Contents lists available atScienceDirect
Industrial Crops & Products
journal homepage:www.elsevier.com/locate/indcropPotential anti-Alzheimer e
ffects of selected Lamiaceae plants through
polypharmacology on glycogen synthase kinase-3
β, β-secretase, and casein
kinase 1
δ
Perihan Gürbüz
a,b,⁎, Ana Martinez
b, Concepción Pérez
c, Loreto Martínez-González
b,
Fatih Göger
d,e,f,
İrem Ayran
gaDepartment of Pharmacognosy, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey bCentro de Investigaciones Biologicas, CSIC, Ramiro de Maetzu 9, 28040 Madrid, Spain cMedicinal Chemistry Institute‐CSIC, Juan de la Cierva 3, 280 06 Madrid, Spain dDepartment of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskişehir, Turkey
eDepartment of Pharmacy, Program in Pharmacy Services, Yunus Emre Vocational School, Eskişehir, Turkey fAnadolu University Medicinal Plants, Drugs and Scientific Research Centre (AUBIBAM), 26470 Eskişehir, Turkey gDepartment of Medicinal Plants, Faculty of Agriculture, Selçuk University, Konya, Turkey
A R T I C L E I N F O Keywords: Amyloidβ Lamiaceae GSK-3β Alzheimer’s disease Origanum onites LC-DAD-ESI-MS/MS A B S T R A C T
The aerial parts of Lamiaceae plants have been used since ancient times all over the world, especially in Mediterranean cultures, for their medicinal and culinary properties. Although some of them have a historical role in the improvement of cognition and memory, the precise mechanism of these plants remains unclear.
This study evaluated the potential anti-Alzheimer effects of some popular cultivated Lamiaceae plants via their inhibitory potential towards glycogen synthase kinase (GSK-3β), β-secretase (BACE-1), and casein kinase 1δ (CK-1δ), enzymes related to AD.
Luminometric-based assays were used for the evaluation of GSK-3β and CK-1δ inhibitory activities in which a fluorescence resonance energy transfer (FRET) assay was used for determining BACE-1 inhibitory activity. The major compounds were identified by high pressure liquid chromatography with diode array detection coupled with electrospray ionization tandem mass spectrometry (LC-DAD-ESI-MS/MS).
Among the extracts tested, Origanum onites L., O. majorana L., and Mentha piperita L. were the most active ones for GSK-3β inhibitory activity, with IC50values under 15μg/mL, while O. majorana and S. officinalis L. were the most active for BACE-1 and O. onites, O. majorana, and Melissa officinalis L. for CK-1δ, with half maximal in-hibitory concentration (IC50) values under 250μg/mL. On the basis of the composition of these active extracts it is apparent thatflavonoids, i.e. apigenin, luteolin, and their glycoside derivatives, along with phenolic acids, i.e. rosmarinic acid and salvianolic acids, are the major principles of these active extracts.
The outcomes of this study supported the traditional use of Lamiaceae plants for cognitive disorders and highlighted their underlying mechanisms, targeting GSK-3β/BACE-1/CK-1δ together for the first time.
1. Introduction
Alzheimer's disease (AD) is a progressive and irreversible neurode-generative disease characterized by the formation of extraneuronal deposition of the amyloid β (Aβ) protein as senile plaques and in-traneuronal aggregation of microtubule-associated protein tau as neu-rofibrillary tangles (NFTs). Amyloid β is formed from sequential pro-teolytic cleavage of amyloid precursor protein (APP) by the aspartic proteaseγ- and β-secretase in the amyloidogenic pathway (Vassar et al.,
1999). BACE is the key rate-limiting enzyme that initiates the formation of Aβ in the amyloidogenic pathway and an increase in BACE-1 activ-ities causes the production and accumulation of neurotoxic forms of Aβ in the brain and leads to neurodegeneration (Hu et al., 2010). Although GSK-3 wasfirst described as an enzyme regulating glycogen synthesis, it was recently found that this enzyme also plays a crucial role in a variety of biological events, including apoptosis and gene expression (Doble and Woodgett, 2003). Activation of GSK-3β, one of the two homologous isoforms of GSK-3 and largely expressed in the central nervous system,
https://doi.org/10.1016/j.indcrop.2019.05.080
Received 18 December 2018; Received in revised form 25 May 2019; Accepted 27 May 2019
⁎Corresponding author at: Department of Pharmacognosy, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey. E-mail address:[email protected](P. Gürbüz).
Available online 30 July 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
has been shown in a variety of studies involving apoptosis and apop-totic neuronal cell death, Aβ toxicity, and tau pathologies ( Eldar-Finkelman and Martinez, 2011). Recently it was demonstrated that another kinase family member; CK-1δ, is also associated with patho-logical accumulation of tau in several neurodegenerative disorders, especially in AD (Schwab et al., 2000; Yasojima et al., 2000). More recent studies also proved that constitutively active CK-1 increases the formation of Aβ (Flajolet et al., 2007).
Since it is understood that GSK-3β, BACE-1, and CK-1δ have a key function at the beginning and during the progression of AD, the search for a compound that acts as an inhibitor of those enzymes has gained importance.
Lamiaceae (the mint family) is the sixth largest family offlowering plants and is one of the most popular families due to the aromatic potential of most species (Li et al., 2017). The family members have a cosmopolitan distribution and there are various economically im-portant culinary herbs, i.e. rosemary, thyme, basil, oregano, sage, and both spearmint and peppermint. There are numerous articles about the neurobiological effects of different Lamiaceae plants, including clinical studies (Perry et al., 2003,2000;Tildesley et al., 2003). Many of the in vitro studies focused on the cholinesterase inhibitory activities of the extracts and were conducted with terpenic and phenolic compositions including different classes of flavonols and their glycosides, and dimers and trimers of caffeic acid derivatives (Savelev et al., 2004). More re-cently Perry et al. reported that of a mixture of sage, rosemary, and melissa for the duration of administration and at the dose chosen was more effective than a placebo in supported verbal episodic memory in healthy subjects under 63 years old (Perry et al., 2018). However, de-spite the promising clinical observations and in vitro enzyme inhibitory results, the precise mechanism for these plants remains unknown. This background prompted us to investigate the neurobiological activities of some popular and cultivated selected Lamiaceae plants through the abovementioned enzyme inhibitory properties related to AD. The aim of the present work was to understand the anti-Alzheimer effects of ten different frequently consumed Lamiaceae species on the targeted en-zymes. The chemical characterization of the extracts was carried out using LC-DAD-ESI-MS/MS.
2. Materials and methods 2.1. Instrumentations and reagents
For the in vitro GSK-3β inhibition assay, human recombinant GSK-3β and prephosphorylated polypeptide substrate were purchased from Millipore (Millipore Iberica S.A.U.). For the in vitro CK-1 inhibition assay, human recombinant CK-1δ was also purchased from Millipore (Millipore Iberica S.A.U.). A Kinase-Glo Luminescent Kit was obtained from Promega (Promega Biotech Ibérica, SL). ATP and all other re-agents were from Sigma-Aldrich (St. Louis, MO, USA). All other cell materials were analytical grade. LC–MS/MS analysis was carried out using an Absciex 3200 Q trap MS/MS detector. The experiments were performed with a Shimadzu 20A HPLC system coupled to an Applied Biosystems 3200 Q-Trap LC–MS/MS instrument equipped with an ESI source operating in negative ion mode. For the enzyme inhibition as-says a FLUOstar Optima (BMG Labtechnologies GmbH, Offenburg, Germany) microplate reader was used to determine the luminescence andfluorescence.
2.2. Plant samples
In 2015, seeds of Salvia officinalis (sage), Lavandula angustifolia Mill. (lavender), Mentha piperita (peppermint), Melissa officinalis (lemon balm), Origanum onites (oregano), Thymus vulgaris L. (thyme), Origanum majorana (marjoram), Origanum vulgare L. (oregano), Sideritis congesta P.H.Davis & Hub.-Mor., and Dorystaechas hastata Boiss. & Heldr. ex Benth. were obtained from the Department of Medicinal Plants and
cultivated under controlled conditions in the Agricultural Faculty, Selçuk University in Konya, Turkey. After that, the aerial parts of the plants were harvested during the fullflowering period in May and June. The plant materials were dried in the shade and stored at +4 °C under controlled conditions until analyzed.
2.3. Extract preparation
The dried and powdered aerial parts of plant samples (15 g) were extracted with 100 mL of MeOH:H2O (70:30, v/v) four orfive times at 37 °C. After evaporation of the solvent, the crude MeOH extracts were lyophilized until used in activity and chromatography assays. Thefinal concentrations of the samples submitted to LC-DAD-ESI–MS/MS and the yields of extracts are given inTable 1.
2.4. GSK-3β, CK-1δ, and BACE-1 Enzyme inhibition assays
For GSK-3β inhibitory activity the method described by Baki et al. (Baki et al., 2007) was followed, with slightly modifications. The Ki-nase-Glo Kit was used to screen compounds for activity against CK-1δ and GSK-3β. The activity is proportional to the difference between the total and consumed ATP. The BACE-1 assay was carried out according to the manufacturer’s protocol from Invitrogen (L0724). The ability to inhibit BACE-1 enzyme activity was investigated by means of a bio-chemical assay performed using the FRET methodology. TDZD-8 and SB 216763 were used as positive controls for enzyme activities.
2.5. LC-DAD-ESI-MS/MS analysis
For the chromatographic separation, a GL Science Intersil ODS 250 × 4.6 mm, i.d., 5μm particle size, octadecyl silica gel analytical column operating at 40 °C was used. The solventflow rate was main-tained at 0.3 mL/min. Detection was carried out with a PDA detector. The elution gradient consisted of mobile phases (A) acetoni-trile:water:formic acid (10:89:1, v/v/v) and (B) acetoni-trile:water:formic acid (89:10:1, v/v/v). The composition of B was in-creased from 10% to 100% in 40 min. The LC-ESI-MS/MS data were collected and processed by Analyst 1.6 software.
3. Results
3.1. GSK-3β, BACE-1, and CK-1δ inhibitory activities
All inhibitory enzyme activities of the Lamiaceae extracts men-tioned above are summarized inTable 2.
GSK-3β Inhibition. All of the extracts possessed promising GSK-3β inhibitory activity below 75μg/mL. Among the Lamiaceae extracts tested, Origanum onites, Mentha piperita, O. majorana, Thymus vulgaris, O. Table 1
Extract yields andfinal concentrations submitted to LC-DAD-ESI-MS/MS* ana-lyzes.
Sample Yield (w/w %) C**(mg/mL)
Sideritis congesta (SC) 13.7 6.7 Mentha piperita (MP) 27.4 14.0 Lavandula angustifolia (LA) 14.5 18.9 Salvia officinalis (SO) 28.8 16.8 Dorystoechas hastata (DH) 15.0 12.4 Melissa officinalis (MO) 24.1 8.9 Thymus vulgaris (TV) 19.4 7.8 Origanum majorona (OM) 20.9 9.1 Origanum vulgare (OV) 16.0 10.0 Origanum onites (OO) 19.8 12.8
* Liquid chromatography with diode array detection coupled with electro-spray ionization tandem mass spectrometry.
vulgare, Melissa officinalis, and Lavandula angustifolia had the overall highest inhibitory activity on GSK-3β. In particular, OO (IC50, 7.94μg/ mL), MP (IC50, 8.84μg/mL), OM (IC50, 12.75μg/mL), TV (IC50, 21.80μg/mL), OV (IC50, 24.20μg/mL), MO (IC50, 27.59μg/mL), and LA (IC50, 40.35μg/mL) all inhibited GSK-3β activity by at least 50% at concentrations of 50μg/mL or lower.
CK-1δ Inhibition. Three of the Origanum species along with Thymus vulgaris and Melissa officinalis moderately inhibited the enzyme, with IC50values between 150μg/mL and 250 μg/mL, while the remaining extracts slightly inhibited the CK-1δ, with IC50above 250μg/mL.
BACE-1 inhibition. None of the tested extracts effectively inhibited BACE-1, with IC50above 200μg/mL. O. majorana and Salvia officinalis slightly inhibited the enzyme, with IC50 values of 220μg/mL and 229μg/mL, respectively.
3.2. Phytochemical analysis
The phenolic constituents of the extracts, which were assigned using LC-DAD-ESI-MS/MS, are summarized in Table 3. The phytochemical analysis showed that the compounds detected in the ten Lamiaceae species are in agreement with the published literature for these species. The major group of compounds detected in the extracts comprised aglycones and glycosides of flavones and caffeic acid derivatives. In particular, apigenin and the luteolin derivatives were the main class of flavonoidal compounds and salvianolic acids were the majority of caf-feic acid derivatives. These compounds were assigned based on their retention times and observed [M−H]− ions, with supportive UV spectra and MS/MS interpretation comparing with the relevant litera-ture. With considering UV chromatograms at 330 nm, apigenin glu-curonide, luteolin gluglu-curonide, and rosmarinic acid were the pre-dominant compounds for O. onites and O. majorana, with eriocitrin, rosmarinic acid, and salvianolic acid E/B isomer for Mentha piperita. 4. Discussion
Amyloid deposits, NFT formation, and neuronal cell loss are the major hallmarks of AD. Since it is understood that overexpression of GSK-3β, BACE-1, and CK-1δ is associated with different neuronal pa-thological conditions related to AD, the search for inhibitors of GSK-3β, BACE-1, and CK-1δ represents a promising approach for prevention and therapy of AD. Based on a retrospective review of the historical role of a number of European herbs in the improvement of cognition and memory, it was shown that culinary herbs, i.e. Salvia sp. and Melissa sp., might potentially provide a natural treatment for AD (Howes et al., 2003;Perry et al., 1999). Moreover, there are various clinical, enzyme inhibitory, i.e. acetylcholine (AchE) and butyrylcholine esterase, and
cell-related neurobiological activity studies on different Lamiaceae plants regarding especially Melissa, Salvia, Rosmarinus, and Lavandula species (Orhan et al., 2008;Perry et al., 1999,2003). However, despite the promising clinical observations and in vitro enzyme inhibitory re-sults, the precise mechanism for these plants remains unknown. This background prompted us to investigate the neurobiological activities of some popular cultivated Lamiaceae plants through the abovementioned enzyme inhibitory properties related to AD.
In a remarkable number of studies it is reported that the neurotoxic effect of Aβ42 is mediated by GSK-3β activation and therefore inhibi-tion of GSK-3β can reduce Aβ42-induced neurotoxicity (Avrahami et al., 2012;Koh et al., 2008;Sofola et al., 2010).
There are few cell-based assays to measure the expression of GSK-3β protein levels in order to understand the neuroprotective effects of Salvia officinalis. In one of those studies S. officinalis inhibited the ex-pression of GSK-3β protein levels and its active metabolite, rosmarinic acid, was shown to possess neuroprotective effects through different mechanisms (Iuvone et al., 2006). Herein all of the extracts tested showed GSK-3β inhibitory activity, including considerably active ex-tracts, i.e. Origanum onites, O. majorana, and Mentha piperita.
Origanum onites displayed the best inhibition towards GSK-3β, with a 7.94 ± 1.51μg/mL IC50value. The LC/DAD/ESI-MS analysis led to the separation and identification of the majority of its constituents. Overall, 14 compounds were identified in the OO extract (Table 2), most of them being common with other Origanum species. According to the UV chromatogram at 330 nm, apigenin O-glucuronide, luteolin 7-O-glucuronide, and rosmarinic acid were the major principles of O. onites and O. majorana. Flavones, i.e. apigenin, luteolin, and related compounds found in nonaromatic fractions, have been reported to have many neuroprotective and anti-inflammatory activities through dif-ferent pathways related to AD (Balez et al., 2016;Rezai-Zadeh et al., 2008). An earlier study indicated that this group of compounds play a role in reducing binding energy to the enzyme and inhibiting GSK-3β, with IC50values of 1.5μM for luteolin, 1.9 μM for apigenin, 2.0 μM for quercetin, and 3.5μM for kaempferol (Johnson et al., 2011). In our study the constituents, including especially theflavones, were not so-lely tested in the GSK-3β enzyme assay, but it is thought that this group of compounds should contribute to the GSK-3β inhibitory activity of the extracts. In the same study, it was also stated that when comparing the nonglycosylated Citrusflavonoids with glycosylated ones conjugation of sugar groups toflavonoid agylcones decreased the inhibitory capacities of the testedflavonoids, with an IC50value for eriocitrin of more than 100 μM. This implied that compounds other than apigenin-7-O-glu-curonide and luteolin-7-O-gluapigenin-7-O-glu-curonide also contribute to the inhibitory effect against GSK-3β.
When looking deeply at the neuroprotective effects of Mentha spe-cies in the literature, it is obvious that there is a lack of activity studies related to AD. Only a few enzyme inhibitory activity studies have been performed to understand the effectiveness of Mentha species. In a recent study, Mentha piperita was shown to preventβ amyloid and aging-in-duced amnesia in mice (Joshi and Bhadania, 2014), but no action of mechanism was suggested for this neuroprotection. In another study, linarin, an apigenin diglycosidic derivative, was shown to possess dose-dependent inhibitory activity towards AChE isolated from Mentha ar-vensis (Oinonen et al., 2006). Inhibitory effects of the essential oil of different Mentha species on AChE were also demonstrated (Miyazawa et al., 1998).
In our study, the nonaromatic fraction of MP showed the second best inhibition, with an IC50value of 8.84 ± 2.04μg/mL towards GSK-3β. When considering the phytochemical data, the major compounds of MP were as follows: eriocitrin, rosmarinic acid, and salvianolic acid E/B isomer. There are numerous studies on the neuroprotective activities of salvianolic acid B, including anti-inflammatory (Wang et al., 2010), Aβ fibril formation inhibition, and fibril destabilizing in vitro (Durairajan et al., 2008;Tang and Zhang, 2001) and in vivo (Lee et al., 2013). In a recent study, it was also demonstrated that the phenolic acids Table 2
Glycogen synthase kinase-3β, β-secretase and casein kinase 1δ inhibitory ac-tivities of the extracts.
Sample IC50*(μg/mL)
GSK-3βb BACE-1b CK-1δb
Sideritis congesta (SC) 73.33 ± 4.71 > 250 > 250 Mentha piperita (MP) 8.84 ± 2.04 > 250 > 250 Lavandula angustifolia (LA) 40.35 ± 2.48 > 250 > 250 Salvia officinalis (SO) 54.97 ± 2.10 229 ± 29 > 250 Dorystoechas hastata (DH) 53.11 ± 1.30 > 250 > 250 Melissa officinalis (MO) 27.59 ± 4.23 > 250 222.21 ± 12.32 Thymus vulgaris (TV) 21.80 ± 2.26 > 250 237.86 ± 5.18 Origanum majorona (OM) 12.75 ± 2.29 220 ± 32 203.69 ± 5.22 Origanum vulgare (OV) 24.20 ± 1.94 > 250 226.44 ± 10.87 Origanum onites (OO) 7.94 ± 1.51 > 250 157.35 ± 10.39
GSK-3β: Glycogen synthase kinase-3β; BACE-1: β-secretase; CK-1δ: casein kinase 1δ inhibitory.
danshensu and salvianolic acid B existing in Salvia miltiorrhiza roots also possess protective properties against Aβ-induced cytotoxicity (Zhou et al., 2011). In the present study, it can be suggested that salvianolic acid E/B isomer strongly contributed to the GSK-3β inhibitory activity of the MP extract.
The inhibitory activity of medicinal plants on CK-1δ, one of the other key enzymes for tau-related pathologies, was not distinctly ex-hibited. In a recent study, caffeic acid, the monomer of salvianolic acids, was reported to decrease tau phosphorylation induced by Aβ (Sul et al., 2009). More recently it was demonstrated that rosmarinic acid prevents fibrillization and diminishes vibrational modes associated with theβ sheet in tau protein linked to AD (Cornejo et al., 2017). To the best of our knowledge, the current study is thefirst on the inhibitory
properties of some medicinal Lamiaceae plants on CK-1δ. Herein all Origanum extracts, Thymus vulgaris, and Melissa officinalis showed moderate inhibitory activities on the enzyme. Based on the UV chro-matogram of Melissa officinalis at 330 nm, rosmarinic acid is the most predominant compound, followed by salvianolic acids. Regarding the CK-1δ inhibitory properties and phytochemical data of the mentioned extracts, it could be suggested that the moderate inhibition in addition to the GSK-3β inhibitory activities of the extracts also contribute to the anti-Alzheimer effects of the abovementioned Origanum species and Melissa officinalis.
According to the literature none of these Lamiaceae plants had been tested for BACE-1 inhibition in vitro as crude methanolic extracts. In a recent study essential oil of Lavandula luisieri showed inhibition to Table 3
Phenolic compounds tentatively identified in the investigated Lamiaceae speciesa.
Tentative identification Rt (min) [M-H]− MS2(m/z) Sample Ref.
6, 8-C-dihexosylapigenin 15.6 593 503, 473, 243, 353 OM, OV (Dias et al., 2013)
5-Caffeoylquinic acid 16.4 353 191, 173 SC (Dias et al., 2013)
Medioresinol 16.7 387 207, 163 LA, TV (Ozarowski et al., 2013)
Quercetin rutinoside 17.2 609 301 DH Std.
Luteolin diglucuronide 17.4 637 461, 311, 285 MP, LA, OV (Hanafy et al., 2017) Roseoside (adduct with formic acid) 17.4 431 385, 223,205, 161, 153 DH (Spínola et al., 2015) Quercetin O-hexoside 18.1 463 301 OO, SO (Martins et al., 2014) Trihydroxycinnamic acid-O-glucoside 18.5 357 195, 151, 136 LA (Farag et al., 2016) Eriocitrin 18.5 595 459, 287, 269, 151, 135 MP (Hanafy et al., 2017) Quercetin glucuronide 18.7 477 301, 283 SO, OO (Spínola et al., 2015) Luteolin rutinoside 18.8 593 447, 327, 285 SO, MP (Hanafy et al., 2017)
Apigenin derivative 18.9 621 351, 269 OV (Hanafy et al., 2017)
Verbascoside 18.9 623 461,179, 161 SC (Petreska et al., 2011)
Isoscutellarein 7-O-[6”'-O-acetyl]-allosyl (1-2) glucoside 19.0 651 609, 531, 447, 285 SC (Petreska et al., 2011)
Caffeic acid 19.1 179 135 SO Std.
Kaempferol rutinoside 19.3 593 285 DH (Spínola et al., 2015)
Isorhamnetin-rutinoside 19.4 623 315, 300 DH (Spínola et al., 2015) Quercetin acetylglucoside 19.7 505 301 SO, TV (Dias et al., 2013) Rosmarinic acid hexoside 19.7 521 359, 323, 179, 161 TV (Vogelsang et al., 2006) 4-[[(2',5'Dihydroxybenzoyl) oxy] methyl] phenyl
O-β-D-glucopyranoside
19.8 421 259, 153 OO (Martins et al., 2014)
Luteolin glucoside 20.1 447 327, 285, 256, 151, 133 SO, OO, TV, DH Std.
Quercetin glucoside 20.2 463 301, 271, 255 DH (Spínola et al., 2015) Salvianolic acid H/I/J 20.3 537 493, 339, 295, 203 MP (Hanafy et al., 2017) Hypolaetin 7-O-[6”'-O-acetyl]-allosyl (1-2) glucoside 20.4 667 625, 301 SC (Petreska et al., 2011) Luteolin 7-O-glucuronide 20.5 461 285, 175, 133 SO, LA, OO, OM, OV, MP,
TV
Std.
Isorhamnetin hexoside 20.7 477 462, 315, 299 DH (Dias et al., 2013)
Luteolin pentoside 21.3 417 285 OO (Desta et al., 2017)
Salvianolic acid E/B isomer 21.4 717 519, 475, 339, 295 MO (Hanafy et al., 2017) Diosmetin rutinoside 21.4 607 299, 284 DH (Mitreski et al., 2014) Sagerinic acid 21.6 719 539, 521, 495, 359, 341, 197,
179, 161
DH (Farag et al., 2016)
Luteolin acetylglucoside 21.8 489 447, 285 SO (Moharram et al., 2006) Apigenin glucoside 21.9 431 311, 269 OV, LA (Dias et al., 2013) 4'-O-Methylisoscutellarein 7-O-[6”'-O-acetyl]-allosyl (1-2)
glucoside
22.2 665 623, 299, 284 SC (Petreska et al., 2011)
Isorhamnetin glucoside 22.3 477 433, 315, 300 SO (Dias et al., 2013) Salvianolic acid E/B isomer 22.3 717 519, 475, 339, 321, 295 MP, OO, OM, OV (Hanafy et al., 2017) Hispidulin/diosmetin glucoside 22.7 461 299, 283,255 DH (Spínola et al., 2015) Apigenin 7-O-glucuronide 22.7 445 269, 175, 113 SO, OO, OM, OV (Hanafy et al., 2017) Kaempferol glucuronide 23.2 475 299, 284, 175, 113 OO (Martins et al., 2014) Rosmarinic acid 23.5 359 197, 179, 161, 135 SO, LA, OO, TV, OM, OV,
MP, MO
Std.
Kaempferol hexoside 24.2 447 285, 137 OO (Martins et al., 2014) Salvianolic acid C 24.7 491 311, 267, 135 MP (Hanafy et al., 2017) Salvianolic acid A 24.8 493 359, 295, 197, 161 MO, TV (Hanafy et al., 2017) Salvianolic acid B derivative 26.7 715 535, 491, 311, 179, 131 MO (Hanafy et al., 2017)
Eriodictiol 27.7 287 151, 135 MP (Martins et al., 2014)
Luteolin 28.0 285 133 MP, OO Std.
Apigenin 31.0 269 225, 149, 117 LA, OO, OM, OV Std.
Rosmanol 31.1 345 301,283 DH (Ozarowski et al., 2013)
Naringenin 31.5 271 151, 119 OO, OM, OV (Martins et al., 2014)
Epirosmanol 31.9 345 301,283 DH (Ozarowski et al., 2013)
Rosmanol isomer 33.1 345 284 DH (Ozarowski et al., 2013)
a SO: Salvia officinalis; LA: Lavandula angustifolia; MP: Mentha piperita; MO: Melissa officinalis; OO: Origanum onites; TV: Thymus vulgaris; OM: Origanum majorona; OV: Origanum vulgare; SC: Sideritis congesta; DH: Dorystoechas hastata.
BACE-1, with an IC50 value of 90μg/mL, and a necrodane mono-terpenoid was identified as the compound responsible from the activity (Videira et al., 2013,2014). In our study nonaromatic extract of LA did not show remarkable BACE-1 inhibition. Only OM and SO slightly in-hibited the BACE-1, with IC50values of 220 ± 32 and 229 ± 29, re-spectively. In some studies Salvia officinalis was shown to protect cells from Aβ peptide-induced neurotoxicity (Iuvone et al., 2006), and S. miltiorrhiza, one of the most important TCM plants used in CNS dis-orders, was also reported to have neuroprotective effects against Aβ-induced neuronal apoptosis via different mechanisms inhibiting oxi-dative stress and attenuating the mitochondria-dependent apoptotic pathway (Yu et al., 2014). Based on the present study, it can be sug-gested that BACE-1 inhibition in addition to the GSK-3β inhibitory properties of SO extract also plays a role in the protection of neurons from Aβ-induced toxicity.
As a consequence, when all the activity and phytochemical data were combined, it is apparent that phenolic compounds including a rich pool offlavones and related compounds together with phenolic acid derivatives play a role in the anti-Alzheimer effects of the extracts tested.
5. Conclusion
To the best of our knowledge, this is thefirst report on the inhibition of GSK-3β, BACE-1, and CK-1δ by the abovementioned Lamiaceae plants evaluated biochemically. To sum up, it is thought that wide-spread consumption of these popular culinary and medicinal plants should protect neurons via different pathways especially via GSK-3β inhibition. However, an important limitation of our study is that the findings are not of physiologic relevance at this time and future studies should be carried considering the blood–brain barrier permeability, bioavailability, and metabolism of the extracts.
Conflict of interest None.
Acknowledgement
The authors are indebted to the Scientific and Technological Research Council of Turkey (BAYG-2219; Grant No: 1059B191501512) forfinancially supporting the project.
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