Abstract
The ethyl acetate and methanol extracts prepared from the fruits, aerial parts, and roots of Heptaptera anatolica (Boiss.) Tutin, (Umbelliferae), H. anisoptera (DC.) Tutin, H. cilicica (Boiss. & Balansa) Tutin (endemic), and H. triquetra (Vent.) Tutin were tested for their acetylcholinesterase (AChE) inhibitory and antioxidant activities. AChE inhibition was evaluated using ELISA microplate reader at 500, 1000, and 2000 μg mL−1. Antioxidant activity was determined by 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test and Fe+2-ferrozine test system for metal chelating power at the same concentrations. Total phenol contents of the extracts were determined using Folin-Ciocalteu reagent. At 2000 μg mL−1, only the aerial parts and fruits of H. anatolica showed moderate anti-AChE effect (61.97% and 49.80%, respectively), while the aerial parts and fruits of H. triquetra had the highest DPPH scavenging effect (80.48% and 86.19%, respectively). All of the methanol extracts exhibited significant ferrous ion-chelating effect varying between 72.97% and 92.36%, whereas only four of the ethyl acetate extracts exerted chelating effect over 70%. These results indicate that Heptaptera species could be a good source for antioxidant compounds.
Introduction
The genus Heptaptera (Umbelliferae) possesses ten species in the world mainly distributed from Europe to the Middle East including Italy, Balkans, Turkey, Syria, and Palestine (CitationBoissier, 1872; CitationTutin et al., 1968; CitationZohary, 1972). It is represented by four species in Turkey, one of which is endemic; H. anatolica (Boiss.) Tutin; H. anisoptera (DC.) Tutin; H. cilicica (Boiss. & Balansa) Tutin (endemic), and H. triquetra (Vent.) Tutin (CitationDavis, 1972, Citation1988; CitationGüner et al., 2000). Although limited phytochemical study has been so far carried on the Heptaptera species, the genus has been reported to be rich in coumarin derivatives; i.e., mangiferin, colladin, colladonin, badrakemin, 5,7-dihydroxycoumarin, and umbelliprenin derivatives (CitationSimova et al., 1986; CitationAppendino et al., 1992a, Citation1992b, Citation1993).
In one of our former studies, we screened several coumarin derivatives including umbelliferone, 4-methylumbelliferone, 4-hydroxycoumarin, scopoletin, 8-methoxypsoralen, bergapten, and iso-bergapten, and found some of them having moderate acetylcholinesterase inhibitory activity (CitationOrhan et al., 2008). Although no traditional use of this genus has been reported up to date, the genus is known to contain coumarins in major amounts, which directed us to test them for their antioxidant potential. Since Alzheimer’s disease (AD) is strongly associated with oxidative stress and neurodegeneration as well, and AChE is a key enzyme associated with the pathogenesis of AD. Nowadays, inhibition of this enzyme is one of the most accepted strategies in AD treatment. Since our bibliographical survey showed a lack of any biological activity data on Heptaptera as well as its richness in coumarins, consequently, we decided to examine the relevant biological activities (AChE inhibition and antioxidant activity) of all of the Heptaptera species growing in Turkey. For this purpose, the ethyl acetate and methanol extracts prepared from the fruits, aerial parts, and roots of these species were tested in vitro for their acetylcholinesterase (AChE) inhibitory and antioxidant activities at 500, 1000, and 2000 μg mL−1 concentrations. AChE inhibitory effect was evaluated by spectrophotometric method of CitationEllman et al. (1961) using an ELISA microplate reader at 500, 1000, and 2000 μg mL−1 concentrations. Antioxidant activity of the extracts was determined by 2,2-diphenyl-1-picrylhydrazyl radical scavenging and ferrous ion-chelating power tests at the same concentrations. Total phenol contents (TPC) of the extracts were determined using Folin-Ciocalteu reagent. Existence of several coumarins was detected in the extracts by thin layer chromatography (TLC) and preliminary phytochemical screening tests were also carried out in the extracts.
Materials and methods
Plant materials
H. anisoptera from Erzincan province (east Anatolia), H. anatolica from Muğla province (west Anatolia), and H. triquetra from Tekirdağ province (northwest Anatolia) were collected during July and August in the year of 2006. The sample of H. cilicica (south Anatolia) was gathered from Mersin province in July, 2007. The plant species were identified by one of us (G.Y.) and the voucher specimens were deposited in the Herbarium of the Faculty of Pharmacy of Ankara University (H. anisoptera-AEF 23720; H. anatolica-AEF 23819; H. cilicica-AEF 23717; H. triquetra-AEF 23723).
Extraction
The samples of each species were dried in the shade, powdered and soaked successively firstly in ethyl acetate for 3 days and secondly in methanol for another 3 days. The organic layers were filtrated and evaporated in vacuo until dryness to give the ethyl acetate and methanol extracts.
Coumarin standards
Among the coumarin standards used in TLC, umbelliferone (U7626), (M1381), 4-hydroxycoumarin (H2253), scopoletin (S2500), and 8-methoxypsoralen (M3501) were purchased from Sigma (St. Louis, MO). The other two coumarins; imperatorin and osthol were kindly donated by Maksut Coşkun from the Department of Pharmaceutical Botany, Faculty of Pharmacy, Ankara University, Ankara, Turkey. Purity of the standards was checked by TLC prior to TLC analysis of the extracts.
AChE inhibitory activity
AChE inhibition was assayed by the spectrophotometric method of CitationEllman et al. (1961). Electric eel AChE (Type-VI-S, EC 3.1.1.7, Sigma) was employed as the enzyme source, while acetylthiocholine iodide (Sigma) as substrate and 5,5′-dithio-bis(2-nitrobenzoic)acid (DTNB) were also used in the anti-AChE activity determination. All reagents and conditions were the same as in our previous publications (CitationOrhan et al., 2007a, Citation2007b). In brief, 140 μl of 0.1 mM sodium phosphate buffer (pH 8.0), 20 μl of DTNB, 20 μl of test solution and 20 μl of AChE solution were added by multichannel automatic pipette (Eppendorf, Germany) 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 acetylthiocholine iodide. The hydrolysis of acetylthiocholine iodide 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). The measurements and calculations were evaluated by using Softmax PRO 4.3.2.LS software. Percentage of inhibition of AChE was determined by comparison of rates of reaction of samples relative to blank sample (ethanol in phosphate buffer pH=8) using the formula (E-S)/E x 100, where E is the activity of enzyme without test sample and S is the activity of enzyme with test sample. The experiments were performed in four parallel sets. Galanthamine purchased from Sigma was the reference in this study.
Antioxidant activity
Antioxidant activity of the extracts was evaluated by DPPH radical scavenging and ferrous-ion chelating activity tests. Gallic acid, a natural phenolic-type antioxidant and butylated hydroxyanisol (BHA), a widely used synthetic antioxidant were employed as references in the tests.
DPPH radical scavenging activity test
The stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) (257621, Sigma-Aldrich Co., Steinheim, Germany), radical scavenging activity was determined by CitationBlois’s method (1958). The samples and references dissolved in ethanol (75%) were mixed with DPPH solution (1.5 × 10−4 M). Remaining DPPH amount was measured at 520 nm using a Unico 4802 Unico 4802 UV-visible double beam spectrophotometer (Dayton, NJ, USA) UV-visible double beam spectrophotometer (USA). Gallic acid (G7384, Sigma Co., St. Louis, MO, USA), and butylated hydroxyanisol (BHA) (W218308, Sigma-Aldrich Co., Steinheim, Germany), were employed as the references. Inhibition of DPPH in percent (I%) was calculated as given below:
where Ablank is the absorbance of the control reaction (containing all reagents except the test sample), and Asample is the absorbance of the extracts/reference.
Fe+2-ferrozine test system for metal chelating
The ferrous ion-chelating effect of the extracts by Fe+2-ferrozine test system was estimated by the method of CitationChua et al. (2008). Briefly, 740 μL of methanol and the samples (200 μL) were incubated with 20 μL of 2 mM FeCl2 solution. The reaction was initiated by the addition of 40 μL of 5 mM ferrozine into the mixture 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 follows:
The control contained only FeCl2 and ferrozine. Analyses were run in four replicates and expressed as average values with standard error mean (SEM).
Determination of total phenolic content
Phenolic compounds were assayed according to the Folin-Ciocalteu method (CitationSingleton & Rossi, 1965). In brief, the samples (150 μL) in test tubes were mixed with 750 μL of Folin-Ciocalteu reagent and 600 μL of sodium carbonate (7.5%). The tubes were then vortexed and incubated at 40°C for 30 min. Afterward absorption was measured at 760 nm. The total phenolic contents of the extracts were expressed as gallic acid equivalents (mg/g extract).
Preliminary phytochemical screening of the extracts
An aliquot of each sample was spotted onto the silica gel plate (Silica gel coated TLC plates; Merck) with a developing solvent system of butanol:methanol:ethyl acetate:water (65:35:4:1). The spots were checked under a UV detector at 254 nm and 366 nm. Plates were visualized under UV light. Presence of flavonoids in the extracts was proved by Shinoda color reaction in test tubes. On the other hand, the extracts were found not to contain alkaloid, anthocyanin, cyanogenetic heteroside, tannin, and cardioactive heterosides using general color tests using experimental tubes.
Results and discussion
The results obtained with the ethyl acetate and methanol extracts of four Heptaptera species and references in AChE inhibitory and antioxidant activity tests as well as total phenol contents (TPC) are given in . Among them, only the ethyl acetate extracts of the fruits and aerial parts of H. anatolica exerted modest activity towards AChE, having 49.8% and 61.97% of inhibition at 2000 μg mL−1, while the rest had activity below 40%, which is considered to be insignificant (). The methanol extracts of the fruits and aerial parts of H. triquetra had the best scavenging effect against DPPH at 2000 μg mL−1 (86.19 and 80.48%, respectively), whose TPCs were also the highest among the extracts studied (190.68 and 112.33 mg g−1 extract expresses as gallic acid equivalent). All of the extracts belonging to the four species screened displayed significant ferrous ion-chelating effect in a dose-dependent manner ().
Table 1. Acetylcholinesterase (AChE) inhibitory activity of the extracts of the Heptaptera species.
Table 2. Total phenol contents, coumarin analysis by TLC, and DPPH radical scavenging activity (inhibition %±S.E.M.) and of the extracts of the Heptaptera species.
Table 3. Ferrous ion-chelating percentage±S.E.M. of the extracts of the Heptaptera species.
On the other hand, our TLC analysis showed that especially the ethyl acetate extracts of the Heptaptera species investigated herein were richer in coumarin derivatives as compared to the methanol extracts (). The methanol extracts of H. triquetra were found not to have the coumarin standards analyzed.
There is growing interest in using and discovering new antioxidants from natural sources since synthetic antioxidants possess some unwanted effects. Therefore, a huge amount of research is being carried out on plants in order to find new antioxidant alternatives. On the other hand, Alzheimer’s disease is a kind of neurodegeneration which is also connected with oxidative mechanism. However, in this study, the extracts of all four Turkish species exhibited insignificant anti-AChE effect, while they had better antioxidant activity.
Heptaptera species are known to be rich in coumarin derivatives, sesquiterpene coumarin ether types in particular (CitationCrowden et al., 1969; CitationAppendino et al., 1992b, Citation1993). Coumarins having a basic benzo-2-pyrone skeleton are one of the simplest groups of phenolic compounds and have been reported to have strong antioxidant effect by various mechanisms including DPPH and hydroxyl radical scavenging activity, inhibition of lipid peroxidation, metal chelating, etc. (CitationNgi et al., 2000; CitationTorres et al., 2006; CitationSurveswaran et al., 2007; CitationWu et al., 2007). Therefore, it is most likely that coumarin-type of compounds in Heptaptera species could be responsible for antioxidant and mild anticholinesterase activity. In fact, we have recently published a research article about anticholinesterase activity of several coumarins (umbelliferone, 4-methylumbelliferone, 4-hydroxycoumarin, scopoletin, 8-methoxypsoralen, bergapten, and iso-bergapten) and only 4-methylumbelliferone, scopoletin, and bergapten showed inhibition against AChE over 50% at 1 mg mL−1 (CitationOrhan et al., 2008). However, synergistic interactions can frequently occur in a single herb due to existence of dozens of bioactive compounds and therefore, it is very important to elucidate the active component(s).
In conclusion, our results showed that H. triquetra showed the best radical scavenging and iron chelating effects among the extracts and could be a potential source for antioxidant compounds; while H. anatolica may be worthy of further phytochemical characterization for its anticholinesterase effect. To the best of our knowledge, this is the first report on anticholinesterase and antioxidant activities of Heptaptera species.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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