Skip to main content

International Journal of Phytomedicine and Phytotherapy

Neurobiological evaluation of thirty-one medicinal plant extracts using microtiter enzyme assays



Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) related to Alzheimer’s disease as well as tyrosinase (TYR) relevant to Parkinson’s disease is an important approach to find novel drug candidates for these diseases.


The extracts from fourteen plant species in various polarities were subjected to high-throughput screening against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and tyrosinase (TYR), the key enzymes related to Alzheimer’s and Parkinson’s diseases. The extracts were subjected to the microtiter enzyme inhibition assays at 100 μg mL-1. Antioxidant effect of the extracts was tested for their scavenging activity against DPPH, DMPD, and NO radicals as well as their ferric- (FRAP) and phosphomolibdenum-reducing power (PRAP) and metal-chelation capacity. Total phenol and flavonoid quantities in the extracts were determined spectrophotometrically. HPLC analysis was performed on Atriplex lasiantha, Conringia grandiflora, and Vaccaria hispanica.


The active extracts inhibiting AChE over 50 % were Centaurium erythraea subsp. rhodense (51.33 ± 3.35 %) and Posidonia oceanica (61.88 ± 2.23 %), while BChE was inhibited most effectively by the root extract of P. oceanica (82.55 ± 2.14 %), followed by Origanum haussknechtii (66.88 ± 0.17 %), which also had the highest inhibition toward TYR (35.28 ± 1.90 %). The extracts from Zostera noltii, P. oceanica, and Ricotia carnosula possessed the best DPPH scavenging activity, whereas Z. noltii caused the highest NO scavenging activity (70.19 ± 0.43 %) and FRAP (1.326 ± 0.065). Atriplex lasiantha and Ecballium elaterium had the strongest effect in PRAP and metal-chelation assays, respectively. Besides, A. lasiantha was found to be a rich source of rutin.


Among the screened plants, Centaurium erythraea subsp. rhodense and Origanum haussknechtii, and the roots of Posidonia oceanica seems to deserve further investigation for their neuroprotective potential.


Alzheimer’s disease (AD) is a progressive neurodegenerative disorder affecting senior individuals and their families. The evidence has proven that pathophysiology of the disease is quite complex with a multifactorial nature including inflammation, oxidative stress, abnormal protein formation, neurotransmitter deficits [1]. Low levels of acetylcholine (ACh), vital for cerebral functions related to memory, have been found in the brains of AD patients, while butyrylcholinesterase (BChE, EC, known as pseudocholinesterase or acylcholine acylhydrolase, may also hydrolyze most of the choline esters including Ach, which is mainly hydrolyzed by acetylcholinesterase (AChE, EC Thus, inhibition of cholinesterases has been an attractive target for treatment of AD [2] and cholinesterase inhibitors have become the extensively used therapeutic tools towards AD.

On the other hand, tyrosinase (TYR; polyphenol oxidase or oxygen oxidoreductase, EC catalyzes the rate-limiting oxidation of tyrosine to melanin, which plays a critical role in pigmentation of skin related to melanoma, undesirable browning of fruits and vegetables, molting process of insects, and dopamine toxicity in Parkinson’s disease (PD) as well as neuronal death in PD and Huntington’s disease [3, 4]. Therefore, inhibition of TYR is a new target and quite important for treatment of the aforementioned diseases.

As well-known, screening of medicinal plants and natural products for their pharmacological activities desired for human health is an essential step for discovery of novel drug candidates. Since our extensive studies on discovery of new cholinesterase and TYR inhibitors from herbal sources have been conducted since the year of 2000, the current stage, we have aimed to screen randomly selected fourteen plant species [Atriplex lasiantha Boiss. (AL), Ecballium elaterium (L.) A. Rich. (EE), Centaurium erythraea subsp. rhodense (Boiss & Reut.) Melderis (CER), Centaurium erythraea subsp. turcicum (Velen.) Melderis (CET), Centaurium maritimum (L.) Fritsch (CM), Centaurium spicatum L., Centaurium tenuiflorum (Hoffmans. & Link) Fritsch, Ricotia carnosula Boiss. & Heldr. (RC), Conringia grandiflora Boiss. & Heldr. (CG), Vaccaria hispanica (Mill.) Rauschert (VH), Origanum haussknechtii Boiss. (OH), Zostera noltii Hornem. (ZN), Zostera marina L. (ZM), and Posidonia oceanica (L.) Delile (PO) with medicinal and industrial importance for their AChE, BChE, and TYR inhibitory activities by ELISA microtiter assays at 100 μg mL-1. Among the plant species screened, Atriplex species in Bulgaria [5], Ecballium elaterium (squirting cucumber) in Italy [6], and Centaurium species [7] in southern Italy are consumed as food, while Ricotia carnosula and Conringia grandiflora, the endemic species to Anatolia, along with Vaccaria hispanica (cow cockle) have been recently cultivated to be used as ingredients in nutraceutical industry in our country and these three species have also potential to be used for ornamental purposes. The marine seagrass (or Neptun grass), Posidonia oceanica, is an endemic species to the Mediterraneaen Sea, which forms wide meadows underwater, while Zostera species (eelgrass) forms dense sea grass beds. All these three marine species are considered to be ecologically imperative species making critical habitat and hunting zones for plentiful fish and invertebrates.

Since oxidative stress, free radical formation, and metal accumulation are strongly associated with pathogenesis of neurodegenerative diseases including AD and PD [8], the extracts obtained from the plants mentioned above were subjected to several antioxidant assays adapted to high-throughput screening methods using microtiter plates.


Plant materials

The plant species screened in the present study were collected throughout Turkey between 2008-2012 as listed in Table 1. Among the species used herein, the voucher specimens of the Centaurium species were kept at Herbarium of Faculty of Pharmacy, Ankara University (Ankara, Turkey), while Ecballium elaterium, Atriplex lasiantha, and Origanum haussknechtii were deposited at Herbarium of Faculty of Pharmacy, Gazi University (Ankara, Turkey). The voucher specimens of Ricotia carnosula, Conringia grandiflora, and Vaccaria hispanica were preserved at the Herbarium of Akdeniz University (Antalya, Turkey), while the samples of the segrass species; Zostera noltii, Zostera marina, and Posidonia oceanica were deposited at the Herbarium of Faculty of Pharmacy, Ege University (Izmir, Turkey). All voucher specimens are available upon request for those who would like to see them.

Table 1 Families, collection places, years, and botanical authenticator of the plant species

Extraction procedure

The corresponding parts of the plants screened were dried in shade at room temperature and powdered to a fine grade by using a laboratory scale mill. The corresponding solvents for each species are given in Table 2 [dichloromethane (DCM), ethyl acetate (EtOAc), ethanol (80 %) (EtOH), and methanol (MeOH)]. Each plant species was extracted with its corresponding solvents by maceration, and following filtration, the combined extracts were evaporated to dryness in vacuo to give the crude extracts. Preparation of the aqueous extract of OH was as follows: 5 g of the powdered plant material were boiled with 100 mL of distilled water for 30 min. The aqueous extract was filtered when hot, and, then the resultant extract was lyophilized.

Table 2 AChE, BChE, and TYR inhibitory effects of the extracts at 100 μg mL-1

Microtiter assays for enzyme inhibition

AChE and BChE inhibitory activity assays

AChE and BChE inhibitory activity of the samples was determined by modified spectrophotometric method of Ellman et al. [9]. Electric eel acetylcholinesterase (Type-VI-S, EC, Sigma) and horse serum butyrylcholinesterase (EC, Sigma) were used as the enzyme sources, while acetylthiocholine iodide and butyrylthiocholine chloride (Sigma, St. Louis, MO, USA) were employed as substrates of the reaction. 5,5'-Dithio-bis(2-nitrobenzoic)acid (DTNB, Sigma, St. Louis, MO, USA) was used for the measurement of the cholinesterase activity. All the other reagents and conditions were the same as described in our previous publication [10]. In brief, 140 μL of 0.1 mM sodium phosphate buffer (pH 8.0), 20 μL of 0.2 M DTNB, 20 μL of sample solutions and 20 μL of 0.2 M acetylcholinesterase/butyrylcholinesterase solution were added by multichannel automatic pipette (Gilson pipetman, France) in 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. The hydrolysis of acetylthiocholine iodide/butyrylthiocholine chloride was monitored by the formation of the yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with thiocholines, catalyzed by enzymes at a wavelength of 412 nm utilizing a 96-well microplate reader (VersaMax, Molecular Devices, USA). Galanthamine, the anticholinesterase alkaloid-type of drug isolated from the bulbs of snowdrop (Galanthus sp.), was purchased from Sigma (St. Louis, MO, USA) and was employed as reference.

TYR inhibitory activity assay

Inhibition of TYR (EC, 30 U, mushroom tyrosinase, Sigma) was determined using the modified dopachrome method with L-DOPA as substrate [11]. Assays were conducted in a 96-well microplate using ELISA microplate reader (VersaMax Molecular Devices, USA) and absorbance was measured at 475 nm. An aliquot of the samples dissolved in DMSO with 80 μL of phosphate buffer (pH 6.8), 40 μL of tyrosinase, and 40 μL of L-DOPA was put in each well. Results were compared with control (DMSO) and α-kojic acid (Sigma, St. Louis, MO, USA) employed as the reference for this assay.

Data processing for enzyme inhibition assays

The measurements and calculations were evaluated by using Softmax PRO 4.3.2.LS software. Percentage inhibition of AChE/BChE and TYR was determined by comparison of rates of reaction of test samples relative to blank sample (ethanol in phosphate buffer pH = 8 for AChE/BChE and DMSO for TYR). Extent of the enzymatic reaction was calculated based on the following equation: E = (C-T)/C × 100, where E is the activity of the enzyme. E value expresses the effect of the test sample or the positive control on the enzyme activity articulated as the percentage of the remaining activity in the presence of test sample or positive control. C value is the absorbance of the control solvent (blank) in the presence of enzyme, where T is the absorbance of the tested sample (plant extract or positive control in the solvent) in the presence of enzyme.

Data are expressed as average inhibition ± standard error mean (S.E.M.) and the results were taken from at least three independent experiments performed in triplicate.

Antioxidant activity assays

DPPH radical scavenging assay

The hydrogen atom or electron donation capacity of the corresponding samples was computed from the bleaching property of the purple-colored methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH). The stable DPPH radical scavenging activity of the extracts was determined by the method of Blois [12]. The samples (2700 μL) dissolved in ethanol (75 %) were mixed with 300 μL of DPPH solution (1.5 × 10-4 M). Remaining DPPH amount was measured at 520 nm using a Unico 4802 UV-visible double beam spectrophotometer (Dayton, NJ, USA). The results were compared to that of gallic acid employed as the reference.

DMPD radical scavenging assay

Principal of the assay is based on reduction of the purple-colored radical DMPD+ (N,N-dimethyl-p-phenylendiamine) [13]. According to the method, a reagent comprising of 100 mM DMPD, 0.1 M acetate buffer (pH = 5.25), and 0.05 M ferric chloride solution, which led to formation of DMPD radical, was freshly prepared and the reagent was equilibrated to an absorbance of 0.900 ± 0.100 at 505 nm. Then, the reagent (1000 μL) was mixed up with 50 μL of the sample dilutions dissolved in ethanol (75 %) and absorbance was taken at 505 nm using a Unico 4802 UV-visible double beam spectrophotometer (USA). Quercetin was employed as the reference and the experiments were done in triplicate.

Nitric oxide (NO) radical scavenging activity

The scavenging activity of the samples against NO was assessed by the method of Marcocci et al. [14]. Briefly, the extract dilutions were mixed with 5 mM sodium nitroprusside and left to incubation for 2 h at 29 °C. An aliquot of the solution was removed and diluted with Griess reagent (1 % sulfanilamide in 5 % H3PO4 and 0.1 % naphthylethylenediamine dihydrochloride). Absorbance of the occurred chromophore was measured at 550 nm using a Unico 4802 UV-visible double beam spectrophotometer (USA).

Fe+2-ferrozine test system for metal-chelation

The metal-chelating effect of the samples by Fe+2-ferrozine test system was estimated in consistent with Chua et al.’s method [15]. Accordingly, 740 μL of ethanol and 200 μL of the samples dissolved in ethanol (75 %) were incubated with 2 mM FeCl2 solution. The reaction was initiated by the addition of 40 μL of 5 mM ferrozine solution into the mixture, shaken vigorously, and left standing at ambient temperature for 10 min. The absorbance of the reaction mixture was measured at 562 nm. The ratio of inhibition of ferrozine-Fe2+ complex formation was calculated as given in “Data processing for antioxidant activity assays” and ethylenediaminetetraacetic acid (EDTA) was employed as the reference in this assay.

Ferric-reducing antioxidant power (FRAP) assay

The ferric-reducing antioxidant power (FRAP) of the extracts and reference was tested using the assay of Oyaizu [16] based on the chemical reaction of Fe(III) = > Fe(II). Different concentrations of the extracts dissolved in ethanol (75 %) were added into 2500 μL of phosphate buffer (pH 6.6) and 2500 μL of potassium ferricyanide [K3Fe(CN)6] (1 %, w/v). Later, the mixture was incubated at 50 °C for 20 min and then 2500 μL of trichloroacetic acid (10 %) was added. After the mixture was shaken vigorously, this solution was mixed with 2500 μL of distilled water and FeCl3 (100 μL, 0.1 %, w/v). After 30 min incubation, absorbance was read at 700 nm using a Unico 4802 UV-visible double beam spectrophotometer (Dayton, NJ, USA). Analyses were achieved in triplicate. Chlorogenic acid was the reference in this assay.

Phosphomolibdenum-reducing antioxidant power (PRAP) assay

In order to perform PRAP assays on the extracts, each dilution of the samples was mixed with 10 % phosphomolybdic acid solution in ethanol (w/v) [17]. The solution was subsequently subjected to incubation at 80 °C for 30 min and the absorbance was read at 600 nm using a Unico 4802 UV-visible double beam spectrophotometer (USA) and compared to that of quercetin as the reference.

Data processing for antioxidant activity assays

Inhibition of DPPH, DMPD, and nitric oxide (NO) radicals and metal-chelation capacity was calculated as given below and the results were expressed as percent inhibition (I%):

I% = [(Ablank -Asample)/Ablank] × 100, where Ablank is the absorbance of the control reaction (containing all reagents except the test sample), and Asample is the absorbance of the extracts. Analyses were run in triplicate and the results were expressed as average values with S.E.M. (Standard error of the mean). For FRAP and PRAP assays, the analyses were also achieved in triplicate and increased absorbance of the reaction meant increased reducing power in both assays.

Statistical analysis of data

Data obtained from in vitro enzyme inhibition and antioxidant experiments were expressed as the mean standard error (±SEM). Statistical differences between the reference and the sample groups were evaluated by ANOVA (one way). Dunnett’s multiple comparison tests were used as post hoc tests. p < 0.05 was considered to be significant [*p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001].

Determination of total phenol and flavonoid contents in the extracts

Phenolic amount of the extracts was determined in accordance with Folin-Ciocalteau’s method [18]. In brief, a number of dilutions of gallic acid dissolved in ethanol (75 %) were obtained to prepare a calibration curve. The extracts and gallic acid dilutions were mixed with 750 μL of Folin-Ciocalteau’s reagent and 600 μL of sodium carbonate in test tubes. The tubes were then vortexed and incubated at 40 °C for 30 min. afterward, absorption was measured at 760 nm at a Unico 4802 UV-visible double beam spectrophotometer (USA). Total flavonoid content of the extracts was calculated by aluminum chloride colorimetric method [19]. To sum up, a number of dilutions of quercetin dissolved in ethanol (75 %) were obtained to prepare a calibration curve. Then, the extracts and quercetin dilutions were mixed with 95 % ethanol, aluminum chloride reagent, 100 μL of sodium acetate as well as distilled water. Following incubation for 30 min at room temperature, absorbance of the reaction mixtures was measured at wavelength of 415 nm with a Unico 4802 UV-visible double beam spectrophotometer (USA). The total phenol and flavonoid contents of the extracts were expressed as gallic acid and quercetin equivalents (mg g-1 extract), respectively.

Quantification of rutin in the extracts of AL, CG, and VH by HPLC

Analyses were performed using an Agilent Technologies 1200 Series high pressure liquid chromatography (HPLC), including a binary pump, a vacuum degasser, an autosampler, and a diode array detector. Chromatographic separations were performed on ACE-5-C18 column (150 mm × 4.6 mm, 5 μm). A mobile phase consisting of two eluents (acetonitrile and 40 mM formic acid) was used for separation with a gradient elution at a flow rate of 1 mL min-1. All solvents were filtered through a 0.45 μm Millipore filter before use. Detection wavelength was set at 254 nm for rutin and 330 nm for vitexin. The injection volume was 20 μL for each sample and standard solutions. Identification of rutin and vitexin was made by comparing their retention times and UV spectra of the peaks of pure standards. Standard solution was then added to the samples; increase in the intensity of the peaks verified the identification. All the calculations concerning the quantitative analyses were performed with external standardization by measurement of peak areas. Each injection was achieved in triplicate to see the reproducibility of the detector response at each concentration level.

Results and discussion

Enzyme inhibitory effect of the extracts

The inhibitory effect of thirty-one extracts obtained from fourteen plant species listed in Table 1 was screened against AChE, BChE, and TYR at 100 μg mL-1. Among them, only two extracts, which are the EtOH extract of the aerial parts of CER (51.33 ± 3.35 %) and the root EtOH extract of PO (61.88 ± 2.23 %), exhibited the highest inhibition against AChE (Table 2). The root EtOH extract of PO (82.55 ± 2.14 %) was also the most effective one in BChE inhibition, followed by the MeOH extract of OH (66.88 ± 0.17 %) that inhibited TYR in the highest rate (35.28 ± 1.90 %), which can be considered as moderate (Table 2). Rest of the extracts was found to be either ineffective or with low inhibition below 50 %.

Antioxidant effect of the extracts

Radical scavenging activity of the extracts was tested against three radicals i.e. DPPH, DMPD, and NO at 1000 μg mL-1 (Table 3). The EtOH extracts of the wild sample of RC (84.99 ± 1.09 %), ZN (84.96 ± 0.35 %) as well as the roots of PO (82.77 ± 0.77 %) showed the highest scavenging activity towards DPPH radical (Table 3), whereas the extracts displayed from none to very low scavenging effect (up to 15.18 ± 3.07 %) against DMPD radical. On the other hand, the EtOH extract of ZN (70.19 ± 0.43 %) possessed higher activity than other extracts. According to our findings, this extract (1.326 ± 0.065) exerted the best FRAP, followed by the EtOH extract of the wild sample of RC (1.036 ± 0.061), while most of the extracts showed a mild level of activity in PRAP assay (Table 4). The fruit juice of EE was found to have the highest metal-chelation capacity (55.86 ± 4.11 %).

Table 3 Radical scavenging activity of the extracts against DPPH, DMPD, and NO at 1000 μg mL-1
Table 4 FRAP, PRAP, and metal-chelation activities (± S.E.M.a) of the extracts at 100 μg mL-1

Total phenol and flavonoid contents and HPLC analyses of the extracts

The amount of total phenol and flavonoid contents ranged from none to 79.67 ± 2.09 for total phenols as expressed in mg equivalent of gallic acid to 1 g of extract, whilst total flavonoid amount was found to differ from none to 83.63 ± 2.38 as expressed in mg equivalent of quercetin to 1 g of extract (Table 5). As stated by our results, the most abundant total phenol and flavonoid amounts were determined in the EtOH extract of ZN and the EtOH extract of the wild sample of RC.

Table 5 Total phenol and flavonoid amounts (± S.E.M.) of the extracts

Our HPLC analysis conducted on the AL, CG, and VH extracts, whose phytochemical contents have been analyzed for the first time herein, indicated that the EtOH extracts of the fruits and aerial parts of the plant contained 0.9522 ± 0.1731 and 1.1094 ± 0.0384 mg g-1 of rutin (Fig. 1), whereas DCM and EtOAc extracts of AL did not contain rutin at all (Table 6). On the other hand, CG was found to possess 0.26 % of rutin, while VH had 0.71 % of vitexin. The other flavonoid (quercitrin, luteolin, luteolin-7-glucoside, apigenin, quercetin, and kaempferol) along with some phenolic acid derivatives including rosmarinic acid, gallic acid, and p-coumaric acid were also searched in all extracts of AL by HPLC, nevertheless, none of these compounds were found in these plant species.

Fig. 1

Representative HPLC chromatograms of the EtOH extract of the aerial parts of AL (a) and rutin (b)

Table 6 Rutin and vitexin percentages (w/w) in the ethanol extracts in AL, CG, and VH by HPLC

Screening of plant extracts for their enzyme inhibitory effect is a quite popular pharmacological tool as it gives a preliminary idea about efficiency of a plant towards the targeted disease. On this purpose, we have obtained some promising results out of our screening with thirty-one extracts from fourteen plants species selected for the present study. Besides, our literature survey revealed that none of the plants screened herein has been reported with any of these enzyme inhibitory activities up to date. The genus Atriplex, commonly known as saltbush or orache, consists of mostly halophytic species with edible property such as AL. To our knowledge, cholinesterase or TYR inhibitory activity of AL has not been studied so far, whereas another species, Atriplex halimus L., was demonstrated to have a significant AChE inhibition by Benamar et al. [20]. Particularly, its chloroform extract had 74.60 ± 1.45 % of inhibition at 125 μg mL-1, while the crude extract of the plant exhibited 66.03 ± 1.10 % of inhibition at 250 μg mL-1 which was shown to contain a very low amount of flavonoids consistent with our data. Therefore, the authors stated that some other type of compounds rather than flavonoids may produce the strong AChE-inhibitory effect of Atriplex halimus. Consistently, we have found by HPLC analysis that AL lacks of flavonoids except for rutin, which was actually shown to be inactive in AChE/BChE inhibition assays in our earlier publication [21]. Thus, one can make comment that none to very low inhibitory effect of the AL extracts in the enzyme inhibition assays are likely associated with the absence of flavonoid derivatives such as quercetin (Table 2). EE, a medicinal species also called squirting cucumber or exploding cucumber, has been suggested to be used against memory deficiency amongst the local people in Turkey (personal communication). Despite of this information, our experiments revealed that neither the fruit juice, nor the EtOH extracts from different parts of EE, was capable of inhibiting any of these enzymes in a good level (Table 2). This may lead such a comment that EE should be further tested by other mechanisms related to neurodegeneration.

Among the Centaurium species, especially Centaurium erythraea Rafin., known as small centaury, has been reported to be used for antiflatulent, digestive, gastritis, antidiabetic, and stimulant purposes in Serbia [22] and for treatment of stomach ache and ulcer in Turkish folk medicine [23]. The five species studied herein displayed insignificant enzyme inhibition as well as low to moderate radical scavenging and antioxidant effect. In fact, abundant amount of xanthones and secoiridoids were identified in many Centaurium species, which seem to cause little antioxidant activity [24]. Therefore, stumpy level of antioxidant activity of the Centaurium species studied herein might be explained by this fact. Conversely, it has been known that higher total phenol content in plant extracts usually causes higher antioxidant activity [25], as described in several Centaurium species subjected to some antioxidant assays such as DPPH, hydroxyl, and superoxide radical scavenging and xanthine oxidase inhibitory activity [26]. Hence, this is presumably not the case in our samples of Centaurium since they have low to moderate amount of total phenols and flavonoids.

Up to date, no phytochemical or bioactivity data has been reported on any Ricotia species and CG. In our study, which is the first one on Ricotia sp., the EtOH extracts of the wild and cultivated samples of RC did not exert inhibitory action against the enzymes tested in the present work, whereas the wild sample of the plant was found to exhibit remarkable DPPH radical scavenging activity and FRAP as compared to its cultivated counterpart (Tables 3 and 4). This is an interesting to note that the wild sample of RC may possibly contain more antioxidant substances than that of the cultivated one probably due to some agricultural factors including soil type, rain amount, and other climatic and ecological factors. On the other hand, the only phytochemical study carried out was on the essential oil composition of OH which was shown to contain p-cymene (15.56 %) and borneol (14.24 %) in major quantities [27]. In our study, it was the most active to inhibit BChE and TYR, while several other Origanum species have been demonstrated to display inhibitory property on cholinesterases to some extent such as Origanum syriacum, Origanum ehrenbergii [28, 29], Origanum vulgare var. glandulossum [30], and Origanum majorana [31, 32]. In connection with this data, ursolic acid isolated from Origanum majorana was stated to inhibit AChE in competitive/noncompetitive manner [31]. In our previous study [33], we identified selective BChE-inhibiting effect of the essential oils of Origanum onites, Origanum vulgare, Origanum munitiflorum, and Origanum majorana, which is in accordance with our current data on OH. In fact, Origanum species have been demonstrated to have a rich phenolic content, especially rosmarinic acid as the major constituent [34], which we earlier pointed out to its marked anti-BChE activity [35]. Therefore, it could be speculated that the BChE-inhibiting effect of OH might be related to its rosmarinic acid content, while its antioxidant effects seems to be concomitant with the phenolics found in this plant.

Considering the sea grasses studied, the root EtOH extract of PO exerted notable inhibition towards AChE and BChE (Table 2). In our former study [36], the leaf EtOH extract of PO was found to be ineffective against AChE, which is consistent with our present findings. Although the rich phenolic compounds have been shown to be present in the leaves of the plant such as ferulic acid, phloridzin, phloroglucinol, p-anisic acid, acetosyringone, sinapic acid, phenol, p-hydroxybenzoic acid, p-coumaric acid, and cinnamic acid, 4-hydroxybenzoic acid, 4-coumaric acid, trans-cinnamic acid, and caffeic acid, chicoric acid, vanillin, and gentisic acid [3739], a very little investigation was performed on the roots of this marine plant from the view point of phytochemistry reporting only presence of phenylmethane derivaties [40], which may have some influence on high cholinesterase-inhibiting effect of PO. On the other hand, the copious phenolic content in Zostera species such as flavones and sulfated phenols was revealed by McMillan et al. [41], while ferulic, vanillic, p-hydroxybenzoic, caffeic, gallic, protocatechuic, and gentisic acids were identified in ZM [41] and rosmarinic acid in ZN [42, 43]. Clearly, the plentiful phenolic contents in two Zostera species screened herein prospectively seem to contribute to their antioxidant capacity, which is in accordance with their total phenol and flavonoid amounts determined in our current study.


In summary, our findings obtained from screening of various extracts from fourteen plant species reveal that Centaurium erythraea subsp. rhodense and Origanum haussknechtii, and the roots of Posidonia oceanica possess a marked cholinesterase inhibitory activity, which deserve further investigation in order to identify the compound(s) responsible inhibiting cholinesterases. Our results with antioxidant assays pointed out that Ricotia carnosula, Zostera noltii, and Posidonia oceanica have high antioxidant potential acting by different mechanisms. Consequently, we herein disclose the first the study on AChE, BChE, and TYR inhibitory effects of the abovementioned fourteen plant species, three of which may be useful for cognitive impairment by means of enzyme inhibition as well as antioxidant activity. This is also first report on flavonoid and phenolic acid contents of AL, CG, and VH.


  1. 1.

    Orhan IE. Current concepts on selected plant secondary metabolites with promising inhibitory effects against enzymes linked to Alzheimer’s disease. Curr Med Chem. 2012;19:2252–61.

    CAS  PubMed  Google Scholar 

  2. 2.

    Ghezzi L, Scarpini E, Galimberti D. Disease-modifying drugs in Alzheimer’s disease. Drug Des Dev Ther. 2013;7:1471–9.

    Google Scholar 

  3. 3.

    Orhan IE, Khan MTH. Flavonoid derivatives as potent tyrosinase inhibitors – A survey of recent findings between 2008-2013. Curr Topic Med Chem. 2014;14:1486–93.

    CAS  Article  Google Scholar 

  4. 4.

    Mendes E, Perry MJ, Francisco AP. Design and discovery of mushroom tyrosinase inhibitors and their therapeutic applications. Exp Op Drug Disc. 2014;9:533–54.

    CAS  Article  Google Scholar 

  5. 5.

    Nedelcheva A. An ethnobotanical study of wild edible plants in Bulgaria. EurAsian J BioSci. 2013;7:77–94.

    Article  Google Scholar 

  6. 6.

    Di Tizio A, Łuczaj LJ, Quave CL, Redžić S, Pieroni A. Traditional food and herbal uses of wild plants in the ancient South-Slavic diaspora of Mundimitar/Montemitro (Southern Italy). J Ethnobiol Ethnomed. 2012;8:21–4.

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Pieroni A, Quave CL, Santoro RF. Folk pharmaceutical knowledge in the territory of the Dolomiti Lucane, inland southern Italy. J Ethnopharmacol. 2004;95:373–84.

    Article  PubMed  Google Scholar 

  8. 8.

    Hadzhieva M, Kirches E, Mawrin C. Review: Iron metabolism and the role of iron in neurodegenerative disorders. Neuropathol Appl Neurobiol. 2014;40:240–57.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Ellman GL, Courtney KD, Andres V, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88–95.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Orhan I, Senol FS, Kartal M, Dvorska M, Zemlicka M, Smejkal K, Mokry P. Cholinesterase inhibitory effects of the extracts and compounds of Maclura pomifera (Rafin.) Schneider. Food Chem Toxicol. 2009;47:1747–51.

  11. 11.

    Masuda T, Yamashita D, Takeda Y, Yonemori S. Screening for tyrosinase inhibitors among extracts of seashore plants and identification of potent inhibitors from Garcinia subelliptica. Biosci Biotechnol Biochem. 2005;69:197–201.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Blois MS. Antioxidant determinations by the use of a stable free radical. Nature. 1958;181:1199–200.

    CAS  Article  Google Scholar 

  13. 13.

    Schlesier K, Harvat M, Bohm V, Bitsch R. Assessment of antioxidant activity by using different in vitro methods. Free Rad Res. 2002;36:177–87.

    CAS  Article  Google Scholar 

  14. 14.

    Marcocci I, Marguire JJ, Droy-Lefaiz MT, Packer L. The nitric oxide scavenging property of Ginkgo biloba extract EGb 761. Biochem Biophys Res Commun. 1994;201:748–55.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Chua MT, Tung YT, Chang ST. Antioxidant activities of ethanolic extracts from the twigs of Cinnamomum osmophleum. Biores Technol. 2008;99:1918–25.

    CAS  Article  Google Scholar 

  16. 16.

    Oyaizu M. Studies on products of browning reactions: Antioxidative activities of browning products of browning reaction prepared from glucosamine. Jap J Nutr. 1986;44:307–15.

    CAS  Article  Google Scholar 

  17. 17.

    Falcioni G, Fedeli D, Tiano L, Calzuola I, Mancinelli L, Marsili V, Gianfranceschi G. Antioxidant activity of wheat sprouts extract in vitro: Inhibition of DNA oxidative damage. J Food Sci. 2002;67:2918–22.

  18. 18.

    Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic- phosphotungstic acid reagents. Am J Enol Viticult. 1965;16:144–58.

    CAS  Google Scholar 

  19. 19.

    Woisky R, Salatino A. Analysis of propolis: some parameters and procedures for chemical quality control. J Apicult Res. 1998;37:99–105.

    CAS  Google Scholar 

  20. 20.

    Benamar H, Rached W, Derdour A, Marouf A. Screening of Algerian medicinal plants for acetylcholinesterase inhibitory activity. J Biol Sci. 2001;10:1–9.

    Google Scholar 

  21. 21.

    Khan MTH, Orhan I, Senol FS, Kartal M, Sener B, Dvorska M, Smejkal K, Slapetova T. Cholinesterase inhibitory activities of some flavonoid derivatives chosen xanthone and their molecular docking studies. Chemicobiol Interact. 2009;181:382–9.

    Google Scholar 

  22. 22.

    Zlatković BK, Bogosavljević SS, Radivojević AR, Pavlović MA. Traditional use of the native medicinal plant resource of Mt. Rtanj (Eastern Serbia): Ethnobotanical evaluation and comparison. J Ethnopharmacol. 2014;151:704–13.

    Article  PubMed  Google Scholar 

  23. 23.

    Polat R, Satil F. An ethnobotanical survey of medicinal plants in Edremit Gulf (Balıkesir – Turkey). J Ethnopharmacol. 2012;139:626–41.

    Article  PubMed  Google Scholar 

  24. 24.

    Kumarasamy Y, Nahar L, Sarker SD. Bioactivity of gentiopicroside from the aerial parts of Centaurium erythraea. Fitoterapia. 2003;74:151–4.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Lee MR, Hou JG, Begum S, Xue JJ, Wang YB, Sung CK. Comparison of constituents, antioxidant potency, and acetylcholinesterase inhibition in Lentinus edodes, Sparassis crispa, and Mycoleptodonoides aitchisonii. Food Sci Biotechnol. 2013;22:1747–51.

    CAS  Article  Google Scholar 

  26. 26.

    Valentão P, Fernandes E, Carvalho F, Andrade PB, Seabra RM, Bastos ML. Antioxidant activity of Centaurium erythraea infusion evidenced by its superoxide radical scavenging and xanthine oxidase inhibitory activity. J Agric Food Chem. 2001;49:3476–79.

    Article  PubMed  Google Scholar 

  27. 27.

    Baser KHC, Kurkcuoglu M, Tumen G. Composition of the essential oil of Origanum haussknechtii Boiss. J Ess Oil Res. 1998;10:227–8.

    CAS  Article  Google Scholar 

  28. 28.

    Salah SM, Jager AK. Screening of traditionally used Lebanese herbs for neurological activities. J Ethnopharmacol. 2005;97:145–9.

    Article  PubMed  Google Scholar 

  29. 29.

    Loizzo MR, Menichini F, Conforti F, Tundis R, Bonesi M, Saab AM, Statti GA, de Cindio B, Houghton PJ, Menichini F, Frega NG. Chemical analysis, antioxidant, antiinflammatory and anticholinesterase activities of Origanum ehrenbergii Boiss and Origanum syriacum L. essential oils. Food Chem. 2009;117:174–80.

  30. 30.

    Orhan IE, Belhattab R, Senol FS, Gulpinar AR, Hosbas S, Kartal M. Profiling of cholinesterase inhibitory and antioxidant activities of Artemisia absinthium, A. herba-alba, A. fragrans, Marrubium vulgare, M. astranicum, Origanum vulgare subsp. glandulossum and essential oil analysis of two Artemisia species. Ind Crops Prods. 2010;32:566–71.

    CAS  Article  Google Scholar 

  31. 31.

    Chung YK, Heo HJ, Kim EK, Kim HK, Huh TL, Lim Y. Inhibitory effect of ursolic acid purified from Origanum majorana L. on the acetylcholinesterase. Mol Cells. 2001;11:137–43.

    PubMed  Google Scholar 

  32. 32.

    Mossa ATH, Nawwar GAM. Free radical scavenging and antiacetylcholinesterase activities of Origanum majorana L. essential oil. Human Exp Toxicol. 2011;30:1501–13.

    CAS  Article  Google Scholar 

  33. 33.

    Orhan I, Kartal M, Kan Y, Sener B. Activity of essential oils and individual components against acetyl- and butyrylcholinesterase. Z Naturforsch. 2008;63:547–53.

    CAS  Google Scholar 

  34. 34.

    Ozkan G, Ozcan MM. Some phenolic compounds of the extracts obtained from Origanum species growing in Turkey. Environ Monitoring Assess. 2014;186:4947–57.

    CAS  Article  Google Scholar 

  35. 35.

    Orhan I, Aslan S, Kartal M, Sener B, Baser KHC. Inhibitory effect of Turkish Rosmarinus officinalis L. on acetylcholinesterase and butyrylcholinesterase enzymes. Food Chem. 2008;108:663–8.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Kartal M, Orhan I, Abu-Asaker M, Senol FS, Atici T, Sener B. Antioxidant and anticholinesterase assets and liquid chromatography-mass spectrometry preface of various fresh-water and marine macroalgae. Pharmacog Mag. 2009;5:291–7.

    Article  Google Scholar 

  37. 37.

    Cuny P, Serve L, Jupin H, Boudouresque CF. Water soluble phenolic compounds of the marine phanerogam Posidonia oceanica in a Mediterranean area colonized by the introduced chlorophyte Caulerpa taxifolia. Aquatic Bot. 1995;52:237–324.

    CAS  Article  Google Scholar 

  38. 38.

    Dumay O, Costa J, Desjobert JM, Pergent G. Variations in the concentration of phenolic compounds in the seagrass Posidonia oceanica under conditions of competition. Phytochemistry. 2004;65:3211–20.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Haznedaroglu MZ, Zeybek U. HPLC Determination of chicoric acid in leaves of Posidonia oceanica. Pharm Biol. 2007;45:745–8.

    CAS  Article  Google Scholar 

  40. 40.

    Heglmeier A, Zidorn C. Secondary metabolites of Posidonia oceanica (Posidoniaceae). Biochem System Ecol. 2010;38:964–70.

    CAS  Article  Google Scholar 

  41. 41.

    McMillan C, Zapata O, Escobar L. Sulphated phenolic compounds in seagrasses. Aquatic Bot. 1980;8:267–8.

    CAS  Article  Google Scholar 

  42. 42.

    Quackenbush RC, Bunn D, Lingren W. HPLC determination of phenolic acids in the water-soluble extract of Zostera marina L. (eelgrass). Aquatic Bot. 1986;24:83–9.

    CAS  Article  Google Scholar 

  43. 43.

    Achamlale S, Rezzonico B, Grignon-Dubois M. Rosmarinic acid from beach waste: Isolation and HPLC quantification in Zostera detritus from Arcachon lagoon. Food Chem. 2009;113:878–83.

    CAS  Article  Google Scholar 

Download references


F.S. Senol would like to express her genuine thanks to the Scientific and Technological Research Organization of Turkey (TUBITAK) for the scholarship provided during her Ph.D. studies. The grant presented to I.E. Orhan through Young Woman Scientist Award in Life Sciences provided by L’Oreal-Turkish Academy of Sciences for bioactivity and phytochemical studies carried on Atriplex lasiantha is gratefully acknowledged.

Author information



Corresponding author

Correspondence to Ilkay Erdogan Orhan.

Additional information

Competing interests

No competing interests exist among the authors of this paper.

Authors’ contributions

IEO and FSS designed the study, performed the research, collected and analyzed the data and written the manuscript. IEO collected the plant from Chenopodiaceae and prepared the extract. MZH and HK collected and prepared the extracts of three marine species, while SAE carried out all HPLC analyses. GY, MC, and AEY collected the plants from Gentianaceae and prepared their extracts. EA collected the plants from Brassicaceae and Caryophyllaceae and prepared their extracts. NK and GT collected the plant species from Lamiaceae and Cucurbitaceae, respectively. All authors read and approved the final manuscript.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Orhan, I.E., Senol, F.S., Haznedaroglu, M.Z. et al. Neurobiological evaluation of thirty-one medicinal plant extracts using microtiter enzyme assays. Clin Phytosci 2, 9 (2017).

Download citation


  • Medicinal plant
  • Neurobiology
  • Alzheimer’s disease
  • Enzyme inhibition
  • Cholinesterase
  • Tyrosinase