*4.4. Excretion*

Around 99% of PTS is excreted through non-renal pathways, while 0.219% goes through the hepatic pathway [8]. A very small fraction of it was found to be excreted in urine [8]. When comparing intravenous doses of resveratrol and PTS in male rats, the clearance of PTS was much less than that of resveratrol, indicating a longer therapeutic availability [3]. Interestingly, the analysis of the urine samples in cannulated rats identified the parent PTS and its glucuronidated metabolite, which had been previously identified only in the systemic circulation [8]. Increasing the dose of PTS from 2.5 mg/kg to 25 mg/kg decreased the clearance rate by almost half, which is attributed to the saturation or partial saturation of PTS metabolism [24].

#### *4.5. Toxicity*

The administration of PTS, even in high doses, was observed to be nontoxic in mouse models. Four groups of mice were fed PTS doses ranging from 0 to 3000 mg/kg body weight/day for 4 weeks, and there were no significant alterations in the consumption of food or water or in weight gain [23]. Additionally, the in vivo administration of PTS attenuated tumorigenesis and metastasis with negligible toxicity [29]. Pharmacologically, PTS, when intravenously administered, has been noted to be safe, as the compound did not exhibit toxicity specific to any organ [30]. In humans, PTS has been observed to exhibit safety at doses up to 250 mg/day [31].

#### **5. Major Pathways Associated with PTS**

#### *5.1. Antioxidative Pathway: Activation of Nrf2 Signaling*

The antioxidant activity of PTS has been extensively studied and implicated in anticarcinogenesis, the modulation of neurological disorders, the attenuation of vascular diseases, and diabetes management [7]. Extensive evidence has indicated that PTS reduces oxidative stress by attenuating the production of the superoxide anion and hydrogen peroxide, which are implicated in the initiation and progression of various pathogenic processes [7]. Nrf2, a nuclear transcription factor, is one of the major players in regulating cytoprotective and antioxidant genes, which also includes phase II metabolic and antioxidant enzymes [32]. The stimulation of Nrf2 signaling has been identified to produce anti-cancer, anti-diabetic, cardioprotective, and neuroprotective effects [33]. Kelch-like ECH-associated protein 1 (Keap-1) is a negative regulator of Nrf2 and targets the transcription factor for ubiquitylation and degradation. Our lab investigated the protective properties of PTS in pancreatic β-cell apoptosis through an Nrf2-mediated mechanism [32] (Figure 1). We found that PTS activates the Nrf2 pathway, thereby triggering the expression of Nrf2 downstream target genes to facilitate cellular protection in INS-1E cells. In particular, we demonstrated that PTS binds to the arginine residues of Keap-1 and facilitates its disassociation from Nrf2 [34]. Interestingly, PTS has also been reported to mediate the recruitment and interaction of the P62 autophagic cargo with Keap-1, thereby inducing the autophagic degradation of Keap-1 [35]. The major downstream targets activated following PTS administration were antioxidative enzymes, including HO 1, SOD, catalase, and GPX. Moreover, we observed the upregulation of anti-apoptotic gene expression, with the associated downregulation of the expression of the pro-apoptotic mediators Bax (Bcl-2 associated X protein) and caspase-3. Our evidence was strongly suggestive of the protective effect of PTS administration against hyperglycemia-induced oxidative damage in pancreatic β-cells [32] (Figure 1).

Our lab inferred that PTS protects β-cells in Streptozotocin (STZ)-induced diabetic mice, an effect accompanied by the induction of Nrf2 and the consequent upregulation of its target genes. We also investigated the anti-peroxidative role of PTS in the STZ-induced diabetic model. In addition, PTS normalized the circulatory concentration of VLDL and LDL while reducing lipid peroxidation in STZ-induced diabetic mice. Notably, the livers of diabetic mice indicated collapsed hepatic microvesicles on H&E staining due to altered lipid metabolism [36]. PTS administration reduced structural and functional alterations in the hepatic tissue, indicating its protective function in diabetic dyslipidemia mediated via Nrf2 activation [37] (Figure 1).

By countering oxidative damage, PTS treatment inhibited human retinal endothelial cell proliferation and delayed the progression of diabetic retinopathy [38]. In the livers of IUGR piglets, PTS attenuated liver injury caused by Nrf2 activation and the consequent induction of the antioxidant response [39]. Notably, adrenocorticotropic hormone (ACTH) was observed to interfere with Nrf2 signaling in metastatic cells. PTS reduced ACTH activity and was effective against various melanoma cell lines, including MelJuso, A2058, and MeWo [30]. PTS blocked cellular inflammation and oxidative stress in azoxymethaneinduced colon carcinogenesis, thereby reducing tumorigenesis. Through the activation of Nrf2, PTS countered the induction of NF-κB (nuclear factor-kappaB) and diminished the levels of oxidative stress mediators, including inducible nitric oxide synthase (iNOS), Cyclo-oxygenase-2 (COX-2), and aldolase reductase in an AOM-induced colon cancer rodent model [40]. Furthermore, by maintaining glutathione, catalase, SOD, and GSH peroxidase activity through the Nrf2–antioxidant response, PTS showed anti-cancer activity in a UVB-stimulated skin cancer model [41]. In the innate immune system, neutrophils produce reactive oxygen species (ROS) to destroy pathogens with the help of NADPH oxidase, which produces a superoxide anion. The overproduction of ROS can cause tissue damage that is observed in diseases such as rheumatoid arthritis and ischemic injury. PTS lowered the neutrophil count in arthritic animals and facilitated a mild decrease in ROS production, with a limited effect on neutrophil activity [42].

In a dose-dependent manner, PTS has been identified to exhibit a potent antioxidant effect against several free radicals, including 2,2-Diphenyl-1-picryl-hydrazyl (DPPH), 2,2 -Azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), hydroxyl, superoxide, and hydrogen peroxide. Furthermore, PTS treatment is associated with increased antioxidant enzymes, such as SOD and GPX, via Nrf2 activation in neuronal cells [43]. In Alzheimer's disease models, PTS increased PPAR-α, a modulator of neural antioxidant activities [43].

**Figure 1. Nrf-2-mediated antioxidant pathway of pterostilbene:** Activation and phosphorylation of Nrf-2 signaling is the major mechanism through which the antioxidative response is induced by PTS. Ubiquitination mediated by Cullin-3 (CUL-3) leads to the proteasomal degradation of Nrf-2. PTS inhibits the ubiquitin–proteasome system, thereby increasing the accumulation of Nrf-2. PTS also enables the phosphorylation of Nrf-2, which is critical in the nuclear translocation of the transcription factor. Moreover, PTS phosphorylates and activates the ERK signaling pathway, which mediates the dissociation of Keap-1, resulting in Nrf-2 activation. Furthermore, PTS stimulates the binding of Keap-1 and p62, which enhances the activation of Nrf2. Following its activation and nuclear translocation, Nrf-2 binds to ARE and induces the expression of antioxidant enzymes, which in turn critically attenuate oxidative damage in host cells.

#### *5.2. Pro- and Anti-Apoptotic Pathways*

PTS inhibited cell proliferation and acted as an active inducer of apoptosis in certain cancerous cell lines [8]. Moreover, PTS treatment induced caspase release and O2 − production, which depolarizes the mitochondrial membrane, triggering the intrinsic mitochondrialderived apoptosis of cancerous cells [44,45]. Chakraborty et al. identified that PTS modified markers associated with mitochondrial apoptosis and improved the expression of the antioxidant enzymes GPx, GR, and GSH in an in vitro prostate cancer model [46]. Moreover, PTS induced apoptosis in gastric adenocarcinoma cells through the increased upregulation of cytochrome C, Bad, Bax, and caspases [47]. Genomic analysis revealed that PTS treatment in pancreatic cancer upregulated pro-apoptotic genes and anti-proliferative markers [48].

Interestingly, PTS was reported to inhibit the effects of apoptosis in vascular endothelial cells [49]. Apoptosis induces plaque instability in atherosclerosis, where oxLDL (oxidized low-density lipoprotein) triggers the apoptosis of VEC by activating lectin-like oxLDL receptor-1. PTS inhibits the apoptosis induced by oxLDL and stimulates cytoprotective autophagic cell death in VECs, thereby dampening the atherosclerotic effect of oxLDL [50]. Notably, this effect was achieved by increasing the accumulation of intracellular calcium, followed by the subsequent activation of the AMPK α1 subunit (AMPKα1) [50]. PTS also suppressed the oxidative damage induced by oxLDL by reducing the mitochondrial membrane potential and lowering the levels of pro-apoptotic proteins such as Bax and p53 [49]. PTS administration to cochlear cells obtained from STZ-induced diabetic rats demonstrated the protection of the cochlea from ototoxicity through the inhibition of apoptosis [51]. Interesting work from our lab deduced that cytoprotection by PTS against cytokine-induced cellular damage in MIN6 mouse pancreatic cells involves the activation of Nrf2 signaling, associated with the inhibition of pro-apoptotic signaling through the attenuation of the BAX/Bcl-2 ratio and the reduced activity of caspase-3 [36]. Our evidence indicates that the PTS-mediated anti-apoptotic effect is also a consequence of Nrf-2 activation (Figure 2).

**Figure 2. Anti-apoptotic pathway of pterostilbene:** PTS mediates its cytoprotective effect mainly through the activation of Nrf-2, which in turn protects the mitochondrial functioning, thereby causing a reduction in the induction of pro-apoptotic factors such as cytochrome C, BAX, and caspase-3. In vascular endothelial cells, PTS protects against the initiation of apoptotic signaling by countering the effect of oxLDL in activating its receptor lectin-like oxidized low-density lipoprotein (LOX-1), thereby preventing the accumulation of P53 as well as the activation of NFκB. Moreover, PTS increases the intracellular calcium levels and promotes the cytoprotective autophagy of the cell, consequently preventing the deleterious effect of apoptosis.
