Milk Thistle’s Secret Weapon: Thromboelastometry Reveals How Silybin Modulates Coagulation in Human Plasma In Vitro

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes an in vitro ROTEM study evaluating how silybin (10, 50, 100 µM) modulates coagulation parameters in normal human plasma; however, it needs improvement in a few areas.

There are several formatting issues (e.g., in the abstract). Please correct them.

Please provide citations for this statement: “However, most of the available evidence derives from in vitro and cell-based studies…”.

The authors repeatedly state the effects are “clearly concentration-dependent” and “in a concentration-dependent manner.” However, several key effects appear strongest at 10–50 µM and do not translate at 100 µM (suggesting plateauing or reversal). The wording should be toned down, and the authors should provide a clear discussion of why the effect is not monotonic at 100 µM.

Plasma vs in vivo framing: The experiments are performed in normal human plasma (platelet-poor), yet many parts of the discussion address broader hemostasis in vivo. The authors acknowledge this limitation, but it is significant enough that the title/claims should be tightened (e.g., “in plasma” should be prominent), and the conclusions/discussion should be more cautious about extrapolation.

The Methods/Abstract state each measurement was performed four times, but the Results also state there were 192 ROTEM measurements (64 per assay). It is not clear what this means for the true n per condition, or whether these are technical repeats from a single commercial plasma lot versus multiple lots/donors. The authors should clarify plasma sourcing (commercial lot vs multiple lots) and clearly define n for each group/assay.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents novel and potentially significant data on the effect of silybin on coagulation, as assessed by rotational thromboelastometry (ROTEM). However, the interpretation of the findings requires further clarification and a more in-depth mechanistic discussion. Several methodological details also need to be specified to allow proper assessment of the physiological relevance of the results:

1) In the Introduction, the authors describe anticoagulant effects of silybin, whereas in the Results section they report a shortening of clotting time (CT), which rather suggests a procoagulant effect. Please formulate the initial hypothesis more explicitly and clarify how these apparently contradictory findings can be reconciled with the existing body of evidence. In the Discussion, it is suggested that CT shortening may reflect accelerated initiation of fibrin formation; however, this interpretation conflicts with published data demonstrating inhibition of coagulation enzymes by silybin. In this regard, please expand the discussion to consider possible alternative mechanisms.

2) The authors appropriately identify the use of platelet-poor plasma as a limitation, given that the use of plasma instead of whole blood substantially alters the physiological interpretation of ROTEM parameters. Nevertheless, the authors are requested to specify more precisely which aspects of the results can and cannot be extrapolated to in vivo hemostasis, and to temper the statement that ROTEM “reflects in vivo hemostasis” within the context of the present experimental design.

3) It is not specified whether the plasma used in the experiments was fresh or frozen. In addition, please provide information on the number of donors and clarify whether the measurements were performed using plasma obtained from a single individual or from multiple donors.

4) Please comment on whether the concentrations of silybin used in this study (up to 100 μM) are pharmacologically relevant. Relevant literature data on plasma concentrations of silybin in humans should be added.

5) Please clarify whether parameters such as maximum clot firmness (MCF) or lysis indices (LI30/LI60) were measured, and if not, provide a rationale for their exclusion.

6) Please carefully revise the manuscript for typographical and minor linguistic errors.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I consider that you have thoroughly and appropriately addressed my comments, thereby substantially improving the transparency and informativeness of the submitted manuscript.

In its present form, I find the manuscript suitable for publication in the journal.

Kind regards,