HPTLC Fingerprinting and Cholinesterase Inhibitory and Metal-Chelating Capacity of Various Citrus Cultivars and Olea europaea

Alzheimer’s disease (AD) is a neurological disorder and the most common cause of dementia. It is a slowly progressive and degenerative disease that leads to loss of memory, decline in thinking and language skills. The pathogenesis of AD has been so far explained by cholinergic and amyloid hypotheses, and it has been stated that only drugs acting on cholinergic system have moderate, but steady eff ects in clinical trials (1). The cholinergic hypothesis is based on insuffi ciency of the acetylcholine (ACh) level in the brains of AD patients, which is hydrolyzed by acetylcholinesterase (AChE, EC 3.1.1.7) (2). On the other hand, the evidence has shown that butyrylcholinesterase (BChE, EC 3.1.1.8), also known as pseudocholinesterase, takes a slight part in hydrolyzing brain ACh levels in addition to its possible action in the etiology and progression of the disease (3). Thus, inhibition of both AChE and BChE is an important target for the development of new anti-Alzheimer drugs. Since AD is multi-faceted disease, it is also strongly associated with metal ion dyshomeostasis and oxidative stress (4). Dysregulation of some transition metals such as iron, aluminum or copper has been very important in formation of oxidative stress and cellular damage relevant to some neurodegenerative diseases including AD and Parkinson’s disease (5,6). Therefore, it is strategically advantageous to have a drug candidate with both cholinesterase inhibition and metal-chelating properties for the treatment of AD as the need for development of novel drugs remains. ISSN 1330-9862 original scientifi c paper


Introduction
Alzheimer's disease (AD) is a neurological disorder and the most common cause of dementia.It is a slowly progressive and degenerative disease that leads to loss of memory, decline in thinking and language skills.The pathogenesis of AD has been so far explained by cholinergic and amyloid hypotheses, and it has been stated that only drugs acting on cholinergic system have moderate, but steady eff ects in clinical trials (1).The cholinergic hypothesis is based on insuffi ciency of the acetylcholine (ACh) level in the brains of AD patients, which is hydrolyzed by acetylcholinesterase (AChE, EC 3.1.1.7)(2).On the other hand, the evidence has shown that butyrylcholinesterase (BChE, EC 3.1.1.8), also known as pseudocholinesterase, takes a slight part in hydrolyzing brain ACh levels in addition to its possible action in the etiology and progression of the disease (3).Thus, inhibition of both AChE and BChE is an important target for the development of new anti-Alzheimer drugs.Since AD is multi-faceted disease, it is also strongly associated with metal ion dyshomeostasis and oxidative stress (4).Dysregulation of some transition metals such as iron, aluminum or copper has been very important in formation of oxidative stress and cellular damage relevant to some neurodegenerative diseases including AD and Parkinson's disease (5,6).Therefore, it is strategically advantageous to have a drug candidate with both cholinesterase inhibition and metal-chelating properties for the treatment of AD as the need for development of novel drugs remains.
Citrus species (Rutaceae) bearing widely consumed fruits of nutritional and industrial importance are rich in fl avonoid derivatives, i.e. hesperidin, naringin and polymethoxylated fl avonoids, whose distributions diff er in Citrus tissues including albedo, fl avedo, pericarp (peel) and seed (7).Earlier studies have revealed that the phenolic compounds exist in higher amounts in Citrus peel than in other parts of Citrus fruit, such as seeds, leaves or fl owers (8).Olea europaea L. (olive) from Oleaceae family is an evergreen tree particularly native to the Mediterranean region.In addition to being edible, olive tree is also considered as a medicinal plant.Due to nutritional and health aspects of Citrus sp. and O. europaea, their edible tissues have been extensively investigated for their various biological activities and phytochemical content.Nevertheless, there have been relatively few reports on health-promoting values of non-edible tissues of Citrus fruits and O. europaea.Hence, in the present study, att empts have been made to investigate inhibitory eff ect of 31 ethanol extracts from 17 Citrus cultivars from Turkey as well as the bark and leaves of olive trees growing in Turkey and Cyprus against AChE and BChE using ELISA microtiter assays.Additionally, high-performance thin layer chromatography (HPTLC) fi ngerprinting of the extracts was done using hesperidin and caff eic acid standards, two phenolic substances commonly found in Citrus species.

Plant materials and extraction
The samples of 17 Citrus cultivars were obtained from The West Mediterranean Agricultural Research Institute (BATEM, Antalya Province, Turkey) in 2011, where they are cultivated in the experimental fi elds.The bark and leaf samples of Olea europaea were collected from Denizli Province (western Turkey) and the leaf samples from Mehmetcik village at Famagusta town (northern Cyprus) in 2012.The pericarps (peels) of the Citrus fruits were peeled and carefully separated into their albedo (whitish) and fl avedo (yellow or orange) parts.All plant samples were dried at room temperature in shadow and coarsely ground prior to the extraction.The dried and powdered samples were extracted at room temperature by percolation with ethanol (80 %; Carlo Erba, Val de Reuil, France).All of the extracts were concentrated using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) in vacuo until a solid extract of each sample was obtained.

Microtitre assays for anticholinesterase activity
AChE and BChE inhibitory potential of the extracts was determined by modifi ed spectrophotometric method of Ellman et al. (9) as described in our previous publication (10).Electric eel acetylcholinesterase (TypeVI-S, EC 3.1.1.7)and horse serum butyrylcholinesterase (EC 3.1.1.8)were used as the enzyme sources purchased from Sigma--Aldrich (St. Louis, MO, USA).Acetylthiocholine iodide and butyrylthiocholine chloride (Sigma-Aldrich) were employed as the substrates for the reaction, while 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB; Sigma-Aldrich) was the colouring agent.A volume of 140 μL of 0.1 mM sodium phosphate buff er (pH=8.0),20 μL of 0.2 M DTNB, 20 μL of the sample solutions and 20 μL of 0.2 M AChE/ BChE solution were added with multichannel automatic pipett e (Gilson S.A.S., Villiers le Bel, France) to a 96-well microplate and incubated for 15 min at 25 °C.The reaction was then initiated with the addition of 10 μL of 0.2 M acetylthiocholine iodide/butyrylthiocholine chloride.Formation of the yellow 5-thio-2-nitrobenzoate anion resulted from the reaction of DTNB with thiocholines aft er hydrolysis of acetylthiocholine iodide/butyrylthiocholine chloride, which was monitored at 412 nm utilizing a 96--well microplate reader VersaMax™ (Molecular Devices, Sunnyvale, CA, USA).Galanthamine, purchased from Sigma-Aldrich, was employed as the reference.

Data processing for enzyme inhibition assays
The measurements and calculations were evaluated by using Soft max ® PRO v. 4.3.2.LS soft ware (Molecular Devices).Percentage inhibition (I) of AChE/BChE was determined by comparison of reaction rates of test samples with the blank sample (ethanol in phosphate buff er, pH=8), and calculated using the equation given below: where A blank is the absorbance of the control reaction (containing all reagents except the test sample), and A sample is the absorbance of the extracts.Data was expressed as average inhibition±standard error of the mean (SEM), which were obtained from three independent experiments.

Determination of metal-chelating capacity by Fe 2+ -ferrozine test system
The metal-chelating capacity of the extracts was estimated with the method of Chua et al. (11) using Fe 2+ -ferrozine test system followed by ELISA method.In brief, the samples (200 μL each) dissolved in ethanol (75 %) were incubated with 2 mM FeCl 2 solution (Sigma Chemical Co., Steinheim, Germany).The reaction was started aft er the addition of 40 μL of 5 mM ferrozine (Sigma Chemical Co.) solution, which was shaken gently and left to rest for 10 min at ambient temperature.The absorbance of the reaction mixture as well as ethylenediaminetetraacetic acid (EDTA; Sigma Chemical Co.) as the reference was measured at 562 nm using a Unico 4802 UV/Vis spectrophotometer (Dayton, NJ, USA).Metal chelation capacity was measured as a percentage inhibition of Fe 2+ -ferrozine complex calculated using Eq. 1.The experiments were run in triplicate and the results were expressed as average values with SEM.

HPTLC analysis
HPTLC analysis was performed on a CAMAG (Muttenz, Switzerland) apparatus equipped with automatic TLC sampler 4, twin trough chamber (20 cm×10 cm), chromatogram immersion device III, TLC plate heater III, automatic development chamber ADC2 and visualizer.The extracts and standards, i.e. caff eic acid (Carl Roth GmbH, Zurich, Switzerland) and hesperidin (Acros Organics, Basel, Switzerland) at the concentrations of 5 and 0.2 mg/mL, respectively, were dissolved in methanol of ultra gradient HPLC grade (Carl Roth GmbH).The volume of each sample was 10 or 15 μL.Development conditions were as fol-lows: relative humidity RH=33 %, solution saturation 20 min, developing distance from application position/lower edge of the plate of 62/70 mm, developing solvent ethyl acetate (Acros Organics, Basel, Switzerland)/formic acid (Acros Organics)/water (CAMAG) in a ratio of 75:15:10, developing time 20 min, and plate drying time 5 min.The plates used were HPTLC glass Si 60 F254 (20 cm×10 cm, model HX308464; Merck Co., Darmstadt, Germany), while visualization of the spots was achieved with AlCl 3 and natural product polyethylene glycol (PEG) 400 (Merck Co.).

Cholinesterase inhibitory and metal chelation activities of extracts
The results of enzyme inhibition tests show that Citrus extracts at the concentration of 500 μg/mL did not affect AChE activity, while Olea europaea leaf extracts slightly inhibited (below 16 %) both enzymes (Table 1).On the other hand, quite variable inhibition rates of Citrus extracts (between (7.72±0.71)and (70.28±1.12)%) were observed against BChE.Caff eic acid and hesperidin, tested in the same manner but at the concentration of 100 μg/mL, had (6.0±2.5) and (27.3±1.2) % of AChE inhibition as well as (24.1±0.9) and (17.7±4.2) % of BChE inhibition, respectively.The extracts had either no or very low (below 13 %) metal-chelating capacity (Table 1), while caff eic acid and hesperidin were not tested in this assay.

HPTLC profi le of the extracts
According to the HPTLC profi les of the extracts screened in the current study, the fl avedo extracts had more intense zones at 366 nm than the albedo extracts.Looking at the results of white light refl ectance transmittance, it can be observed that the yellow zones of the albedo samples are more intense than the corresponding yellow zones of the fl avedo samples.Our fi ndings indicate that it is possible to discriminate Citrus aurantium, C. deliciosa, C. limon, C. maxima/C.paradisi and C. sinensis/C.reti culata.The fi ngerprints of C. maxima and C. paradisi look very similar, thus, these two species cannot be discriminated.This is also the case for C. sinensis and C. reticulata.As seen in Fig. 1, the fi ngerprints of the albedo and fl avedo extracts of C. aurantium look very similar to each other under white light, with a strong red zone at retention factor (R F ) of approx.0.36 and several yellow zones above it.All extracts belonging to C. limon species had a similar fi ngerprint with a characteristic red zone at R F of approx.0.39 under white light.The fi ngerprints of the fl avedo and albedo samples of C. maxima each had a yellow zone with a higher R F value than hesperidin.A parallel fi ngerprint was observed for all extracts of C. paradisi cultivars that contained hesperidin, while the extracts obtained from C. reticulata and C. sinensis 'Navelina' resemble each other (Fig. 2).Among the analyzed extracts in Fig. 3, all the C. sinensis cultivars had two yellow zones, one of them with the same R F value as hesperidin.The leaf extracts of O. europaea collected from the two locations exhibited quite analogous phytochemical profi les, whereas the bark extract of this plant had a diff erent fi ngerprint than those of the leaf extracts.
On the other hand, the leaves of Citrus cultivars have been examined in a very few studies, one of which was performed with the leaf essential oil of C. aurantifolia.The leaf oil was revealed to have IC 50 values of (139±35) and (42±5) μg/mL on AChE and BChE, respectively (19), while Loizzo et al. (20) investigated the leaf hexane extract of C. aurantifolia with a marked AChE inhibitory eff ect.In the present study, the leaf ethanol extract of C. aurantium inhibited (46.37±0.54)% of BChE.
The computational studies described stronger AChE inhibitory eff ect of naringin than hesperidin (24), which inhibited (27.33±1.16)% of AChE in the current study.Without any doubt, hesperidin, present in most of the Citrus extracts screened in the HPTLC analysis, could be one of the compounds contributing somewhat to BChE inhibitory eff ects of the extracts.Caff eic acid, a dominant phenolic compound available in many Citrus cultivars (25,26), was reported earlier to exhibit either low inhibitory activity ((11.05±1.03)% at 100 μg/mL) against AChE (27) and IC 50 ≥200 μM (28) or no inhibition (29), which is consistent with our result ((6.01±2.50)% at 100 μg/mL).
According to our literature survey, AChE inhibitory eff ects of the leaf extracts of the stored and fresh samples of O. europaea were (69.2±6.0) and (85.4±3.4) % at 1 μg/mL, respectively (30).Nevertheless, the leaf and bark extracts from O. europaea used in this study had insignifi cant eff ect on both AChE and BChE as compared to that of the reference (galanthamine).
Although a few studies demonstrated metal-chelating capacity of Citrus cultivars at various levels (31,32), the Citrus extracts studied herein exerted either very low

Conclusions
The present study demonstrated that, among the seventeen cultivars of Citrus and the leaf and bark samples of Olea europaea, the maximum BChE inhibition was caused by the fl avedo extract of C. limon 'Cyprus', followed closely by the seed extract of C. maxima.Our results indicated that the albedo and fl avedo extracts showed diverse levels of BChE inhibition, which might mainly depend on the diff erence in their phytochemical content.The screened Citrus extracts had a selective BChE inhibitory and no AChE inhibitory activity at all.The extracts had either no or low metal-chelating capacity.To the best of our knowledge, this is the fi rst study disclosing cholinesterase inhibitory and metal-chelating activities of the Citrus species cultivated in Turkey as well as of O. europaea growing in Turkey and Cyprus, and also the fi rst HPTLC fi ngerprinting of these plants.Overall, the aforementioned Citrus extracts could be considered as BChE inhibitors rather than AChE inhibitors.

Table 1 .
Cholinesterase inhibitory and metal-chelating activities of ethanol extracts of Citrus sp. and Olea europaea at mass concentration of 500 μg/mL (unless otherwise stated) Results are expressed as mean value±standard error of the mean (N=3), b no inhibition/activity, c tested at 100 μg/mL, d not tested, e reference for AChE and BChE inhibition assays at 100 μg/mL, f reference for metal-chelating capacity assay at 100 μg/mL.
a *