Turkish Journal of Agriculture and Forestry Turk J Agric For (2018) 42: 82-87 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-1711-18 Neuroprotective potential of the fruit (acorn) from Quercus coccifera L. 1, 2 3 4 1 Fatma Sezer ŞENOL *, Nazım ŞEKEROĞLU , Sevgi GEZİCİ , Esra KILIÇ , İlkay ERDOĞAN ORHAN 1 Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara, Turkey 2 Department of Food Engineering, Faculty of Engineering and Architecture, Kilis 7 Aralık University, Kilis, Turkey 3 Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Kilis 7 Aralık University, Kilis, Turkey 4 Department of Biology, Faculty of Arts and Sciences, Kilis 7 Aralık University, Kilis, Turkey Received: 03.11.2017 Accepted/Published Online: 12.12.2017 Final Version: 26.04.2018 Abstract: Neurodegenerative diseases such as Alzheimer disease and Parkinson disease are among the deadly disorders affecting the elderly population. Unfortunately, effective treatments and medications are still needed to combat these diseases. The ethanol extracts of the raw peeled acorns and acorn coffees from Quercus coccifera prepared by two different methods (boiled-roasted ground and roasted ground) were tested against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and tyrosinase (TYR), the enzymes associated with neurodegeneration. The acorns used in the present study were harvested from wild Quercus coccifera L. trees in Kilis Province, located in the Eastern Mediterranean region of Turkey in November 2016. Cholinesterase (ChE) and TYR inhibitory activity of the ethanol extracts prepared from raw material and two coffee samples were screened using an ELISA microtiter assay at 2 mg/mL stock concentration. Antioxidant effects of the extracts were also tested for their scavenging activity against DPPH, while total phenol and flavonoid quantities of the extracts were determined spectrophotometrically. Our results indicated that all of the extracts had notable AChE and BChE inhibition, while they were inactive against TYR. The most active extract against ChEs was prepared from the raw material, which led to 65.94 ± 3.59% and 85.04 ± 3.83% inhibition against AChE and BChE, respectively. Our data indicate that the fruit extract from Quercus coccifera is a source of promising ChE inhibitors and deserves future study. Key words: Alzheimer disease, cholinesterase inhibition, neurodegeneration, Quercus coccifera 1. Introduction in the Mediterranean phytogeographic region. Based Forests have played an important role for mankind for on ethnobotanical studies, the use of different parts of centuries. Humans depend on forests for survival, from the kermes oak has been recorded against hemorrhoids, air we breathe to the wood we use. Besides providing habitats diabetes, diarrhea, and kidney stones as well as for its for animals and livelihoods for humans, forests also offer hypotensive, abortive, and wound-healing properties watershed protection, prevent soil erosion, and mitigate in Anatolian folk medicine (Agelet and Velles, 2003; climate change (Çalışkan and Boydak, 2017; Kahveci, Tuzlacı and Sadıkoğlu, 2007; Ugurlu and Secmen, 2008; 2017; Razzaghi et al., 2017). Güneş et al., 2017). Q. coccifera has been so far reported The genus Quercus (Fagaceae), known as “meşe” in to have several biological effects such as antiulcerogenic, Turkish, is represented by 32 taxa, 5 of which are endemic antibacterial, antifungal, antihelmintic, and antioxidant to Turkey, and it is vital for the Anatolian landscape activity (Alkofahi and Atta, 1999; Şöhretoğlu et al., 2007; (Güner et al., 2012). Quercus species are mostly used Manolaraki et al., 2010; Buzayan and El-Garbulli, 2012; for production of furniture, agricultural instruments, Genç et al., 2012). Additionally, previous studies have and building materials, while these plants are also a shown that Q. coccifera contains phenolic compounds source of fodder for animals (Özcan and Bayçu, 2005). such as tannins and flavonoids, besides tocopherols and Moreover, 26.34% of the forestland in Turkey consists of fatty acids (Ito et al., 2002; Genç et al. 2012; Şöhretoğlu oak trees. Fruits (acorns) of this species have also been et al., 2014; Akcan et al., 2017). In our previous study, we used as a coffee substitute like racahout, which is a spicy analyzed minerals of the same samples and found that Turkish acorn drink similar to hot chocolate (Bainbridge, processing affected their mineral composition as well as 1986; Rakic et al., 2006). Q. coccifera L. (kermes oak) heavy metal concentration, which were below acceptable is one of the evergreen species especially distributed limits (Sekeroglu et al., 2017). * Correspondence: [email protected] 82 ŞENOL et al. / Turk J Agric For Alzheimer and Parkinson diseases are progressive 2.2. Preparation of the coffee samples and extracts neurodegenerative disorders, which are especially affecting The plant materials were air-dried at room temperature the elderly population. Today, Parkinson disease affects and peeled. Acorn coffees were prepared with two different approximately 10 million people worldwide, whereas methods: boiled, roasted, and then ground; and only Alzheimer disease is responsible for about 60%–70% of roasted and then ground. The raw materials were ground dementia cases (http://www.neurodegenerationresearch. in a mechanical grinder. The coffee samples and ground eu/about/what/). Both of these diseases have no complete raw materials were weighed accurately in a digital balance cure now, but drugs only for symptomatic treatment (Shimadzu, Japan) and extracted with ethanol (80%) for 2 based on a cholinergic hypothesis are available clinically. days. The ethanol phases were filtered and removed using The majority of commercially available medications that a rotary evaporator (Buchi, Switzerland) until dryness proved to be effective in cognitive-related disorders or to give the crude extracts. The extract yields (w/w%) are were approved by the US Food and Drug Administration given in the Table. to treat symptoms are mostly plant-derived molecules 2.3. Enzyme inhibition assays (D’onofrio et al., 2017). In our ongoing studies for finding new cholinesterase 2.3.1. AChE and BChE inhibitory activity (ChE) and TYR inhibitors of natural origin, we recently AChE and BChE inhibitory activity of the samples was explored the neuroprotective properties of several measured by the slightly modified spectrophotometric commercial brands of Turkish coffee sold in Turkey and method of Ellman et al. (1961). Electric eel AChE (EC Northern Cyprus (Aslan Erdem et al., 2016), a number 3.1.1.7, Sigma, St. Louis, MO, USA) and horse serum BChE of traditional herbal coffees consumed in Turkey such (EC 3.1.1.8, Sigma) were used, while acetylthiocholine as terebinth coffee (Orhan et al., 2012), and some other iodide and butyrylthiocholine chloride (Sigma) were herbal coffees prepared from the different parts of carob, employed as substrates of the reaction. 5,5’-Dithiobis(2- black cumin, dates, and tumble thistle. Our previous nitrobenzoic)acid (DTNB, Sigma) was used for the results revealed that date samples (Phoenix dactylifera) measurement of the anticholinesterase activity. All exerted the highest acetylcholinesterase (AChE) and reagents, conditions, and calculations were the same as butyrylcholinesterase (BChE) inhibition among the tested described in our previous publication (Senol et al., 2016). extracts (Sekeroglu et al., 2012), and taking these promising The measurements and calculations were evaluated by results into consideration, we have now decided to screen using Softmax PRO 4.3.2.LS software. The experiments the neuroprotective potential of acorns and coffees from were done in quadruplicate. Galanthamine hydrobromide Q. coccifera. (Sigma) was used as the reference drug. 2.3.2. TYR inhibitory activity 2. Materials and methods TYR (EC 1.14.18.1, mushroom tyrosinase, Sigma) 2.1. Plant material inhibitory activity of the samples was determined The fruits of Quercus coccifera L. were collected from Kilis spectrophotometrically by the slightly modified method (Turkey) in November 2016 and were identified by the of Masamoto et al. (1980) with L-DOPA (Sigma) as the second author. A voucher specimen was preserved in the substrate (Lee et al., 2009). All reagents and conditions were herbarium of Kilis 7 Aralık University (Kilis, Turkey). the same as described in our previous publication (Aslan Table. Extraction yields (w/w) and inhibitory activity of the extracts against AChE, BChE, and TYR. Inhibition against AChE Inhibition against BChE Inhibition against TYR Yield % (Inhibition% ± SDa), (Inhibition% ± SD), (Inhibition% ± SD), (w/w) 200 µg/mLb 200 µg/mLb 133.33 µg/mLb 65.94 ± 3.59 85.04 ± 3.83 Raw material 5.17 10.13 ± 3.22 (IC50 = 60.51 ± 3.29 µg/mL) (IC50 = 19.11 ± 0.17 µg/mL) Roasted 1.03 30.22 ± 3.13 47.66 ± 3.13 4.84 ± 0.75 57.40 ± 3.23 Boiled-roasted 1.30 39.91 ± 2.51 2.95 ± 1.93 (IC50 = 56.82 ± 3.96 µg/mL) Reference 97.12 ± 2.89c 86.77 ± 2.86c 83.97 ± 1.68d aStandard deviation, bfinal concentration, cgalanthamine hydrobromide - 100 µg/mL, dkojic acid - 133.33 µg/mL. 83 ŞENOL et al. / Turk J Agric For Erdem et al., 2016) The assays were performed in a 96-well obtained to prepare a calibration curve. Absorbance of the microplate using an ELISA microplate reader (VersaMax reaction mixtures was measured at a wavelength of 415 nm Molecular Devices, USA) by measuring their absorbance using a 96-well microplate reader (VersaMax