engleski

Novel approach for determination of antioxidative activity of aminosalicylates using DPPH assay method

The inflammatory bowel disease (IBD) involves chronic inflammation of digestive tract and it is determined either as Crohn’s disease, that takes place anywhere on the linings of the digestive tract or ulcerative colitis, that causes inflammation mainly in the colon. Etiology of IBD remains unclear, but it is known that oxidative damage to biological membranes plays an important role in tissue damaging. Aminosalicylates have been in use for over 60 years in treatment of IBD and their most known representative, sulfasalazine, was introduced in late 1930s for treatment of rheumatoid arthritis. Later on, it was discovered that its good therapeutic impact is related to 5- aminosalicylic acid (5-ASA). Due to many side effects caused by sulfasalazine, other drugs were introduced, such as balsalazide and olsalazine, but also 5-ASA that is commonly known as mesalamine. Although precise mechanism of action of 5-ASA is still not known, it has been shown that it possesses antioxidative properties and is a potential scavenger of free radicals that are responsible for the pathogenesis of IBD. Thus, the aim of this work was to determine the antioxidative power of 5-ASA and its prodrugs. The antioxidative potential of the drugs was determined using DPPH assay method. RP-HPLC-DAD method was developed and used for monitoring of DPPH assay. XBridge C18 column (3.5µm particle size, 4.6x150mm) was used as the stationary phase, with 25°C as the column temperature. Mobile phase consisted of 80% methanol and 20% Millipore water and isocratic elution was used at flow rate of 1 ml/min. DPPH assay was monitored with diode array detector at 517 nm and retention time of DPPH peak was 4.25 minutes. Scavenging strength of compounds was shown using TROLOX as standard antioxidant and it was expressed as TROLOX equivalent antioxidant capacity (TEAC). It was calculated through the obtained calibration curve which presented linearity between 0.01mM and 0.14 mM range (R2 = 0.9964). It was observed that 0.1 mM 5-ASA showed 140 to 310 times stronger antioxidative power than 1mM sulfasalazine and balsalazide, whilst oxidative nature of 1mM olsalazine was not detected. These results imply that majority of the antioxidative power of aminosalicylates originates from free 5-ASA.

DPPH ; HPLC ; IBD

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