Citation/Atıf: Can Z, Kara Y, Kolayli S, Çakmak İ. Determination Of Anti-Urease Activity Of Propolis From The Marmara Region Of Turkey (Extended abstract in Turkish). U. Arı B. / U. Bee J. 2022, 22: DOI: 10.31467/uluaricilik.1068885 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE DETERMINATION OF ANTI-UREASE ACTIVITY OF PROPOLIS FROM THE MARMARA REGION OF TURKEY Türkiye'nin Marmara Bölgesinden Elde Edilen Bazı Propolislerin Anti-üreaz Aktivitesinin Belirlenmesi Zehra CAN1 2 2, Yakup KARA , Sevgi KOLAYLI , İbrahim ÇAKMAK3 1Bayburt University, Faculty of Applied Sciences, Department of Emergency Aid and Disaster Management, 69010, Bayburt, TÜRKİYE, Yazışma Yazarı/Corresponding author E-posta: zehracan61@gmail.com, ORCID No. 0000 0002 7156 4941 2Karadeniz Technical University, Faculty of Science, Department of Chemisty, 61080 Trabzon, TÜRKİYE, E-posta: yaakupkara@gmail.com, ORCID NO. 0000 0003 3121 5023, E-posta: skolayli61@yahoo.com, ORCID NO. 0000 0003 0437 6139 3Bursa Uludag University, Beekeeping Development, Application and Research Center-AGAM, Faculty of Agriculture, Department of Animal Science, 16059 Bursa, TÜRKİYE, E-posta: icakmak@uludag.edu.tr, ORCID NO. 0000 0002 8000 5770 Geliş Tarihi / Received: 06.02.2022 Kabul Tarihi / Accepted: 01.03.2022 DOI: 10.31467/uluaricilik.1068885 ABSTRACT Propolis is a natural product collected by honeybees from plants especially flowers and buds by mixing with wax and resin and used for many purposes in hive. It is known that propolis has been used in the treatment of various diseases in traditional medicine for many years and has biological activities such as antioxidant, antimicrobial, antiulcer, antitumor, anti-inflammatory. There is increasing interest in alternative approaches to inhibit Helicobacter pylori (H. pylori) and thereby treat many gastric diseases. This study investigated various properties of raw propolis samples from the Marmara region of Turkey. Anti-urease activity was investigated in propolis samples. The anti-urease activity of IC50 values ranged from 1.110 to 5.870 mg/mL. As a result, it can be said that propolis extract is a good inhibitor that can be used in the treatment of H. pylori to improve human health. Keywords: Propolis, urease, enyzme inhibition ÖZ Propolis, bal arıları tarafından bitkilerden özellikle çiçek ve tomurcuklardan balmumu ve reçine ile karıştırılarak toplanan ve kovanda birçok amaç için kullanılan doğal bir üründür. Propolisin uzun yıllardır geleneksel tıpta çeşitli hastalıkların tedavisinde kullanıldığı ve antioksidan, antimikrobiyal, antiülser, antitümör, antiinflamatuar gibi biyolojik aktiviteleri olduğu bilinmektedir. Helicobakter pilori'yi (H. pylori) inhibe etmeye ve böylece birçok mide hastalığını tedavi etmeye yönelik alternatif yaklaşımlara artan bir ilgi vardır. Bu çalışma, Türkiye'nin Marmara bölgesinden alınan ham propolis örneklerinin çeşitli özelliklerini araştırmıştır. Propolis örneklerinde anti-üreaz aktivitesi araştırıldı. IC50 değerlerinin anti-üreaz aktivitesi 1.110 ila 5.870 mg/mL aralığındaydı. Sonuç olarak propolis ekstraktının H. pylori tedavisinde insan sağlığını iyileştirmek için kullanılabilecek iyi bir inhibitör olduğu söylenebilir. Anahtar Kelimeler: Propolis, üreaz, enzim inhibisyonu Uludağ Arıcılık Dergisi – Uludag Bee Journal 2022, 22 (1): 25-30 25 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE GENİŞLETİLMİŞ ÖZET (2016b), tüm EPE'lerin H. pylori üreazını 0.260 ila Giriş: Mide ülserine genellikle asidik ortamlarda 1.525 mg/mL gibi geniş bir inhibitör aralıkla inhibe yaşayan ve büyüyen H. pylori'nin neden olduğu ettiğini bildirmiştir. Literatürdeki veriler mevcut bilinmektedir. H. pylori üreaz enzimi salgılar, üreyi çalışmadaki verilerden daha farklı görülmektedir. amonyağa dönüştürür ve ürettiği amonyak ile mide Bunun nedeni propolis örneklerinin alındığı asidinin etkilerinden kendini korur. H. pylori ve bölgeden ve toplanma şeklinden kaynaklandığını üreazı, kronik gastrit, peptik ülser, mide kanseri ile söyleyebiliriz. güçlü bir şekilde ilişkilidir. Üreaz enzimi (E.C. H. pylori'ye karşı ortaya çıkan antibiyotik direnci, 3.5.1.5.), ürenin karbon dioksit ve amonyağa araştırmacıları ülser iyileşmesini hızlandırmak ve hidrolizini katalize eden çok spesifik bir enzimdir. enfeksiyonu ortadan kaldırmak için bir ajan olarak Amaç: Propolis, bal arılarının (Apis mellifera L.) propolisi keşfetmeye teşvik ediyor. Yapılan ağaç, bitkilerin tomurcuklarından topladıkları öz çalışmalar 15 farklı propolisin üreaz enziminin sularını, kendisinde bulunan salgı bezlerinden aktivitesini önemli ölçüde inhibe ettiğini göstermiştir salgılanan enzimlerle işleyerek oluşturdukları açık (Baltas ve ark., 2016a). Üreaz inhibitörlerinin bazı sarı renginden koyu rengine kadar değişim gösteren toksik etkileri göz önüne alındığında, doğal bir ürün yapışkan doğal bir üründür. Yapılan araştırmalarda olan propolis, H. pylori ile ilişkili mide-duodenal propolisin antioksidan, antibakteriyal, enfeksiyonu baskılamak ve kontrol etmek için çok antienflamatuar, antifungal, antitumor ve antiülser güvenli bir kaynak olma potansiyeline sahiptir. gibi biyolojik aktiviteye sahip olduğu bildirilmiştir. Bu çalışmada, Türkiye’nin Marmara Bölgesinin faklı Çoğu hastalığın tedavisi onunla ilişkili olan illerinden temin edilen propolis örneklerinin farklı enzimlerin inhibisyonu ile mümkündür. Bu oranlarda üreaz enziminin aktivitesini engellediği çalışmada, Marmara bölgesinin farklı illerindeki tespit edildi. Hergeçen gün önemi daha çok artan propolislerin potansiyel üreaz inhibitörü olup propolisin, Helikobakteri pilori bakterisinin sebep olamayacağı incelendi. olduğu enflamasyon hastalıklarına karşın doğal bir Gereç-Yöntem: 2018-2019 yılları arasında inhbitör olarak kullanılarak ilaçların oluşturmuş Türkiye’nin Marmara bölgesinin farklı yerlerinden olduğu yan etkilerini ortadan kaldırabileceğini (Bursa Uludağ Üniversitesi Kampüsü, Bursa (Merkez), Kapıdağ peninsula (Balıkesir), Bandırma, İznik, Marmara adası, İstanbul, Çanakkale, INTRODUCTION Tekirdağ, Yalova, Sakarya) Arı Yetiştiricileri Bee products such as honey, polen, propolis have Birliklerinden propolis örnekleri temin edildi. Herbir been used for traditional and complementary propolis örneği %70’lik etanol çözücüsünde treatment since ancient times (Sahin, 2016). In ekstraktlar hazırlandı. Hazırlanan propolis particular, propolis, one of the bee products, has örneklerinde üreaz enzim inhibisyonuna karşı antioxidant, antimicrobial, anti-inflammatory and inhibitor etkisi araştırıldı. Standart olarak tiyoüre anticancer properties due to the presence of kullanıldı. phenolic acids, flavonoids (Catchpole et al. 2015). Bulgular: Etanolik propolis ektraktlarının üreaz The chemical composition of propolis varies enzim varlığında IC 1.110 to 5.870 mg/mL. depending on the season, geographical and 50 değerleri arasında tespit edildi. Propolis botanical origins, and the mechanisms used to ekstraktlarının IC değeri rakamsal olarak ne kadar collect the material (Bankova, 2005, Ulloa et al. 50 düşük ise enzimi inhibe etme oranı da o derece 2017). Until this time, the chemical contents of yüksektir. propolis samples from different parts of the world have been investigated and most of them have been Tartışma ve Sonuç: Bu çalışmada polifenoller found to have a unique and region-specific açısından zengin propolis örnekleri için inhibisyon phytochemical profile (Stavropoulou et al. 2021). etkileri oldukça güçlü olduğu görülmektedir. Önceki The characteristic constituents of propolis are çalışmada, çeşitli Türk propolis numunelerinin anti- favonoids such as chrysin, galangin, pinocembrin, üreaz aktivitesi 0.080 ile 1.560 mg/mL arasında and pinobanksin. Caffeic acid phenethyl ester is a değişmekteydi ve yüksek fenolik içerikli numuneler major constituent of propolis with broad biological daha yüksek anti-üreaz aktiviteleri sergiledi (Baltaş activities (Alanazi et al. 2021). There are about 250 ve ark., 2016a). Başka bir çalışmada Baltaş ve ark. 26 U.Arı D. – U.Bee J. 2022, 22 (1): 25-30 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE and even more chemicals in propolis and and most MATERIAL AND METHODS of them are not known sufficiently. Chemicals It is now known that gastric ulcer is generally caused by H. pylori, which survives and grows in acidic Jack bean urease, urea, acetohydroxamic acid, environments (Dunn et al. 1997, Mégraud et al. sodium nitroprusside and organic solvent were 1999, Amin et al. 2013). H. pylori secretes urease purchased from Sigma-Aldrich (St. Louis, MO, USA) enzyme, converts urea into ammonia and protects and Merck. itself from the effects of stomach acid with the Propolis Samples ammonia it produces. H. pylori and its urease are strongly associated with chronic gastritis, peptic Propolis samples from 11 different areas in the ulcers, gastric cancer. The urease enzyme (E.C. Marmara region of Turkey were obtained from 3.5.1.5.) is a very specific enzyme that catalyzes the experienced beekeepers between 2018 and 2019. hydrolysis of urea to carbon dioxide and ammonia The samples, sample codes, and their geographic (Mareska et al. 2013, Modakh et al. 2015). origin are given in Table 1. Extraction, approximately 3 g each propolis sample was taken and onto 30 mL H. pylori infection remains a worldwide public health 70% ethanol was added and stirred on a shaker problem, particularly due to current treatment (Heidolph Promax 2020, Schwabach). After 24 inadequacies. This bacterium is an important cause hours of extraction at room temperature, filtration of ulcer and gastric cancer. Antibiotic treatment is was performed. widely used in the treatment of this disease. However, some drugs used in the treatment of this Urease enzyme inhibition assay disease have a toxic effect. Increasing antibiotic Urease enzyme inhibition of propolis samples was resistance is one of the reasons why H. pylori determined in UV-Vis spectrophotometer. eradication has failed in most countries (Baltas et al. (Weatherburn 1967). In summary, 500 µL of buffer 2016a). Therefore, it becomes necessary to search solution (pH 8.2), 200 µL urease enzyme solution for new H. pylori urease inhibitors with improved and 100 µL sample were incubated for 15 min in test stability and low toxicity in natural products such as tube. Then a phenol reagent 500 µL, and alkali plant extracts, honey, pollen, and propolis. reagent 600 µL were added to each tube This study intended to survey the potential inhibitor absorbance reading was performed at 625 nm than activity of the Marmara Region propolis against incubated 50 min. Thiourea was used as standard urease enzyme inhibition, expanding the therapeutic inhibitor. The IC50 results are given in mg/mL. use of propolis and thus consolidating the medicinal properties of propolis type as a possible new therapy for H. pylori eradication. Future studies should be RESULTS focusing on propolis from different regions to treat Anti-Urease Activity different health problems particularly microbial infection problems. The urease inhibition potential of propolis extracts was evaluated in vitro by Jack bean urease assay. The result of urease inhibitor activity of the test samples was shown as IC50 (mg/mL). The propolis samples totaling 11 belonged to 10 different locations. (Figure 1). Exhibited inhibition effects with IC50 values ranged from 1.110 to 5.870 mg/mL in all samples, and with a mean value of 2.493±1.678 mg/mL (Table 1). Uludağ Arıcılık Dergisi – Uludag Bee Journal 2022, 22 (1): 25-30 27 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE Table 1. Code, collected region of propolis samples and enzyme inhibition IC50 values. Code Regions Anti-Urease IC50 mg/mL Pr1 Bursa Uludag University Campus 1.420±0.090 Pr2 Bursa (Central) 2.230±0.110 Pr3 Kapidag peninsula (Balikesir) 1.542±0.100 Pr4 Bandırma 1.534±0.080 Pr5 İznik 1.460±0.130 Pr6 Marmara island 1.900±0.070 Pr7 Istanbul 4.600±0.050 Pr8 Canakkale 5.870±0.700 Pr9 Tekirdag 1.200±0.010 Pr10 Yalova 1.110±0.020 Pr11 Sakarya 4.560±0.130 Mean±SD 2.494±1.678 Range 1.110-5.870 Thioure (µg/mL) 12.110±0.090 Figure 1. Distribution of propolis samples locations. (https://www.alfayapimuhendislik.com/bolge/marmara-bolgesi.html) 28 U.Arı D. – U.Bee J. 2022, 22 (1): 25-30 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE DISCUSSION source for suppressing and controlling gastro- The enzyme urease is a nickel-containing duodenal infection associated with H. pylori. The metalloenzyme belonging to the hydrolase class. future studies should focus on urease inhibitors of Ammonia released as a result of the breakdown of propolis from different regions. urea by urease assists the colonization of H. pylori Conflict of Interest: The author declares no by shifting stomach acid to neutral pH (Baltas et al. possible conflicts of interest. 2016a). Urease provides H. pylori with the opportunity to live in the stomach at low pH, causing Author contribution: Zehra Can: Laboratory part gastritis peptic ulceritus, and especially cancer of the study, evaluation of the results, manuscript (Morishita et al. 2008). A disease caused by writing, submitting to the journal, Yakup Kara: Helicobacter pylori can be prevented through urease Laboratory part of the study, evaluation of the inhibition (Baltas et al. 2016a). Numerous urease results İbrahim Çakmak: Design and planning of the enzyme inhibitors are used as medical drugs, but study, site selection and sample collection, many are costly and have side-effects (Romeno et manuscript editing, and correction, Sevgi Kolaylı: al. 2019, Song et al. 2020). In recent years, many Design and planning of the study investigations have indicated that polyphenol-rich Funding: Bursa Uludag University BAP-AGAM extracts such as bee products (especially propolis) Project no. 2016/2 have substantial inhibitory effects (Manyi- Loh et al. 2012). Aknowledgement: We are most grateful to Meral Kekecoglu for some propolis samples and Aycan The inhibition effects are quite strong for propolis Tosunoglu for their botanical origins. samples which are rich in polyphenols in this study. 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