Oral toxicity assessment of PD fraction
The oral administration of PD fraction at all dose levels did not cause any fatalities or clinical indications of toxicity in rats, in both acute and subacute toxicity trials. No significant body weight variations were observed between the control and the PD test groups and the groups exhibited a typical incremental gain in body weight (Fig.1 a, c). On gross examination of vital organs, no histological alterations or damages were observed, and the relative organ weight was found to be statistically similar (p>0.05) between the control and PD test group (Fig.1 b, d). The hematological and serum biochemical profile of both the toxicity studies is summarized in Table 1, and the results indicated that all hematological parameters are within the normal range in both control and PD test groups. No significant difference was evidenced in liver and kidney function parameter values relative to the control group.
Anti-colitis effects of PD fraction in Trinitrobenzene sulfonic acid induced animal Ulcerative colitis model
Disease activity index (DAI) score
There were no fatalities in the experimental groups. A single-dose of TNBS enema in the colitis model population caused acute colitis, diarrhea and hemoccult, followed by severe weight loss from day 5 to day 20 as observed by the DAI score (Fig. 2a). In the Sulfasalazine treated groups, bloody stools were not observed and stools were intact like pellets, and rats showed less weight loss. DAI score was decreased in the rats fed with PD fraction in a dose- dependent manner as compared to TNBS model rats. Previous studies on oral toxicity of E. superbum extracts and fractions isolated from seeds and pseudostem were found to be non-toxic (LD50 = 3235.9 mg/kg) . In our study the No-Observed Adverse Effect Level (NOAEL) of the PD fraction was valued to be higher than 2000 mg/kg/day in rats. Hence, it can be concluded that PD fraction is safe for oral administration.
Colon tissue assessment
Microscopic examination of the healthy control population's colon tissue showed that it was complete, intact and free of damage with a histological colitis score of zero (Fig 2b, c). In the UC model group, there was a severe disruption of colon tissues with ulceration and inflammation involving the epithelium layer and all intestinal layers. The inflammatory response was consistent with the transmural invasion of inflammatory cells resulting in structural distortion of crypts, epithelial degradation and goblet cell depletion. The UC model had the highest histological scoring for colitis (p<0.001) with corresponding high score for inflammation and crypt damage (Fig. S1 a-d & Fig. 2b, c). However, rats treated with Sulfasalazine and PD fractions showed a gradual healing of colon tissue impairment with a lower colitis score and fewer ulceration and severities (Fig S1 a-d & Fig. 2b, c). This was also evidenced by a lower tissue regeneration score (Fig. S1 d & Fig. 2c). The efficacy of PD on TNBS-induced colonic injury was dose-dependent, with 500 mg/kg being significantly more efficacious than 100 mg/kg (Fig. S1 c-d & Fig. 2c).
Colon tissue redox/oxidative imbalance markers
The function of redox/oxidative imbalance in laboratory animals and human participants with UC has been widely researched, with higher ROS levels coupled by decreasing antioxidant activity in the inflamed mucosa, resulting in greater chronic tissue injury . Thus, the UC mucosa may be in a continual state of redox imbalance, posing a major danger to intestinal tissue homeostasis and increasing endogenous antioxidant demand. Therefore, these mechanisms may provide useful therapeutic strategies for the development of novel medications [18, 19].
Furthermore, suppressing ROS-inducing enzymes, active quenching of ROS, or improving cellular antioxidant reserves are some of the interventions for UC treatment . The dominant cellular antioxidants expressed or released into the blood stream following a disease-related event are superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT). They are often used as biomarkers for inflammation and oxidative stress [21, 22]. Numerous experiments have shown that SOD levels decrease under UC environments. In contrast to these results, studies have also indicated increased SOD activity. The above inconsistent outcomes may be attributed to an active SOD protection mechanism in the disease situation leading to a rise in the activity, and the reduction in activity could be attributed to the utilization of the SOD enzyme to compensate for tissue damage and inflammation [23, 24]. The antioxidant markers analyzed in colon samples are presented as Table 2. In our experiment, Superoxide dismutase (SOD) activity was decreased (p<0.001) in colon tissue samples of the UC model population (4.177 U/mg protein). Whereas, the SOD antioxidant function improved in rats treated with Sulfasalazine (10.5) and PD500 (11.4), with function statistically comparable (p>0.05) to the healthy control group (12.6) (Table 2). Catalase a hydrogen peroxide-degrading enzyme is found in the cytoplasm and peroxisomes of the colon epithelial and lamina propria, and is triggered when hydrogen peroxide levels rise during an inflammatory response . In this study, tissue catalase antioxidant activity is decreased in the UC model population (13.3 U/mg protein), and the difference was statistically significant (p<0.001) as compared to the activity in the normal control rats (50.5). The catalase function in rats treated with sulfasalazine (25.8) and all doses of PD fraction (25.4, 26.3, 26.6) were statistically comparable to each other, with an increase in enzyme activity when compared to the UC model population.
Glutathione is a tripeptide composed of cysteine, glycine, and glutamic acid and occurs as reduced (GSH) and oxidized (GSSG). Glutathione deficit in the intestinal mucosa has been linked to UC, and it is routinely assessed as a sign of in-vivo lipid peroxidation and redox/oxidative imbalance . Glutathione-S-transferases, glutathione peroxidases (GPx), and glutathione reductase (GR) are antioxidant enzymes active in the GSH cycle that shield the intestinal mucosa from redox/oxidative and inflammatory injury . The tissue glutathione reduced, peroxidase and reductase function was shown to be depleted in the UC model population (0.7 nmol/GSH/min/mg protein, 0.11 U/mg protein and 6.9 U/mg protein, respectively), and was statistically significant (p<0.001) to the healthy rat population (2.0, 0.46 and 20.0, respectively). In contrast, the reduced glutathione, glutathione peroxidase, and reductase enzyme functions were increased in the PD500 group (1.3, 0.33 and 13.3, respectively), with statistically (p>0.05) similar results to the Sulfasalazine population (1.4, 0.36 U/mg and 13.6, respectively). Furthermore, membrane lipids in UC are vulnerable to ROS-induced disruption, resulting in lipid peroxidation products reflected in malondialdehyde (MDA) levels . The PD500 treatment caused a marked reduction in tissue MDA levels (0.04µmol MDA/mg protein), indicating that the PD fraction effectively reduced TNBS induced lipid peroxidation, which was greater (p<0.001) in the UC group (0.08). These data demonstrate that PD500's activity and underlying mechanism of protection against UC may be due to its ability to influence redox hemostasis, reducing the severity of tissue damage and inflammation. Additionally, biological samples contain a plethora of antioxidant substances both in the aqueous phase (urate, ascorbate, bilirubin, and thiols) as well as in lipophilic phase (α-tocopherol, carotenoids, and flavonoids) [21, 28]. Previous investigations have highlighted the presence of polyphenols such as quercetin-3-O-rutinoside, 3,5-dimethoxy-4-hydroxybenzoic acid, and 4′,5,7-trihydroxyflavone in the PD fraction . The beneficial properties of polyphenols found in the fraction, as well as their synergistic interactions with the antioxidants present in the tissue, might explain the PD fraction's enhanced antioxidant capacity and efficacy in UC.
Colon tissue inflammation markers
The heme protein myeloperoxidase (MPO), which is naturally generated by phagocytes, particularly neutrophils, is a diagnostic for localized leukocyte aggregation and inflammation. MPO activation has been related to disease severity in UC patients with inflamed colon biopsy [29, 30]. The current study found a substantial increase in MPO activity (1.13 mU/g tissue), indicating an alleviation of colon inflammation in the UC model population. On the other hand, the control population showed residual MPO activity (0.25), which is consistent with the absence of an inflammatory process. The rats treated with PD fraction improved their MPO function in a dose-dependent way, and the effects of PD500 (0.36) were similar to those of Sulfasalazine (0.34) (Table 2). Overall, the drop in colonic MPO function and the histologic observations of reduced cellular infiltration (Fig. 2b, c) following PD500 administration show that it has potent anti-inflammatory effects.