In vitro and in silico insights of Cupressus sempervirens, Artemisia absinthium and Lippia triphylla: Bridging traditional knowledge and scientific validation

Accepted Manuscript Title: In vitro and in silico insights of Cupressus sempervirens, Artemisia absinthium and Lippia triphylla: Bridging traditional knowledge and scientific validation Authors: Gokhan Zengin, Adriano Mollica, Abdurrahman Aktumsek, Carene Marie Nancy Picot, Mohamad Fawzi Mahomoodally PII: DOI: Reference:

S1876-3820(17)30108-7 http://dx.doi.org/doi:10.1016/j.eujim.2017.05.010 EUJIM 684

To appear in: Received date: Revised date: Accepted date:

8-5-2017 29-5-2017 29-5-2017

Please cite this article as: Zengin Gokhan, Mollica Adriano, Aktumsek Abdurrahman, Marie Nancy Picot Carene, Fawzi Mahomoodally Mohamad.In vitro and in silico insights of Cupressus sempervirens, Artemisia absinthium and Lippia triphylla: Bridging traditional knowledge and scientific validation.European Journal of Integrative Medicine http://dx.doi.org/10.1016/j.eujim.2017.05.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

In vitro and in silico insights of Cupressus sempervirens, Artemisia absinthium and Lippia triphylla: Bridging traditional knowledge and scientific validation

Gokhan Zengin1,*, Adriano Mollica2, Abdurrahman Aktumsek1, Carene Marie Nancy Picot3, Mohamad Fawzi Mahomoodally3 1Selcuk

University, Science Faculty, Department of Biology, Campus, 42250, Konya, Turkey

2University

“G. d’Annunzio” of Chieti-Pescara, Department of Pharmacy, , 66100, Chieti-Italy

3University

of Mauritius, Faculty of Science, Department of Health SciencesRéduit, Mauritius

*Corresponding

author. Tel.: +90 332 223 27 81; Fax: +90 332 2410106

E-mail address:

[email protected] (Dr. Gokhan ZENGIN)

Abstract Introduction: The search for new therapeutic agents for the management of diabetes mellitus type 2 (DMT2) and neurodegenerative disorders has attracted much interest coupled with the rising number of patients suffering from these pathologies. Traditionally, extracts from medicinal plants have been used to manage a number of ailments and still remain a potent source of new therapeutic agents. Methods: Therefore, the present study was undertaken to evaluate the in vitro antioxidant and enzyme (acetyl cholinesterase (AChE), butyryl cholinesterase (BChE), tyrosinase, α-amylase, and α-glucosidase) inhibitory potential of three medicinal plants (Cupressus sempervirens, Artemisia absinthium, and Lippia triphylla). The phenolic composition of the ethanolic extracts was also characterized using reversed-phase high-

2

performance liquid chromatography (RP-HPLC). In silico molecular docking was used to investigate the possible interaction between active compounds and the studied enzymes. Results: C. sempervirens showed the highest inhibition rates against AChE, BChE, α-amylase, and α-glucosidase (2.47 mg galantamine equivalents (GALAE)/g extract, 2.98 mg GALAE/g extract, 1.61 mmol acarbose equivalents (ACAE)/g extract, and 1.86 mmol ACAE/g extract for respective enzymes). The plant extracts showed antioxidant power in the following order C. sempervirens> L. triphylla> A. absinthium. Protocatechuic acid, (+)catechin, apigenin, and chlorogenic acid were identified in all the plant extracts. The best docking pose obtained for each bioactive compound against the enzymes was mostly stabilized via hydrogen bonds and pi-pi stacks. Conclusion: This study provides insight into the antioxidant capacity and the inhibitory potential of these medicinal plants against key enzymes linked to DMT2 and neurodegenerative disorders.

Keywords: Cupressus sempervirens; bioactive compounds; diabetes; neurodegenerative disorders; traditional medicine.

3

1. Introduction The use of plants for medicinal purposes precedes recorded history of mankind. Paleoanthropological findings in Iraq have demonstrated that Neanderthals probably used plants for medicinal purposes [1]. Indeed, plants were used for their antiseptic, painkilling and wound healing properties by our ancestors. Over the past decades, the use of medicinal plants, especially secondary metabolites such as phytochemicals, has gained much momentum in drug discovery and development. In fact, the advert of modern technologies combining biology and chemistry along with computational tools has allowed scientists to uncover promising therapeutical compounds from natural resources such as plants[2-4]. Cupressus sempervirens a medium size evergreen plant is a species of cypress native to the Mediterranean region[5]. Traditionally C. sempervirens was used to treat head colds, coughs, bronchitis, haemorrhoids, varicose veins, venous circulation disorders, as astringent, antiseptic, antispasmodic, and bladder toner in urinary incontinence [6]. Artemisia absinthium, a perennial shrubby plant of the Asteraceae family, is the major component of the spirit drink, absinthe[7]. The whole plant was reported to be a tonic and anthelmintic [8]. Lippia triphylla, a flowering plant of the Verbenaceae family native to the South America, was traditionally used to treat diarrhea, dysentery, stomach ache, indigestion, as analgesic, anti-inflammatory, antipyretic, and carminative[9]. The current research was aimed at evaluating the biological potential and identifying phenolic compounds present in the ethanolic extracts of C. sempervirens, A. absinthium, and L. triphylla using reversed-phase high-performance liquid chromatography technique.

4

The antioxidant/reducing/chelating/radical scavenging potentials of the plant extracts were also studied using standard assays. Finally, the inhibitory action of the ethanolic extracts on acetyl cholinesterase (AChE), butyryl cholinesterase (BChE), tyrosinase, αamylase, and α-glucosidase was determined in vitro. It is anticipated that results obtained from this study could be valuable in the development of novel phytopharmaceuticals.

2. Material and Methods 2.1. Plant Material and extraction procedure The plant materials (C. sempervirens seeds, A. absinthium, and L. triphylla aerial parts) were bought from a local market in Konya and air dried at the room temperature for 10 days. Taxonomic identification was confirmed by the senior taxonomist Dr. Murad Aydın Sanda, from the Department of Biology, Selcuk University, Turkey (Voucher numbers: GZ 1023, GZ 1026 and GZ 1028, respectively). The dried plant samples were ground to a fine powder using a laboratory mill. The powdered plant materials (10 g) were macerated in 200 mL of ethanol at room temperature (25°C ± 1°C) for 24 hours. Filtrates were concentrated in vacuo and extracts were stored at 4°C in dark until further analysis.

2.2.

Quantification of phenolic compounds by reversed-phase high-performance liquid

chromatography (RP-HPLC) RP-HPLC (Shimadzu Scientific Instruments, Kyoto, Japan) was employed to identity phenolic compounds present in the plant extract [10]. Detection and quantification were carried out using an LC-10ADvp pump, a Diode Array Detector, a CTO10Avp column heater, SCL-10Avp system controller, DGU-14A degasser and SIL-10ADvp

5

auto sampler (Shimadzu Scientific Instruments, Columbia, MD). Separation was conducted at 30 °C on Agilent® Eclipse XDB C-18 reversed-phase column (250 mm x4.6 mm length, 5 µm particle size). Identification and quantitative analyses were done by comparison with standards. Protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, ()-epicatechin, ferulic acid, benzoic acid, rutin, rosmarinic acid, and apigenin (purity ≥95%) were used as standards. They were purchased from Sigma-Aldrich (St. Louis, MO, USA). The amount of each phenolic compound was expressed as microgram per gram of extract using external matrix-matched calibration curves, which were obtained for each phenolic standard. 2.3. Biological activities evaluation Free radical scavenging (DPPH and ABTS radicals), reducing power (CUPRAC and FRAP), phosphomolybdenum, and metal chelating (ferrozine method) and enzyme inhibitory activities (cholinesterase (Elmann’s method), tyrosinase (dopachrome method), α-amylase (iodine/potassium iodide method) and α-glucosidase (chromogenic PNPG method)) were determined using the methods previously described by Mocan, Zengin, Uysal, Gunes, Mollica, Degirmenci, Alpsoy and Aktumsek [11]. Antioxidant abilities were expressed as equivalents of trolox and EDTA (for metal chelating). The enzyme inhibitory activities of the extracts were expressed as equivalents of standard drugs per gram of the plant extract (galantamine for AChE and BChE, kojic acid for tyrosinase, and acarbose for α-amylase and α-glucosidase inhibition assays). 2.4. Total phenolic and flavonoid content The total phenolic content was determined by Folin-Ciocalteu method [12] with slight modification and expressed as gallic acid equivalents (GAE/g extract), while total

6

flavonoid content was determined using AlCl3 method [11] with slight modification and expressed as rutin equivalents (RE/g extract). 2.5. Molecular Modelling 2.5.1. Receptors preparation The crystalline structure of the selected enzymes together with their inhibitors have been downloaded from the Protein Databank RCSB PDB [13]: acetylcholinesterase (pdb:4X3C)

[14]

in

complex

with

tacrine-nicotinamide

hybrid

inhibitor,

butyrilcholinesterase (pdb:4BDS) [15] in complex with tacrine, amylase (pdb:1VAH) [16] in complex with r-nitrophenyl-α-D-maltoside, glucosidase (pdb:3AXI) [17] in complex with maltose and tyrosinase (pdb:2Y9X) [18] in complex with tropolone. The raw crystal structures have been prepared for the docking experiments as previously reported [11, 19]. Non-catalytic water molecules, inhibitors and all the other molecules present in the pdb files were removed by using UCSF Chimera [20] and the proteins alone were neutralized at pH 7.4 by Epik implemented in Maestro 10.2 suite [21]. Seleno-cysteines and seleno-methionines, if present, were converted respectively to cysteines and methionines. All the missing fragments and other errors present in the crystal structures were automatically solved by the Wizard Protein Preparation implemented in Maestro 10.2 suite [21].

2.5.2. Ligands preparation (+)-Catechin and apigenin, were selected as representative compounds to carry out molecular docking study, as these compounds were present in abundance in the herbs extracts, also chlorogenic acid is present in relevant amount and its docking experiments have been previously reported in our paper [22]. The chemical structure of apigenin and catechin is reported in Figure 1. The three dimensional structures have been downloaded

7

from Zinc databases [23] and used for molecular modeling experiments after preparation. The ligands were prepared by the LigPrep tool embedded in Maestro 10.2, neutralized at pH 7.4 by Epik and minimized by OPLS3 force field [24].

2.6. Molecular Docking Dockings of the representative substances have been performed for each selected enzyme employed for the in vitro enzymatic inhibition tests in this work. Gold 6.0 [25] has been employed for the docking calculations by using the “Gold Score” scoring function for all the enzymes [26]. In all cases, the binding pocket was determined automatically by centering the grid on the crystallographic inhibitor, extended in a radius of 10 Angstroms from the center. The best pose for each compound docked to the selected enzymes was the best ranked among the 200 generated. 2.7. Statistical analysis All the assays were carried out in triplicate. The results were expressed as mean value and standard deviation (mean ± SD). Statistical differences between the extracts were analyzed by using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test (α = 0.05). All the analysis was carried out using SPSS v22.0 software.

3. Results and Discussion

3.1. Identification of phenolic compounds by RP-HPLC RP-HPLC enables the identification of phenolic compounds present in plant extracts. As shown in Table 1, protocatechuic acid, (+)-catechin, apigenin, and chlorogenic acid were identified in all the plant extracts. C. sempervirens contained higher amount of

8

protocatechuic acid (16 µg/g extract) and (+)-catechin (205.4 µg/g extract) compared to A. absinthium and L. triphylla. (-)-Epicatechin and benzoic acid were identified in C. sempervirens only. Rosmarinic acid was not identified in the plant extracts. 3.2. Enzyme inhibitory activity Increasing evidences tend to advocate the link between DMT2 and the occurrence of neurodegenerative disorders [27]. DMT2 sequelae affect the brain over time until cognitive decline becomes clinically apparent [28]. AChE, BChE, and tyrosinase have been targeted for the management of neurodegenerative disorders such as Alzheimer’s disease. On the other hand, α-amylase and α-glucosidase are key players in the control of glycaemic level. Interestingly, phytochemicals have proved to be inhibitors of these key enzymes and have ushered to the development of novel pharmaceuticals [29]. As presented in Table 2, C. sempervirens showed the highest inhibition rates against AChE and BChE (2.47 and 2.98 mg GALAE/g extract for respective enzymes). A similar inhibition pattern was observed against α-amylase and α-glucosidase (1.61 and 1.86 mmol ACAE/g extract for respective enzymes). Both C. sempervirens and A. absinthium showed good inhibitory activity against tyrosinase in vitro (Table 2). RP-HPLC evaluation of C. sempervirens revealed the presence of several phytochemicals including (-)-epicatechin, benzoic acid, apigenin, chlorogenic acid, phydroxybenzoic acid, (+)-catechin, and protocatechuic acid. Uysal, Zengin, Mollica, Gunes, Locatelli, Yilmaz and Aktumsek [19] recently reported (-)-epicatechin bonded to the enzyme pocket of AChE, tyrosinase, α-amylase, and α-glucosidase using in silico methods. Moreover, in vivo studies concluded that fermented legumes rich in (-)-epicatechin reduced α-amylase, α-glucosidase, and AChE activities [30]. (+)-Catechin a naturally occurring flavan-3-ol was also identified in C. sempervirens. Glycated hemoglobin level

9

decreased significantly in DMT2 patients treated with catechin rich drinks over 12 weeks [31]. On the other hand, Farah et al. (2008) reported that the consumption of food rich in chlorogenic acid was associated with a reduced risk for the development of Alzheimer’s disease, a common neurodegenerative disorder. Additionally, chlorogenic acid was reported to cause AChE inhibition in the hippocampus and frontal cortex ex vivo [32]. Italian blueberry cultivars reported to possess chlorogenic acid inhibited different pharmacologically relevant isoforms of carbonic anhydrase associated to obesity and tumor formation [33]. Protocatechuic acid, a major benzoic acid derivative was reported to possess a 10 fold stronger antioxidative power compared to alpha-tocopherol [34]. Indeed, in this study C. sempervirens extract contained the highest amount of protocatechuic acid. Moreover, protocatechuic acid has been shown to exert glycaemic control [34] and to reduce brain oxidative stress [35]. Indeed, brain oxidative stress has been proposed as an underlying mechanism responsible for neurodegenerative disorders [36]. Apigenin, a flavonoid present in C. sempervirens was previously reported to act as antidepressant by promoting neurotransmission and limiting reabsorption of bioamines [37]. 3.3. Molecular docking In vitro experiments of the inhibitory activity of plants do not provide a comprehensive

understanding

regarding

the

binding

orientation

of

active

phytochemicals to the targeted enzymes [38]. Computational modelling namely, in silico molecular docking, is a useful tool to predict the interaction between phytochemicals and enzymes [39]. Evidence from the literature, report the inhibitory action of apigenin and catechin on AChE and BChE [40, 41]. Apigenin and catechin along with chlorogenic acid were also reported to induce moderate inhibitory effect on α-amylase [42, 43]. On the other hand, we have found that the plant extracts possess good inhibition activity toward

10

tyrosinase. This may be attributed to the presence of chlorogenic acid as well as apigenin and cathechin [44]. In light of the observed inhibitory activity, in silico molecular docking simulation was used to investigate the interactions between catechin and apigenin (Fig 1) towards the targeted enzymes. The best and most representative enzyme-ligand complexes were reported in Figures 2-4. The best docking pose obtained for catechin docked to tyrosinase is stabilized by three hydrogen bonds to Gly281, His259, His296 and one bond involving the copper atom Cu400 as shown in Fig 2(A). The best pose of catechin to α-glucosidase involves three hydrogen bonds to Glu197, His438 and Gln67 (Fig 2(B)). From Fig 2(C), it can be observed that catechin docked to AChE enzymatic cavity by forming four hydrogen bonds with Gln69, Asp72, His440, Tyr70 and a π-π stack with the aromatic side chain of Trp84. On the other hand, catechin docked to BChE in an analogous way by forming four hydrogen bonds with Glu197, His438, Gln67. Catechin established four hydrogen bonds and two ππ stacks with α-amylase enzymatic pocket as shown in Fig 4 (B). Fig 3 presents the best poses of apigenin with the studied enzymes. In silico studies showed that apigenin established one hydrogen bond with Met280, three π-π stacks with Phe264, His259, His85 and bonded directly to the copper atom Cu400 of tyrosinase enzymatic cavity (Fig 3 (A)). Apigenin was found to interact with AChE enzymatic cavity by forming one hydrogen bond with Glu199. Additionally, two π-π stacks involving the two aromatic parts of apigenin were formed with Trp84 of AChE active site. Apigenin bonded to α-glucosidase by forming five hydrogen bonds with Glu11, Arg315, Arg442, Arg213, His112, Asp69, and one cation- π interaction with residue Arg315. The interaction of apigenin with BChE was less effective involving one hydrogen bond and one π-π stack as shown in Fig 3 (D). Interaction of apigenin with α-amylase involved three hydrogen bonds with Asp197, His299, Gln63 and two π-π stacks with Tyr62 and Trp59.

11

The docking experiments have shown that both catechin and apigenin established strong interactions with the binding pocket of the selected enzymes, with a preference of tyrosinase and -glucosidase for apigenin and -glucosidase, -amylase and tyrosinase for catechin, and low for both substances towards cholinesterases. It has been demonstrated in our previous paper the central role of chlorogenic acid [22] and rutin in the inhibition of tyrosinase and amylase [11]. However, it is noteworthy to highlight that the plant extracts contain several phenolic compounds. Inhibitory action of the plant extracts on AChE, BChE, tyrosinase, α-amylase and α-glucosidase might be due to the concerted action of several phenolic compounds rather than a single molecule.

3.4. Reducing, antioxidant, radical scavenging, and chelating activities Determination of antioxidant capacity using only one method is not reliable since antioxidants exhibit their protective role by 3 main proposed mechanisms, namely, hydrogen transfer, electron transfer, and metal chelation [45]. In this respect, the antioxidant

potential

of

the

plant

extracts

was

evaluated

using

different

spectrophotometric measurements including, free radical scavenging (DPPH and ABTS), reducing power (CUPRAC and FRAP), phosphomolybdenum and metal chelating [19]. Reducing power reflects the electron donating capacity of natural antioxidants. The FRAP and CUPRAC methods are currently used to measure the reductive ability of plant extracts. The respective assays measure the ability of the plant extracts to reduce Fe (III) to Fe (II) and Cu (II) to Cu (I). As shown in Table 3 C. sempervirens (58.11 and 102.68 mg TE/g extract for CUPRAC and FRAP assay respectively) and L. triphylla (40.22 and 143.54mg TE/g extract for CUPRAC and FRAP assay respectively) showed high reducing power against both CU (II) and Fe (III).

12

A similar pattern was observed against DPPH scavenging. Indeed, the DPPH free radical scavenging assay is a simple and widely used method to determine antioxidant activity of plant extracts. This method is based on the reduction of alcoholic DPPH radical (purple) by gaining one hydrogen atom to form a stable DPPH-H molecules (yellow) [46]. Extracts of L. triphylla showed highest reducing activity on DPPH followed by C. sempervirens and A. absinthium. The plant extracts exhibited a similar scavenging pattern on ABTS radical (Table 3). This radical scavenging method is based on the reduction of blue 2, 2’-azinobis (3-ethylbenzothiazoline sulphonate) radical at 734 nm [47]. Interestingly, the total phenolic content of the plant extracts followed the same order as the radicals scavenging abilities of the plant extracts. Since phenolic compounds are good electron donors [48] they may be responsible of the DPPH and ABTS radicals scavenging activities observed. We evaluated the total antioxidant capacity of the plant extracts using the phosphomolybdenum method by measuring the amount of green phosphate/Mo (V) complex under acidic conditions. Overall, the extracts showed potent antioxidant potential. Transition metals as iron act as catalyst at the initial step of free radical chain reactions. Metal chelators impart antioxidant action at the initial stage of free radical formation [45]. The ferrozine method is currently used for assessing ferrous ion chelating properties of plant extracts. Chelating properties of plant extracts make ferrous ions unavailable and thus prevent the formation of red ferrozine-ferrous ions complex. As noted from Table 3 the chelating power of L. triphylla (17.52 mg EDTAE/g extract) was highest and C. sempervirens (5.40 mg EDTAE/g extract) was least. The total phenolic and flavonoid content of the plant extracts are summarised in Table 4. L. triphylla extract contained the highest phenolic content, followed by C. sempervirens and A. absinthium (Table 4). On the other hand, C. sempervirens showed

13

highest flavonoid content (38.83 mg RE/g extract). The observed enzyme inhibitory activity and antioxidant potential of C. sempervirens might be due to its high flavonoid content.

Conclusion The current study provides an insight into the in vitro antioxidant activity and enzyme (AChE, BChE, tyrosinase, α-amylase, and α-glucosidase) inhibitory potential of C. sempervirens, A. absinthium and L. triphylla. We concluded that the above-noted plant extracts could serve as prospective material for the development of novel plant-based therapeutic agents. Particularly C. sempervirens which showed high inhibitory activity against AChE, BChE, α-amylase, and α-glucosidase, deserves deeper research attention.

Conflict of Interest and Ethical statement The authors declare that there are no conflicts of interest. The paper does not contain any animal studies and hence no ethical approval required.

14

References [1] H.-F. Ji, X.-J. Li, H.-Y. Zhang, Natural products and drug discovery. Can thousands of years of ancient medical knowledge lead us to new and powerful drug combinations in the fight against cancer and dementia?, EMBO Reports 10(3) (2009) 194-200. [2] Ș. Dezsi, A.S. Bădărău, C. Bischin, D.C. Vodnar, R. Silaghi-Dumitrescu, A.-M. Gheldiu, A. Mocan, L. Vlase, Antimicrobial and antioxidant activities and phenolic profile of Eucalyptus globulus Labill. and Corymbia ficifolia (F. Muell.) KD Hill & LAS Johnson leaves, Molecules 20(3) (2015) 47204734. [3] A. Mocan, L. Vlase, O. Raita, D. Hanganu, R. Păltinean, Ş. Dezsi, A.-M. Gheldiu, R. Oprean, G. Crişan, Comparative studies on antioxidant activity and polyphenolic content of Lycium barbarum L. and Lycium chinense Mill. leaves, Pak. J. Pharm. Sci 28(4) (2015) 1511-1515. [4] G. Zengin, A. Uysal, A. Aktumsek, A. Mocan, A. Mollica, M. Locatelli, L. Custodio, N.R. Neng, J.M. Nogueira, Z. Aumeeruddy-Elalfi, Euphorbia denticulata Lam.: A promising source of phytopharmaceuticals for the development of novel functional formulations, Biomedicine & Pharmacotherapy 87 (2017) 27-36. [5] S.A. Emami, J. Asili, M. Rahimizadeh, B.S. Fazly-Bazzaz, M. Hassanzadeh Khayyat, Chemical and antimicrobial studies of Cupressus sempervirens L. and C. horizentalis Mill. essential oils, Iranian Journal of Pharmaceutical Sciences 2(2) (2006) 103-108. [6] A.E. Al-Snafi, Medical importance of Cupressus sempervirens-A review, IOSR Journal of Pharmacy 6(6) (2016) 66-76. [7] F.S. Sharopov, V.A. Sulaimonova, W.N. Setzer, Composition of the essential oil of Artemisia absinthium from Tajikistan, Rec Nat Prod 6(2) (2012) 127-134. [8] W. Ahmad, A. Hasan, A. Abdullah, T. Tarannum, Medicinal importance of Artemisia absinthium Linn (Afsanteen) in Unani medicine: A review, Hippocratic journal of Unani medicine 5(4) (2010) 117-25. [9] M. Pascual, K. Slowing, E. Carretero, D.S. Mata, A. Villar, Lippia: traditional uses, chemistry and pharmacology: a review, Journal of ethnopharmacology 76(3) (2001) 201-214. [10] G. Zengin, C. Sarikurkcu, A. Aktumsek, R. Ceylan, Sideritis galatica Bornm.: a source of multifunctional agents for the management of oxidative damage, Alzheimer's's and diabetes mellitus, Journal of Functional Foods 11 (2014) 538-547. [11] A. Mocan, G. Zengin, A. Uysal, E. Gunes, A. Mollica, N.S. Degirmenci, L. Alpsoy, A. Aktumsek, Biological and chemical insights of Morina persica L.: A source of bioactive compounds with multifunctional properties, Journal of Functional Foods 25 (2016) 94-109. [12] K. Slinkard, V.L. Singleton, Total phenol analysis: automation and comparison with manual methods, American Journal of Enology and Viticulture 28(1) (1977) 49-55. [13] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, The protein data bank, Nucleic acids research 28(1) (2000) 235-242. [14] A. Pesaresi, D. Lamba, TORPEDO CALIFORNICA ACETYLCHOLINESTERASE IN COMPLEX WITH A TACRINE-NICOTINAMIDE HYBRID INHIBITOR. [15] F. Nachon, E. Carletti, C. Ronco, M. Trovaslet, Y. Nicolet, L. Jean, P.-Y. Renard, Crystal structures of human cholinesterases in complex with huprine W and tacrine: elements of specificity for antiAlzheimer's drugs targeting acetyl-and butyryl-cholinesterase, Biochemical Journal 453(3) (2013) 393-399. [16] H. Zhuo, F. Payan, M. Qian, Crystal structure of the pig pancreatic α-amylase complexed with ρ-nitrophenyl-α-d-maltoside-flexibility in the active site, The protein journal 23(6) (2004) 379387. [17] K. Yamamoto, H. Miyake, M. Kusunoki, S. Osaki, Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from Saccharomyces cerevisiae, Journal of bioscience and bioengineering 112(6) (2011) 545-550. [18] W.T. Ismaya, H.J. Rozeboom, A. Weijn, J.J. Mes, F. Fusetti, H.J. Wichers, B.W. Dijkstra, Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone, Biochemistry 50(24) (2011) 5477-5486.

15 [19] A. Uysal, G. Zengin, A. Mollica, E. Gunes, M. Locatelli, T. Yilmaz, A. Aktumsek, Chemical and biological insights on Cotoneaster integerrimus: A new (-)-epicatechin source for food and medicinal applications, Phytomedicine 23(10) (2016) 979-988. [20] W.L. DeLano, The PyMOL molecular graphics system, (2002). [21] S. Maestro, Version 9.2, LLC, New York (2011). [22] A. Mocan, G. Zengin, M. Simirgiotis, M. Schafberg, A. Mollica, D.C. Vodnar, G. Crişan, S. Rohn, Functional constituents of wild and cultivated Goji (L. barbarum L.) leaves: phytochemical characterization, biological profile, and computational studies, Journal of Enzyme Inhibition and Medicinal Chemistry 32(1) (2017) 153-168. [23] J.J. Irwin, T. Sterling, M.M. Mysinger, E.S. Bolstad, R.G. Coleman, ZINC: a free tool to discover chemistry for biology, Journal of chemical information and modeling 52(7) (2012) 1757-1768. [24] J.C. Shelley, A. Cholleti, L.L. Frye, J.R. Greenwood, M.R. Timlin, M. Uchimaya, Epik: a software program for pK a prediction and protonation state generation for drug-like molecules, Journal of computer-aided molecular design 21(12) (2007) 681-691. [25] R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood, T.A. Halgren, P.C. Sanschagrin, D.T. Mainz, Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes, Journal of medicinal chemistry 49(21) (2006) 6177-6196. [26] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and validation of a genetic algorithm for flexible docking, Journal of molecular biology 267(3) (1997) 727-748. [27] L. Custódio, J. Patarra, F. Alberício, N. da Rosa Neng, J.M.F. Nogueira, A. Romano, Phenolic composition, antioxidant potential and in vitro inhibitory activity of leaves and acorns of Quercus suber on key enzymes relevant for hyperglycemia and Alzheimer's disease, Industrial Crops and Products 64 (2015) 45-51. [28] E. Neagu, G. Paun, C. Albu, G.-L. Radu, Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts, Journal of the Taiwan Institute of Chemical Engineers 52 (2015) 1-6. [29] S.-Y. Pan, S.-F. Zhou, S.-H. Gao, Z.-L. Yu, S.-F. Zhang, M.-K. Tang, J.-N. Sun, D.-L. Ma, Y.-F. Han, W.-F. Fong, New perspectives on how to discover drugs from herbal medicines: CAM's outstanding contribution to modern therapeutics, Evidence-Based Complementary and Alternative Medicine 2013 (2013) 1-25. [30] A.O. Ademiluyi, G. Oboh, A.A. Boligon, M.L. Athayde, Dietary supplementation with fermented legumes modulate hyperglycemia and acetylcholinesterase activities in Streptozotocin-induced diabetes, Pathophysiology 22(4) (2015) 195-201. [31] T. Nagao, S. Meguro, T. Hase, K. Otsuka, M. Komikado, I. Tokimitsu, T. Yamamoto, K. Yamamoto, A Catechin‐rich Beverage Improves Obesity and Blood Glucose Control in Patients With Type 2 Diabetes, Obesity 17(2) (2009) 310-317. [32] G. Zengin, A. Karanfil, M.C. Uren, M.S. Kocak, C. Sarikurkcu, H. Gungor, C.M.N. Picot, M.F. Mahomoodally, Phenolic content, antioxidant and enzyme inhibitory capacity of two Trametes species, RSC Advances 6(77) (2016) 73351-73357. [33] A. Mollica, M. Locatelli, G. Macedonio, S. Carradori, A.P. Sobolev, R.F. De Salvador, S.M. Monti, M. Buonanno, G. Zengin, A. Angeli, Microwave-assisted extraction, HPLC analysis, and inhibitory effects on carbonic anhydrase I, II, VA, and VII isoforms of 14 blueberry Italian cultivars, Journal of Enzyme Inhibition and Medicinal Chemistry 31(sup4) (2016) 1-6. [34] R. Harini, K.V. Pugalendi, Antihyperglycemic effect of protocatechuic acid on streptozotocindiabetic rats, J Basic Clin Physiol Pharmacol 21(1) (2010) 79-91. [35] J. Sripetchwandee, Y. Semaming, P. Sa-nguanmoo, H. Pintana, P. Pannangpetch, N. Chattipakorn, S.C. Chattipakorn, Protocatechuic acid attenuates brain oxidative stress and brain mitochondrial dysfunction in insulin-dependent diabetic rats, Alzheimer's & Dementia: The Journal of the Alzheimer's Association 11(7) (2015) P846. [36] E. Mariani, M. Polidori, A. Cherubini, P. Mecocci, Oxidative stress in brain aging, neurodegenerative and vascular diseases: an overview, Journal of Chromatography B 827(1) (2005) 65-75.

16 [37] S. Carradori, M.C. Gidaro, A.l. Petzer, G. Costa, P. Guglielmi, P. Chimenti, S. Alcaro, J.P. Petzer, Inhibition of Human Monoamine Oxidase: Biological and Molecular Modeling Studies on Selected Natural Flavonoids, Journal of Agricultural and Food Chemistry 64(47) (2016) 9004-9011. [38] B. Vijayakumari, V. Sasikala, S.R. Radha, H.Y. Rameshwar, In silico analysis of aqueous root extract of Rotula aquatica Lour for docking analysis of the compound 3-O-acetyl-11-keto-βboswellic acid contents, SpringerPlus 5(1) (2016) 1486. [39] K. Atkovska, S.A. Samsonov, M. Paszkowski-Rogacz, M.T. Pisabarro, Multipose binding in molecular docking, International journal of molecular sciences 15(2) (2014) 2622-2645. [40] M. Katalinić, G. Rusak, J.D. Barović, G. Šinko, D. Jelić, R. Antolović, Z. Kovarik, Structural aspects of flavonoids as inhibitors of human butyrylcholinesterase, European journal of medicinal chemistry 45(1) (2010) 186-192. [41] Y. Xie, W. Yang, X. Chen, J. Xiao, Inhibition of flavonoids on acetylcholine esterase: binding and structure–activity relationship, Food & function 5(10) (2014) 2582-2589. [42] H.-R. Li, M. Habasi, L.-Z. Xie, H.A. Aisa, Effect of chlorogenic acid on melanogenesis of B16 melanoma cells, Molecules 19(9) (2014) 12940-12948. [43] D.R. Wulan, E.P. Utomo, C. Mahdi, Molecular modeling of Ruellia tuberosa L compounds as aamylase inhibitor: an in silico comparation between human and rat enzyme model, Bioinformation 10(4) (2014) 209-215. [44] M.M. de Freitas, P.R. Fontes, P.M. Souza, C.W. Fagg, E.N.S. Guerra, Y.K. de Medeiros Nóbrega, D. Silveira, Y. Fonseca-Bazzo, L.A. Simeoni, M. Homem-de-Mello, Extracts of Morus nigra L. Leaves Standardized in Chlorogenic Acid, Rutin and Isoquercitrin: Tyrosinase Inhibition and Cytotoxicity, PloS one 11(9) (2016) e0163130. [45] H.D.S.M. Perera, J.K.R.R. Samarasekera, S.M. Handunnetti, O.V.D.S.J. Weerasena, In vitro antiinflammatory and anti-oxidant activities of Sri Lankan medicinal plants, Industrial Crops and Products 94 (2016) 610-620. [46] M. Öztürk, F. Aydoğmuş-Öztürk, M.E. Duru, G. Topçu, Antioxidant activity of stem and root extracts of Rhubarb (Rheum ribes): An edible medicinal plant, Food Chemistry 103(2) (2007) 623630. [47] S. Surveswaran, Y.-Z. Cai, H. Corke, M. Sun, Systematic evaluation of natural phenolic antioxidants from 133 Indian medicinal plants, Food Chemistry 102(3) (2007) 938-953. [48] E. Bendary, R. Francis, H. Ali, M. Sarwat, S. El Hady, Antioxidant and structure–activity relationships (SARs) of some phenolic and anilines compounds, Annals of Agricultural Sciences 58(2) (2013) 173-181.

17

Figure 1. Chemical structure of (+)-catechin and apigenin.

Figure 2. 2D interactions of the best pose found for catechin docked to (A) tyrosinase, (B) aglucosidase, (C) AChE, (D) BChE.

18

Figure 3. 2D interactions of the best pose found for apigenin docked to (A) tyrosinase, (B) aglucosidase, (C) AChE, (D) BChE.

19

Figure 4. 2D interactions of the best pose found for (A) Apigenin, (B) catechin docked to αamylase.

20

Table 1. Determination of phenolic compounds from the plant extracts by RP-HPLC, regression parameter, linearity, limit of detection (LOD) and limit of quantification (LOQ) * No Phenolic components Plant extracts Analytical characteristics (µg/g extract) C. sempervirens A. absinthium L. triphylla Linear range R² LOD LOQ (mg L-1) (mg L-1) (mg L-1) 1 2 3

Protocatechuic acid 16±0.1a 1.2±0.1c (+)-Catechin 205.4±14.2a 12.4±1.5c p-Hydroxybenzoic 4.0±0.2a nd acid 4 Chlorogenic acid 10.6±0.2b 35.9±0.6a 5 Caffeic acid nd 3.9±0.2a 6 (-)-Epicatechin 12.8±0.6 nd 7 Ferulic acid nd 1.1±0.1b 8 Benzoic acid 3.4±0.2 nd 9 Rutin nd 15.8±0.3 10 Rosmarinic acid nd nd c 11 Apigenin 7.3±0.1 8.9±0.1b * Data from three repetitions, with mean ± standard deviation; significantly (p < 0.05) different. nd, not detected.

3.5±0.1b 26.2±1.4b 4.4±0.2a 3.0±0.2c 3.5±0.2a nd 1.9±0.1a nd nd nd 13.9±0.1a means with different

0.20-25.0 0.90-113 0.20-25.0

0.9991 0.9988 0.9994

0.086 0.172 0.007

0.260 0.522 0.020

0.35-45.0 0.9988 0.080 0.241 0.16-21.0 0.9993 0.054 0.162 0.50-66.0 0.9990 0.170 0.514 0.12-17.0 0.9993 0.004 0.011 0.85-55.0 0.9998 0.111 0.335 0.40-55.0 0,9990 0.180 0.550 0.40-55.1 0.9989 0.050 0.150 0.17-11.0 0.9997 0.034 0.104 superscript letters in the same column were

21

Table 2. Enzyme inhibitory effects of the plant extracts* Plant extracts

AChE inhibition (mg GALAE/g extract)

BChE Tyrosinase α-Amylase α-Glucosidase inhibition inhibition inhibition inhibition (mmol (mg (mg KAE/g (mmol ACAE/g extract) GALAE/g extract) ACAE/g extract) extract) a a b C. sempervirens 2.47±0.01 2.98±0.13 29.43±1.63 1.61±0.01a 1.86±0.01a A. absinthium 2.03±0.05c 1.76±0.16c 31.06±0.57a 1.41±0.02b 0.40±0.06c b b c c L. triphylla 2.15±0.01 2.12±0.02 15.05±1.63 1.37±0.05 1.00±0.14b GALAE, galantamine equivalents; ACAE, acarbose equivalents; KAE, kojic acid equivalents. * Data from three repetitions, with mean ± standard deviation; means with different superscript letters in the same column were significantly (p < 0.05) different.

22

Table 3. Reducing power, total antioxidant capacity, radical scavenging activity and chelating power of the plant extracts.* mg TE/g extract Plant extracts

CUPRAC

FRAP

mg EDTAE/g extract

Phosphomolydenu DPPH ABTS Metal chelating activity m C. sempervirens 58.11±2.15a 102.68±4.03b 401.04±21.07a 83.01±0.64b 52.60±0.60b 5.40±1.39c c c c c A. absinthium 27.75±0.27 49.47±0.49 378.51±6.00 25.49±1.49 30.18±1.20c 14.67±0.07b L. triphylla 40.22±0.51b 143.54±2.86a 397.04±9.25b 113.77±2.75a 94.36±1.18a 17.52±1.18a TE: trolox equivalents; EDTAE: EDTA equivalents. * Data from three repetitions, with mean ± standard deviation; means with different superscript letters in the same column were significantly (p < 0.05) different.

23

Table 4. Total phenol and flavonoid content of the plant extracts.* Plant extracts

Total phenolic content (mg Total flavonoid content (mg GAE/g extract) RE/g extract) C. sempervirens 83.90±2.06b 38.83±0.08a A. absinthium 33.33±1.48c 12.01±0.06c L. triphylla 88.66±2.71a 13.83±0.08b * GAEs, gallic acid equivalents; REs, rutin equivalents. Data from three repetitions, with mean ± standard deviation; means with different superscript letters in the same column were significantly (p < 0.05) different.