimicrobial coverage, acting on a broad spectrum of microorganisms, being as well considered as moderately toxic. Furthermore, they are believed to act by disrupting membrane transport processes or by inhibiting synthesis of DNA and RNA and some key enzymes, such as ATPases and phosphotransferases (Ma and Marquis, 1996; Młynarczyk et al., 2008; Valkova et al., 2001). In addition, enhanced antimicrobial activity has been ascribed for various natural compounds from plant sources, such as vanillin. In this study, the presence of vanillic acid (the oxidised form of vanillin), a derivative of vanillin, (Yemiş et al., 2011) was also observed, and could be at least partially ascribed for the antimicrobial properties of the extracts. It can be suggested from the data amassed in this study that the methanol and water extracts of B. macrodonta possessed significant antimicrobial activities, as noted in Tables 4 and 5. Both Gram positive and negative bacteria were found to be sensitive against the methanol and water extracts respectively, with MIC ranging from 0.05 to 0.15 mg/mL. The antibacterial activities of some extracts were comparable among some bacteria, and for others (E. coli, E. cloacae, P. aeruginosa, M. flavus, S. aureus, B. cereus, and L. monocytogenes) the activities observed were similar or significantly better than the positive controls streptomycin and/or ampicillin. Additionally, the methanol and water extracts exhibited significant antifungal properties with MIC ranging from 0.0125 to 0.2 mg/mL, with some MICs comparable to the positive controls (Bifonazole and Ketoconazole) for some fungal species. Moreover, it was also observed that some of the extracts were more active than the positive controls (Table 4). Nevertheless, in a previous study the antimicrobial properties of a methanol extract of B. macrodonta against human rotavirus, with EC50 values ranging from 6.9 to 9.7 μg/ml, was reported (Civra et al., 2017).

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