Sinem OKTEM-OKULLU, Nesteren MANSUR, Erkan MOZİOGLU, Meltem KOLGAZİ

Hidroksisinamik Asit Türevlerinin Canlı-Dışı Helicobacter Pylori Karşıtı Etkileri ile Üreaz Enzimini Engelleme Etkinliklerinin Araştırılması

Helicobacter pylori H. pylori , insanlarda, gastrit, peptik ülser, gastrik kanser ve mukoza ilintili lenfoid doku lenfoması gibi ciddi mide hastalıklarına yol açan önemli bir hastalık etkenidir. H. pylori, ürettiği üreaz enzimleri sayesinde üreyi, karbondioksit ve amonyağa parçalayarak mide çeperinin asidik koşullarını normal pH’ya getirip hayatta kalabildiği için bu bakterilerle vücudun savaşımı kolay değildir. H. pylori enfeksiyonları için antibiyotikler mevcut olsa da antibiyotik direnci gelişimi nedeniyle bu tedaviler sonuçsuz kalabilmekte ve yeni antibiyotiklere gereksinim her geçen gün artmaktadır.Hidroksisinamik asit türevleri basit fenolik asitler olup meyvelerde, meyve çekirdeklerinde ve sebzelerde bulunmaktadır. Ferulik asit, kafeik asit, p-kumarik asit, sinapik asit, sözü edilen bu fenolik asit grubuna ait olup antioksidan, anti-inflamatuvar, antimikrobiyal özelliklere sahiptir ve bu nedenle bazı bakteri enfeksiyonlarının tedavisinde, ilaçlara seçenek olarak kullanılmaktadır. Bu çalışmamızda, bu üç fenolik asidin H. pylori üzerindeki antimikrobiyal etkinliği ve ürez enzimini engelleme etkisi araştırıldı.Hidroksisinamik asit türevlerinin anti-H. pylori etkisi H. pylori G27 standart suşu üzerinde test edildi. Minimum inhibisyon konsantrasyonu MİK , değerleri 512 ila 0,5 ug/mL arasında değiştiği seri tüp seyreltme yöntemiyle, minimum bakterisidal konsantrasyon MBK değerleri ise MİK içinde kullanılan aynı konsantrasyonlarda canlı ve ölü bakterilerin nispi oranının hesaplanması ile belirlendi. MİK için CLSI M07-A9, MBC için CLSI M26-A protokolleri kullanıldı. Ureaz inhibisyon aktivitesi Helicheck, üreaz aktivitesine özgü indikatörlü besiyerinde ölçülmüştür.H. pylori’ye karşı test edilen tüm bileşikler için MİK 64 ug/mL ve MBK 128 ug/mL idi. Test edilen bileşiklerin H. pylori tarafından salgılanan üreaz enzimi üzerinde hiçbir inhibisyonu saptanmadı. Nükleotid salma deneyi sonuçlarına göre, hidroksisinamik asit türevlerinin bakteri zarında hasara sebep olması ile zarda oluşan deliklerden dışarı salınma eğiliminde olması beklenen herhangi bir nüklotit miktarı ölçülememiştir.Gerçekleştirilen bu çalışma, literatür taramalarımız doğrulutusunda, hidroksisinamik asit türevlerinin anti-H. pylori aktivitesini gösteren ilk çalışmadır. Bu bileşiklerin anti-H. pylori üzerindeki etki mekanizmasını anlamak için daha ileri analizlere ihtiyaç vardır

In Vitro Anti-Helicobacter Pylori Effects of Hydroxycinnamic Acid Derivatives and Their Urease Inhibition Studies On H. Pylori Urease Enzyme

Helicobacter pylori H. pylori is a human pathogen that can persist in the stomach of infected people for their life spans. It causes chronic gastric inflammation leading to serious gastric diseases such as gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. To colonize in the acidic environment of the stomach, H. pylori produces urease enzyme that catalyzes the hydrolysis of urea to yield into ammonia and carbon dioxide. This enzyme neutralizes the acidic environment of the gastric lumen, and gives the bacteria a short-term survival in this highly acidic environment. There are numerous antibiotic-based therapies for the treatment of H. pylori infection. However, antibiotic resistance has a significant impact on the failure of these treatments.Hydroxycinnamic acid derivatives are simple phenolic acids, found mainly in cereals, fruits, seeds of fruits and vegetables. Ferulic acid, Caffeic acid, p-Coumaric acid, and Sinapic acid belong to this phenolic acid group. These derivatives act as antioxidant, anti-inflammatory, antimicrobial agents and have been used for the treatment of some bacterial infections as alternatives to drugs.The objective of this study was to investigate the anti-H. pylori and urease enzyme inhibitory effects of Ferulic acid, Caffeic acid, p-Coumaric acid and Sinapic acid.The effect of hydroxycinnamic acid derivatives was tested on H. pylori standard train G27. MIC was determined by serial tube dilution method in which the final concentration ranged between 512 to 0.5 µg/ml and MBC was determined by calculating the relative proportion of live and dead bacteria with the same concentration range used in MIC. For MIC, the CLSI M07-A9and for MBC CLSI M26-A protocols were used. Urease inhibitory activity was detected by Helicheck, H. pylori-specific growth media shows urease activity by changing the color of the media. Nucleotide release was measured by spectrophotometry. The MIC was 64 µg/ml and MBC was 128 µg/ml for all compounds and they had no effect on urease enzyme. There was no detectable nucleotide release from the bacterial membrane because of the hydroxycinnamic acid derivatives damage

Keywords:

H. pylori anti-helicobacter pylori effect, hydroxycinnamic acid derivatives, urease inhibition, nucleotide release assay,

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