Acetylcholinesterase inhibitory activity and molecular ...

South African Journal of Botany 106 (2016) 1–4

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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb

Bioactive constituents from roots of Salvia syriaca L.: Acetylcholinesterase inhibitory activity and molecular docking studies Mir Babak Bahadori a, Leila Dinparast b, Hassan Valizadeh a,⁎, Mahdi Moridi Farimani c, Samad Nejad Ebrahimi c a b c

Organic Chemistry and Phytochemistry Research Laboratory, Faculty of Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 29 August 2015 Received in revised form 26 October 2015 Accepted 24 December 2015 Available online xxxx Edited by V Steenkamp

a b s t r a c t Several Salvia species have been used to enhance memory in European traditional medicine. Accordingly, we aimed to evaluate the acetone extract of the roots of Salvia syriaca for anti-Alzheimer constituents. Structures of purified compounds were determined as ursolic acid, corosolic acid, β-sitosterol, urs-12-en-2α,3β-diol and daucosterol (1–5). β-sitosterol and daucosterol exhibited high acetylcholinesterase inhibitory activities (24.1 and 34.3 μg/mL, respectively). The experimental results were also confirmed by docking analysis. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Salvia syriaca Acetylcholinesterase Alzheimer's disease Docking study β-sitosterol Daucosterol

1. Introduction The genus Salvia is the largest member of the Lamiaceae family and comprises over 1000 species (Wu et al., 2012). Salvia species are traditionally used for medicinal purposes worldwide (Farimani et al., 2012; Bahadori and Mirzaei, 2015). Various biological activities such as antimicrobial, cytotoxicity, anti-protozoal, anti-HIV, antioxidant and anti-inflammatory activities have been reported for the genus (Topcu, 2006; Farimani et al., 2015). In addition, Salvia species are used for central nervous system disorders (Karimi et al., 2012). Several species from the genus are used in perfume, food and pharmaceutical industries (Bahadori et al., 2015). A broad spectrum of natural compounds such as terpenoids and phenolics are found in Salvia species (Lu and Foo, 2002; Bautista et al., 2013). Salvia syriaca is used as a forage (Flamini et al., 2007). Previous phytochemical studies on S. syriaca reported the isolation of some flavonoids (Hatam and Yousif, 1992), one sesterterpenoid salvisyriacolide ⁎ Corresponding author at: Kilometer 35, Tabriz/Azarshahr Road, Tabriz, Iran. P.O. Box: 53714-161. Tel./fax: +9841 34327541. E-mail address: [email protected] (H. Valizadeh).

http://dx.doi.org/10.1016/j.sajb.2015.12.003 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

(Rustaiyan and Sadjadi, 1987), a new sesquiterpene named syriacine (Al-Jaber et al., 2012) and four novel seco-ursadiene triterpenoids (Al-Jaber et al., 2012; Al-Aboudi et al., 2015) from the aerial parts of the plant. Ulubelen et al. (2000) reported some di- and triterpenoids, sitosterol and one flavonoid from the roots of the plant with cardiovascular activities. Also, there are some studies on the essential oil of S. syriaca which reported germacrene B, germacrene D and bicyclogermacrene as major volatile compounds (Baser et al., 1996; Flamini et al., 2007). Flora of Iran contains 61 Salvia species of which 17 are endemic (Jamzad, 2012). Our research group reported many structurally interesting and bioactive diterpenoids, sesterterpenoids, triterpenoids and isoprenoids with novel carbon skeletons from Iranian Salvia species in recent years (Farimani et al., 2011, 2012, 2013; Ebrahimi et al., 2014; Farimani et al., 2015). Continuing our studies on the discovery of bioactive constituents from Iranian Salvia species, we studied the acetone extract of the roots of S. syriaca in the present work to find new cognitive enhancers. Herein, we report the isolation and identification of three triterpenoids, one steroid and one steroid-glucoside on the basis of 1D and 2D NMR data (Fig. 1). Acetylcholinesterase inhibitory activity and molecular docking studies of the isolated compounds are also reported.

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M.B. Bahadori et al. / South African Journal of Botany 106 (2016) 1–4

Fig. 1. Structures of isolated compounds (1–5).

2. Experimental 2.1. General experimental procedures NMR spectra were measured on a Bruker Avance III 500 MHz spectrometer (1H: 500 MHz, 13C: 125 MHz). UV/Vis analysis was carried out using a Shimadzu spectrophotometer (2550 UV/Vis). 2.2. Chemicals CDCl3, methanol-d4 and pyridine-d5 for NMR were purchased from Armar Chemicals. Silica gel (mesh 230–400), acetylthiocholine iodide, DTNB and galantamine were obtained from Merck (Germany). 2.3. Plant material The roots of S. syriaca were collected in May 2013 from Urmia, West Azarbaijan province. The plant was identified by Mr. Shahram Bahadori, a taxonomist at the Urmia School of Pharmacy Herbarium and a voucher specimen (USPH-104) has been deposited for the plant. 2.4. Extraction and isolation The powdered roots of the plant (2.2 kg) were extracted using acetone (3 × 10 L) by maceration at room temperature. The extract was concentrated in vacuo to obtain 40 g reddish gummy acetone extract. Fractionation of the extract by column chromatography (5 cm × 80 cm) on silica gel (step gradient n-hexane-EtOAc as eluent) afforded 20 fractions. Fraction 7 (0.8 g) was separated on a silica gel column with CHCl3–MeOH (95:5) to afford four fractions (7a–7d). Fraction 7c was further purified using preparative thin layer chromatography (CHCl3–MeOH (90:10)) to afford compound 1 (8 mg, Rf = 0.6). Fraction 8 (1.1 g) was separated by column chromatography with CHCl3–MeOH (95:5) as eluent to give five fractions (8a–8e). Fraction 8d was recrystallized from CHCl3 to yield compound 2 (3 mg). Fraction 9 (0.4 g) was recrystallized from acetone to afford compound 3 (200 mg). Fraction 12 (0.9 g) was subjected to silica gel column chromatography with CHCl3–MeOH (90:10) as eluent and afforded eight fractions (12a–12 h). Fraction 12e was further purified using preparative thin layer chromatography (CHCl3–MeOH (85:15)) to

afford compound 4 (10 mg, Rf = 0.8). Fraction 15 (1.3 g) was triturated with acetone and the residue was recrystallized from CHCl3–MeOH to yield compound 5 (45 mg). 2.5. Acetylcholinesterase inhibitory activity In vitro acetylcholinesterase inhibitory activities of the extract and purified compounds were determined using a spectrophotometric method described by Ellman et al. (1961) with some modifications using a Shimadzu spectrophotometer (2550 UV/Vis). A partially purified enzyme was prepared for the experiments according to a previously published method (Karimi et al., 2010). The stock solutions of testing compounds were prepared by dissolving them in DMSO. Briefly, 500 μL of sodium phosphate buffer (100 mM, pH = 8.0), 150 μL of DTNB (3.5 mM), 150 μL of substrate (acetylthiocholine iodide) and 150 μL inhibitor solution were added into the reaction mixture in a 1 mL cell and incubated for 15 min at 37 °C. Afterward, the reaction was started by addition of 50 μL of enzyme. The enzyme was kept on ice before the addition to the incubation mixture. After immediate mixing of the reaction mixture, the changing of the absorbance was measured at 412 nm for 10 min. The inhibition rate of the samples on acetylcholinesterase was calculated by the following formula: % Inhibition = [(Absorbancecontrol Absorbancecontrol] × 100.

−

Absorbancesample)/

The IC50 values were calculated from inhibition curves (inhibitor concentration vs. percent of inhibition). The results were expressed as mean ± SEM of three independent experiments. Galantamine was used as the standard drug. 2.6. Molecular docking studies To study the molecular interaction and binding mode of the purified compounds, molecular docking studies were performed using AutoDock 4.2 software (Morris et al., 1998). For this purpose, the crystal structures of human acetylcholinesterase (PDB code: 4PQE) was downloaded from protein data bank (http://www.rcsb.org). Subsequently, all water molecules of acetylcholinesterase were removed from the enzyme structure


3

3. Results and discussion

rivastigmine and huperzine may have undesirable side effects. These alkaloid drugs potentially are toxic (Kennedy et al., 2011). So, discovery of new AChEIs with more activity and less price is needed. The genus Salvia is known as a memory enhancer plant (Culhaoglu et al., 2013) and several Salvia species have been reported to have anticholinesterase activity (Kolak et al., 2009; Loizzo et al., 2010). In the present study, the acetone extract of the roots of S. syriaca showed an inhibitory activity against AChE with an IC50 value of 500 μg/mL. The isolated compounds were tested for their AChE inhibitory potential and exhibited moderate to high activities (Table 1). β-sitosterol (3) showed the highest enzyme inhibition activity with an IC50 value of 24 μg/mL, followed by daucosterol (IC50 = 31 μg/mL). The isolated triterpenoids (ursolic acid, corosolic acid and urs-12-en-2α,3β-diol) showed moderate activities as shown in Table 1. All of the isolates exhibited their inhibitory activity in a concentration dependent manner. Galantamine was used as a standard drug in this study (IC50 = 8.7 μg/mL). Galantamine showed a stronger AChE inhibitory activity than Salvia compounds in this work. The IC50 values are reported in Table 1 with two different units (μg/mL and μM). Orhan et al. (2007) showed that several extracts of some Salvia plants have AChE inhibitory activity at 1 mg/mL (inhibition percent from 12 to 66) but no activity at 0.2 mg/mL. Çulhaoğlu et al. (2013) reported oleanolic acid and ursolic acid from Salvia chrysophylla with anticholinesterase activity. Şenol et al. (2010) studied 55 Salvia species from Turkey with 0%–36% AChE inhibitory activity at 100 μg/mL. Zhou et al. (2011) showed that tanshinone diterpenoids from roots of Salvia miltiorrhiza act as acetylcholinesterase inhibitors. Also, there are some reports on AChE inhibitory potential of monoterpenoids from essential oils of Salvia species (Perry et al., 2002; Duru et al., 2012). Our results and the literature review on Salvia genus indicated that, terpenoid derived phytochemicals are the major responsible compounds for AChE inhibitory activity of these plants.

3.1. Structure elucidation of isolated compounds

3.3. Molecular modeling

The acetone extract of the roots of S. syriaca was subjected to silica gel column chromatography and preparative thin layer chromatography to yield compounds 1–5. The structures of the purified compounds were determined on the basis of 1D and 2D NMR spectra and by comparison of their spectral data with those of the literature (Wang et al., 2012; Esmaeili and Farimani, 2014). The isolated compounds were identified as ursolic acid (1), corosolic acid (2), β-sitosterol (3), urs-12-en-2α,3β-diol (4) and daucosterol (5) (Fig. 1). Compounds 2, 4 and 5 are reported from S. syriaca for the first time.

The molecular docking analysis could provide an important view into ligand–enzyme bindings and conformations (Raeisi, 2013). In order to explore the activities of the present compounds and binding interactions between ligands and acetylcholinesterase, the structures of isolated compounds (1–5) were docked into the active site of AChE (PDB code: 4PQE). Some important results of the in silico studies are summarized in Table 2. The experimental IC50 values of isolates revealed that the better result was obtained for compound 3 followed by compound 5. This is in agreement with the estimated free energies of binding of tested compounds (Table 2). Docking analysis showed that the compounds with high acetylcholinesterase inhibitory activity (3 and 5) created hydrogen bond with amino acids in the active site of AChE (Fig. 2) and had high binding affinity (ΔGBinding = − 11.31 and − 5.22 kcal/mol, respectively). Also, docking studies indicated that

Table 1 AChE inhibitory activity of the acetone extract and purified compounds from roots of S. syriacaa.

Acetone extract Ursolic acid (1) Corosolic acid (2) β-sitosterol (3) urs-12-en-2α,3β-diol (4) Daucosterol (5) Galantamineb a b

IC50 (μg/mL)

IC50 (μM)

499.5 ± 6.8 46.3 ± 1.2 55.2 ± 1.6 24.1 ± 0.7 63.0 ± 1.3 34.3 ± 0.3 8.7 ± 0.2

– 101.4 ± 2.6 116.7 ± 3.3 58.1 ± 1.6 142.4 ± 2.9 59.6 ± 0.5 30.2 ± 0.7

Values are the mean ± SEM of three experiments (p b 0.05). Standard drug.

and the polar hydrogen atoms, Kollman and Gasteiger charges were added to amino acid residues of the protein structure using AutoDock Tools (ADT, version 1.5.6) (Sanner, 1999). The 3D molecular structures of the compounds were optimized at the Hartree–Fock level with 3-21G basis set using Gaussian 03 program. Then, the required AutoDock format (pdbqt) of the receptor and ligands were obtained using AutoDock Tools 1.5.6. The docking analyses of the purified compounds were carried out by 200 runs of AutoDock with the Lamarckian genetic algorithm method (LGA). The population size, maximum number of evaluation (medium) and maximum number of generation were set at 150, 2,500,000 and 27,000, respectively. The grid box was centered on the active site of the enzyme (Pohanka, 2011) with x, y, and z coordinates of −26.138, 27.432, −6.496 Å. The number of points in the x, y and z dimensions was 40 × 40 × 40. The spacing between grid points was set at 0.375 Å. The molecular visualizations were executed in Python 3.4.

3.2. Acetylcholinesterase inhibitory activity The current treatment of AD is based on the inhibition of AChE but common cholinesterase inhibitors like physostigmine, galantamine,

Table 2 Summarizing the important docking results. The H-bond distances have been measured between related atoms. Estimated ΔGBinding (kcal/mol)

Hydrogen bonding

Compound

Interacted ligand functional group

Interacted amino acid

Distance (°A)

Interacted residues

Ursolic acid

+18.98

−

−

−

Corosolic acid

+21.04

−

−

−

β-sitosterol

−11.31

–OH

GLU202

1.832

urs-12-en-2α,3β-diol

+14.96

−

−

−

SER203, HIS447, GLU202, GLY448, TRP86, TYR337, PHE338, PHE295, TYR341,TYR124, TRP286, PHE297, GLY121, GLY122 PHE295, PHE338, TYR337, HIS447, GLY448, GLU202, TRP86, GLY121, GLY122, TYR124, SER203, PHE297 TYR133, TRP86, GLU202, ASP74, TYR124, TYR341, PHE338, TYR337, SER203, GLY121, GLY120, SER125 PHE295, PHE338, TYR341, TYR337, HIS447, GLU202, GLY448, TRP86, GLY120, GLY121, GLY122, TYR124, TRP286, PHE297,TYR341 ILE451, GLY448, HIS447, TRP86, SER125, ASP74, TYR124, TRP286, TYR341, VAL294, PHE336, TYR337, SER203, GLY121, GLY120 SER203, HIS447, TYR337, GLU202, GLY120, TRP86, TYR124, PHE297, GLY121

Daucosterol

−5.22

–O–

TYR341

2.999

Galantamine

−9.30

–O– –OH

TYR124 TYR337

2.599 2.645

4


Fig. 2. Hydrogen bond between hydroxyl group of β-sitosterol and Glu202.

triterpenoid compounds with lower activity (1, 2 and 4) have no hydrogen bond with the active site residues. The estimated free energies of binding and observed hydrogen bonds for tested molecules could confirm the experimental results. In comparison, galantamine (standard anticholinesterase drug) showed two hydrogen bonds and high binding affinity in docking study and also exhibited the highest in vitro inhibitory activity. The results showed that β-sitosterol and daucosterol are better inhibitors than triterpenoids studied in this work. This is in agreement with Pereira et al. (2016) who reported that β-sitosterol and daucosterol have stronger acetylcholinesterase activity than oleanolic acid, ursolic acid and corosolic acid. It seems that sterols and sterol– glucoside compounds (without methyl groups on C-4 and C-14) are stronger inhibitors than triterpenoids which contain methyl groups on C-4 and C-14 moieties. These sterols may have more flexible structures and could create stronger interactions with active site of the enzyme. 4. Conclusion Bioactive constituents were isolated from the roots of S. syriaca. The acetylcholinesterase inhibitory activity of the acetone extract and the isolated compounds (1–5) was investigated for the first time and the IC50 values obtained in micrometer range. In silico study of isolated compounds showed possible binding modes and confirmed the experimental results. Also, the molecular docking analysis confirmed the potential of studied compounds for future drug discovery investigations. Further QSAR and kinetic studies are needed to understand the inhibitory mechanism of corresponding compounds and relative derivatives. Acknowledgments The partial financial assistance from the Research Vice Chancellor of Azarbaijan Shahid Madani University is gratefully acknowledged. References Al-Aboudi, A.M.F., Abu Zarga, M.H., Abu-Irmaileh, B.E., Awwadi, F.F., Khanfar, M.A., 2015. Three new seco-ursadiene triterpenoids from Salvia syriaca. Nat. Prod. Res. 29, 102–108. Al-Jaber, H.I., Abrouni, K.K., Al-Qudah, M.A., Abu Zarga, M.H., 2012. New terpenes from Salvia palaestina Benth. and Salvia syriaca L. growing wild in Jordan. J. Asian Nat. Prod. Res. 14, 618–625. Bahadori, M.B., Mirzaei, M., 2015. Cytotoxicity, antioxidant activity, total flavonoid and phenolic contents of Salvia urmiensis Bunge and Salvia hydrangea DC. Ex Benth. RJP 2, 27–32. Bahadori, M.B., Valizadeh, H., Asghari, B., Dinparast, L., Farimani, M.M., Bahadori, S., 2015. Chemical composition and antimicrobial, cytotoxicity, antioxidant and enzyme inhibitory activities of Salvia spinosa L. J. Funct. Foods 18, 727–736.

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