*2.10. Evaluation of Colitis Treatment*

### 2.10.1. Colitis Activity Index

The degree of inflammation was checked daily by determining the colitis activity index (CAI) which consists of three clinical parameters including weight loss, stool consistency, and anal bleeding [51]. Briefly, the score scale from 0 to 4 indicates the degree of inflammation (from healthy to maximal inflammation). Scores of 0, 1, 2, 3 and 4 correspond to no weight loss, 1–5% weight loss, 5–10% weight loss, 10–20% weight loss and more than 20% weight loss, respectively. Similarly, for consistency of stool, a well-formed pellet gets 0, pasty and semi-formed stools with no sign of stickiness to the animal's anus gets 2, and liquid stools with stickiness to the anus gets 4. In addition, bleeding scores were 0 for no blood, 2 for positive findings, and 4 for gross anal bleeding. The average of these points was reported as CAI.

### 2.10.2. Colon/Body Weight Ratio

To determine the colon/body weight ratio, rats were weighed before scarification. Colon tissue samples were resected, cut longitudinally, and washed with an iced phosphatebuffered solution (pH 7.4). The weight of washed resected colon was also determined and the ratio of the wet colon weight to the body weight of each rat was defined as an index of colonic inflammation [31].

### 2.10.3. Weight/Length Ratio of Colon

The length and wet weight of the washed colon tissue samples was assessed, and the weight/length ratio was determined as a sensitive and reliable indicator of the severity of the colonic inflammation [52].

### 2.10.4. Glutathione Content of the Colon Tissue

The amount of reduced glutathione in the colon tissue was defined spectrophotometrically based on the appearance of yellow color after the addition of DTNB (5,5'-dithiobis- (2-nitrobenzoic acid)) to compounds bearing sulfhydryl groups [53]. In brief, 0.5 mL of tissue homogenate 10% *w*/*v* in phosphate-buffered saline (0.1 M, pH 7.4) was blended with 0.5 mL of 10% *w*/*v* trichloro acetic acid (TCA) and vortexed. The blend was then centrifuged at 2500× *g* for 10 min at 4 ◦C and 0.5 mL of supernatants was withdrawn and mixed with reaction mixtures consisting of 2.5 mL phosphate buffer (pH 8) and 0.5 mL DTNB. The absorbance of the mixture was determined at 412 nm spectrophotometrically (Jenway 6105 UV/Vis, Staffordshire, UK) and GSH contents (nmol/g tissue) were assayed against GSH standard curve prepared in the blank medium [54].

### 2.10.5. Malondialdehyde Content of the Colon Tissue

The malondialdehyde (MDA) concentration of the colon tissue which is related to the severity of colitis was measured [55]. Briefly, colon specimens were frozen in liquid

N2 rapidly after scarification and stored for subsequent evaluation. To determine MDA concentration (nmol/g tissue), 3 mL phosphoric acid (1% *w*/*v*) and 1 mL thiobarbituric acid (TBA) (0.6% *w*/*v*) were introduced to colon tissue homogenate (10% *w*/*v*) in KCl (1.15% *w/v*). The mixtures were heated in a water bath for 45 min. Then, 4 mL of n-butanol was added to the cooled mixture and vortexed for 1 min. The mixture was then centrifuged at 3000× *g* for 10 min. Then, absorbance of the supernatant at 532 nm was determined by a spectrophotometer (Jenway 6105 UV/Vis, Staffordshire, UK) and the MDA concentration was calculated based on the constructed standard curve obtained in the blank medium [56].
