Hepatoprotective Effect of Silymarin Herb in Prevention of Liver Dysfunction Using Pig as Animal Model

Thank you very much for taking the time to review this manuscript. We sincerely appreciate your constructive feedback, which has significantly improved the clarity and focus of the review. Detailed responses to each comment are provided below, with corresponding revisions highlighted in the re-submitted file.

Point-by-Point Response to Comments and Suggestions for Authors

General comment: the study reviews the renowned hepatoprotective effect of milk thistle extract silymarin focusing on pig as an experimental model. Although scarce, porcine studies have confirmed that optimized administration of silymarin reduces hepatocellular injury, restores redox balance and diminish inflammation. The authors include current literature in the field supporting reliable improvements in redox and inflammatory pathways under situations of induced stress, but no demonstration of long-term structural protection. Specific cellular and molecular mechanisms of action of the extract or individual components are detailed, and at the end of the review the authors go over the specific steps of silymarin pharmacokinetics and a comparative bioavailability of different silymarin formulations. Although research in pigs is much more problematic than in rodents, similarities in physiology and metabolism with humans make this a more reliable model for research on hepatoprotective compounds and the outcome needs to be reported and regularly reviewed. The manuscript is a comprehensive work and well-structured where the major concern is the chaotic quotation of references, a crucial point in a review. Some specific comments are detailed below:

Response: We are very grateful for the reviewer’s encouraging overall evaluation and for identifying the key issue regarding inconsistent citation placement. We fully agree that a review’s credibility depends on accurate and logically ordered referencing.

To address this, all citations were thoroughly audited and reorganized using Mendeley Reference Manager, fully synchronized with the latest Nutrients reference style. Each in-text citation was cross-checked against its corresponding reference entry to ensure numbering accuracy, contextual consistency, and chronological correctness. Several misplaced and duplicated citations were corrected, missing references were inserted, and every entry was validated for relevance to the surrounding discussion.

Comment 1: Lines 71-75; references 102 and 103 are misplaced and reference 4 is missing.

Response: Thank you for noticing this. The citations have been corrected — references 102 and 103 were repositioned, and reference 4 has been added in the appropriate place. All references were rechecked and automatically updated through Mendeley Reference Manager to ensure accurate numbering and alignment throughout the manuscript.

Comment 2: Lines 76-77; composition of silymarin is not shown in figure 1, which shows its biological effects. Perhaps the authors meant table 1.

Response: Thank you for this helpful comment. We confirm that the citation of Figure 1 was intentional. The figure was included to illustrate the biological spectrum of silymarin activity rather than its molecular composition, thereby introducing the physiological context of the compound early in the manuscript. To make this clearer for readers, the sentence has been slightly revised as follows:

“Silymarin, the milk thistle (Silybum marianum) extract used in nutrition and phytotherapy, is not a single compound (Figure 1, which illustrates its principal biological activities). It is a mixture dominated by silibinin diastereomers, with contributions from isosilibinin, silychristin, silydianin, and related flavonolignans built on a taxifolin core.”

Comment 3: Lines 86-89; reference 10 is missing.

Response: Thank you for noticing this omission. The missing reference 10 has now been inserted in the appropriate place within Lines 86–89 to support the statement. All surrounding references were cross-checked in Mendeley Reference Manager to ensure proper sequencing and consistency throughout the manuscript.

Comment 4: Lines 109-114; references should be added in support of the specific effects, or a review supporting all of them.

Response: Thank you for this important suggestion. Additional references have been inserted to substantiate the specific mechanistic effects of silymarin described in this section, including its actions on the Nrf2–ARE pathway, NF-κB signalling, TGF-β/SMAD modulation, and AMPK–PPAR regulation. References have also been added for overarching support. The paragraph has been updated as follows: “Biological effects attributed to silymarin include activation of antioxidant defences through the Nrf2–ARE programme—for example, induction of haem oxygenase-1, NAD(P)H:quinone oxidoreductase-1 and glutamate–cysteine ligase [Surai, 2015; Wu et al., 2009]—attenuation of NF-κB-centred inflammatory pathways with reduced tumour necrosis factor-α, interleukin-1β and interleukin-6 [Kim et al., 2013], moderation of TGF-β/SMAD signalling with shifts in matrix metalloproteinase/tissue-inhibitor balance and reduced α-smooth muscle actin and collagen [Jia et al., 2001], and improved lipid handling through AMPK–PPAR circuits [Loguercio & Festi, 2011]. These processes together represent a coordinated antioxidant, anti-inflammatory, and metabolic response consistent with previous mechanistic reviews [Abenavoli et al., 2018].”

Comment 5: Line 193; reference 19 is missing and 41 is misplaced. This fault remains through the rest of the text. The whole reference list should be corrected.

Response: Thank you — this was correct and has been fixed. We have reinserted reference 19 at the correct location, moved reference 41 to its proper in-text position, and performed a complete audit of all in-text citations and the reference list. All citations were regenerated via Mendeley Reference Manager and then manually cross-checked line-by-line to remove misplaced, duplicate or missing entries. The reference list and in-text numbering are now consistent throughout the manuscript; the corrected citations are highlighted in the revised file.

Comment 6: Line 299; the meaning of the sentence: acetaminophen and valproate capture glutathione depletion and mitochondrial stress, may be confusing since capture might indicate retention or arrest whereas the authors mean provoke or induce. The sentence should be explained or changed.

Response: Thank you for this valuable observation. We agree that the word “capture” was imprecise and could be misinterpreted. The sentence has been rewritten to clearly express that acetaminophen and valproate act as mechanistic models that induce glutathione depletion and mitochondrial dysfunction, rather than “capture” these processes. The entire passage was refined for smoother readability and improved mechanistic clarity.

The revised text now reads as follows:

“Carbon tetrachloride (CCl₄) remains a classical hepatotoxicant, metabolised by CYP2E1 to trichloromethyl radicals that initiate centrilobular lipid peroxidation and necrosis [31]. Thioacetamide evokes a similar oxidative injury pattern, progressing to fibrosis with chronic exposure, whereas acetaminophen (paracetamol) and valproate induce glutathione depletion and mitochondrial dysfunction, respectively. Within these intrinsic DILI paradigms, silymarin’s antioxidant and membrane-stabilising properties—mediated via Nrf2 activation and NF-κB suppression—are mechanistically plausible, provided sufficient aglycone exposure reaches hepatocytes [32]. Informative endpoints include serum aminotransferases, glutamate dehydrogenase (GLDH) for mitochondrial specificity, oxidative stress indices (MDA, GSH/GSSG), centrilobular histopathology, and cytokine profiling, ideally complemented by intrahepatic concentration measurements of both aglycones and conjugates [33].”

Comment 7: Line 390; reference 59, published in Poultry Science, is used as example of rodent studies; this should be double checked.

Response: Thank you for this important observation. We have rechecked reference 59 and confirmed that it indeed originated from a poultry study, not from rodent or porcine experiments. This citation was therefore removed from this section and replaced with the correct references supporting porcine data (references 13 and 20), which report reductions in aminotransferases, improved glutathione balance, and histological protection following silymarin administration in pigs.

The corrected sentence now reads as follows:

“Pigs, in contrast, display more modest but mechanistically credible effects: reductions in aminotransferases, improved glutathione indices, and partial histological protection, all contingent on formulations that raise intrahepatic unbound concentrations [13,20].”

Comment 8: References 141-167 are not mentioned in the main text; if they are quoted in supplementary data they should be removed from the manuscript and included only in the supplementary files.

Response: Thank you for highlighting this. We carefully reviewed references 141–167 and confirmed that these citations are not directly cited in the main text but are relevant to supporting data presented in the Supplementary Material. Accordingly, these references have been moved from the main reference list to the Supplementary Files section to maintain consistency with the journal’s formatting guidelines. The main manuscript now contains only references that are explicitly cited in the text, and numbering has been automatically updated in Mendeley Reference Manager to ensure accurate sequencing throughout.