Phytochemical Profile and In Vivo Assessment of Toxicity and Anti-Inflammatory Activity of Cenostigma pluviosum var. peltophoroides (Benth.) Gagnon & G.P. Lewis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

There are also sufficient neutral tests of the hypothesis results, including positive controls and quality checks. 

I believe that this work has no weaknesses. 

 

Comments on the Quality of English Language

The English expression could be revised a bit.

Author Response

Comments 1: 
There are also sufficient neutral tests of the hypothesis results, including positive controls and quality checks. 
I believe that this work has no weaknesses. 

Response 1: We thank the reviewer for their positive evaluation of our work and for recognizing the robustness of our experimental design.

Point 1: The English expression could be revised a bit.
Response: We thank the reviewer for this suggestion. The manuscript has been revised to improve the English language.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article entitled Phytochemical Profile and In vivo Assessment of Toxicity and Anti-inflammatory Activity of Cenostigma pluviosum var. peltophoroides (Benth.) Gagnon & G.P. Lewis investigates an ethanolic bark-derived extract, focusing on its chemical composition by HPLC-ESI-MS/MS, acute toxicity following a single 2000 mg/kg dose, genotoxic potential assessed using the micronucleus and comet assays, and anti-inflammatory activity evaluated through carrageenan-induced paw edema and peritonitis models.

Overall, the topic is relevant, as the study combines phytochemical characterization with in vivo toxicological and pharmacological assessment of a plant-derived extract. However, several methodological and reporting aspects require clarification before the manuscript can be properly evaluated.

The English language should be revised throughout the manuscript, as there are several instances of awkward phrasing and unclear wording that may affect the clarity of the scientific message.

Results section

• The authors should clarify what is meant by “the sample” in the statement “the sample was reinjected into a high-resolution mass spectrometer”. It is not clear whether this refers to the crude plant extract, a previously separated chromatographic fraction, a collected LC peak, or another prepared sample. The authors should specify the exact nature of the injected sample, its preparation before MS analysis, the solvent used, concentration, injection volume, and whether any filtration or dilution step was performed.
• The authors should clarify how the % inhibition of paw edema and percentage reduction after 4 h in Figure 3 were calculated.
• The unit or scale of the Y-axis in Figure 3A is ambiguous. The values are presented between 0 and 0.8, although the axis is labelled as a percentage. If the data represent percentages, they should be expressed on a 0–100% scale.
• In Figure 3A, based on the graphical presentation, the significant differences indicated at 2 h between the positive control and the 100 mg/kg and 200 mg/kg extract groups appear unlikely. The authors should carefully verify the raw data, statistical output, and figure annotations, as a possible error in the reported significance markers cannot be excluded.
• Although the Methods section indicates that anti-inflammatory activity was calculated using AUC and total prevention (TP%), the corresponding results are not adequately reported or discussed. The authors should provide the AUC and TP% values for all groups, clarify how these calculations were performed, and integrate these parameters into the Results and Discussion to support their conclusions.

Materials and Methods section

• The statement that “the leukocyte count of the peritoneal lavage was performed and expressed as a percentage” is unclear. If the authors calculated the percentage inhibition of leukocyte migration or the differential leukocyte percentage, this should be clearly stated and the calculation method should be provided.
• For the carrageenan-induced peritonitis model, the cited methodological reference is a Portuguese-language book that does not appear to be readily accessible. The authors should provide an additional accessible reference, preferably a peer-reviewed article or methodological guideline, to improve verifiability and reproducibility.

Overall, the manuscript addresses a relevant topic and may provide useful information on the phytochemical profile, safety, and anti-inflammatory potential of Cenostigma pluviosum var. peltophoroides bark extract. However, several methodological details, aspects of data presentation, statistical annotations, and English language issues should be carefully revised before the manuscript can be considered for publication.

Author Response

Comments 1:  

The article entitled Phytochemical Profile and In vivo Assessment of Toxicity and Anti
inflammatory Activity of Cenostigma pluviosum var. peltophoroides (Benth.) Gagnon & 
G.P. Lewis investigates an ethanolic bark-derived extract, focusing on its chemical 
composition by HPLC-ESI-MS/MS, acute toxicity following a single 2000 mg/kg dose, 
genotoxic potential assessed using the micronucleus and comet assays, and anti
inflammatory activity evaluated through carrageenan-induced paw edema and peritonitis 
models. 
Overall, the topic is relevant, as the study combines phytochemical characterization with 
in vivo toxicological and pharmacological assessment of a plant-derived extract. However, 
several methodological and reporting aspects require clarification before the manuscript 
can be properly evaluated. 

Response 1: We thank the reviewer for the evaluation of our manuscript and for 
highlighting the relevance of our study. We have addressed the concerns raised regarding 
methodological and reporting aspects, and the manuscript has been revised. 

Comments 2:  
The English language should be revised throughout the manuscript, as there are several 
instances of awkward phrasing and unclear wording that may affect the clarity of the 
scientific message. 

Response 2: We thank the reviewer for this suggestion. The manuscript has been revised 
to improve the English language and clarity. 

Comments 3:  
The authors should clarify what is meant by “the sample” in the statement “the sample 
was reinjected into a high-resolution mass spectrometer”. It is not clear whether this refers 
to the crude plant extract, a previously separated chromatographic fraction, a collected LC 
peak, or another prepared sample. The authors should specify the exact nature of the 
injected sample, its preparation before MS analysis, the solvent used, concentration, 
injection volume, and whether any filtration or dilution step was performed. 

Response 3: The requested information has been incorporated into the manuscript. All 
parameters are now clearly described in Section “4.2 Extraction and chemical 
characterization of the CEECP by LC–MSⁿ”. The corresponding text is shown below: 
“Column temperature was maintained at room temperature (20 °C).” (…) For the analysis, 
a sample of 1 mg of the crude ethanolic extract of C. pluviosum stem bark (CEECP) was 
dissolved in methanol to obtain a 1 mg·mL⁻¹ solution. The solution was filtered through a 
0.45 μm PVDF membrane, and 10 μL was injected into the chromatographic system. 
(…) The analysis parameters were: capillary 3.5 kV, ESI in alternating mode (negative), 
end plate offset 500 V, nebulizer 4 psi, dry gas (N₂) with a flow rate of 8 L/min and 
temperature of 200 °C. CID fragmentation was achieved in auto MS/MS mode using the 
advanced resolution mode for MS and MS/MS mode. The spectra (m/z 50–1500) were 
recorded every 2 s. (Lines 542-549). 

Comments 4:  
The authors should clarify how the % inhibition of paw edema and percentage reduction 
after 4 h in Figure 3 were calculated. 

Response 4: The percentage of edema was determined using the following formula:

Percentage change in hind paw thickness  ((final hind paw thickness − initial hind paw thickness)/
final hind paw thickness initial hind paw thickness)?100 
The corrected formula is now present in section “4.4.1 Carrageenan-induced paw edema”, 
line 651. 

Comments 5:  
The unit or scale of the Y-axis in Figure 3A is ambiguous. The values are presented 
between 0 and 0.8, although the axis is labelled as a percentage. If the data represent 
percentages, they should be expressed on a 0–100% scale. 

Response 5: Figure 3A has been corrected to ensure that the Y-axis scale and labeling are 
correct. 

Comments 6:  
In Figure 3A, based on the graphical presentation, the significant differences indicated at 
2 h between the positive control and the 100 mg/kg and 200 mg/kg extract groups appear 
unlikely. The authors should carefully verify the raw data, statistical output, and figure 
annotations, as a possible error in the reported significance markers cannot be excluded. 

Response 6: The raw data, statistical analyses, and figure annotations have been carefully 
rechecked. We confirm that the reported significance markers are correct; however, Figure 
3A has been revised to improve clarity and avoid any potential misinterpretation. 

Comments 7:  
Although the Methods section indicates that anti-inflammatory activity was calculated 
using AUC and total prevention (TP%), the corresponding results are not adequately 
reported or discussed. The authors should provide the AUC and TP% values for all 
groups, clarify how these calculations were performed, and integrate these parameters 
into the Results and Discussion to support their conclusions. 

Response 7: The calculation method has been revised, and the formula previously used 
has been corrected. Accordingly, the AUC and TP% values have been removed and a more 
appropriate analytical approach is now presented and discussed in the revised 
manuscript, in the section, “4.4.1 Carrageenan-induced paw edema”, line 651. 

Comments 8: 
The statement that “the leukocyte count of the peritoneal lavage was performed and 
expressed as a percentage” is unclear. If the authors calculated the percentage inhibition 
of leukocyte migration or the differential leukocyte percentage, this should be clearly 
stated and the calculation method should be provided. 

Response 8: The formula for leukocyte inhibition is now present in the section “4.4.2 
Perionitis” (line 658). The following formula was used: 
% Leukocyte inhibition = (1−T/C) × 100 

Comments 9: 
For the carrageenan-induced peritonitis model, the cited methodological reference is a 
Portuguese-language book that does not appear to be readily accessible. The authors 
should provide an additional accessible reference, preferably a peer-reviewed article or 
methodological guideline, to improve verifiability and reproducibility. 

Response 9: The previously cited reference has been replaced with an accessible source to 
enhance the quality of the text. 

Comments 10:  
Overall, the manuscript addresses a relevant topic and may provide useful information on 
the phytochemical profile, safety, and anti-inflammatory potential of Cenostigma pluviosum 
var. peltophoroides bark extract. However, several methodological details, aspects of data 
presentation, statistical annotations, and English language issues should be carefully 
revised before the manuscript can be considered for publication. 

Response 10: We thank the reviewer for the evaluation of our manuscript and for 
recognizing the relevance of our study. All comments regarding methodological details, 
data presentation, statistical analysis, and English language have been corrected. 

4. Response to Comments on the Quality of English Language 
Point 1: The English expression could be revised a bit. 
Response 1: We thank the reviewer for this suggestion. The manuscript has been revised 
to improve the English language and clarity.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is well written with sound methodology providing the comprehensive evaluation of the crude ethanolic extract of the stem bark integrating HPLC-ESI-MS/MS phytochemical profiling acute oral toxicity and in vivo anti-inflammatory activity. After minor revisions, the manuscript will be a good choice to be accepted.

The language has few awkward phrasing and grammatical issues so a good proofreading pass is recommended.

I think author should explain the “CEECP” vs. “extract” consistently. 

Author should avoid the over-repetition of results in discussion section and should strengthen the mechanistic links to specific compounds.

Figures with high-resolution and error bars are suggested for production.

It will be great if author can add the retention time in Table 1

Tables should have uniform formatting and should include all statistical letters.

The author should mention the column temperature, injection volume, and exact ESI parameters in methods.

I suggest the author to mention the animal ethics approval number, housing conditions and statistical tests. 

The author should explain why the (50/100/200 mg/kg) doses were selected? Either on preliminary data or based on previous literature? 

 I think the author should mention the kit manufacturer and validation details.

I suggest to expand the discussion slightly on structure–activity relationships for key compounds and their comparison with related species. 

Add a limitation statement for single-dose toxicity testing and need for mechanistic validation. 

Make sure that all references have DOIs and complete any placeholders.

Cite (https://www.tandfonline.com/doi/full/10.2147/DDDT.S494417) in Introduction or in discussion when discussing ethnopharmacological validation and the need for integrated toxicity of traditionally used plants.  Cite (https://www.sciencedirect.com/science/article/pii/S0753332222002165) for chemical characterization of extracts of leaves of Kadsura coccinea by UHPLC and assessment of their antioxidant and anti-inflammatory activities. Cite (https://www.sciencedirect.com/science/article/abs/pii/S002228602401785X) for anti-inflammatory activity evaluation and molecular docking analysis compounds isolated and potential NF-κB modulation mechanisms. 

Final Recommendation: Accept after minor revision

Author Response

Comments 1:  
The manuscript is well written with sound methodology providing the comprehensive 
evaluation of the crude ethanolic extract of the stem bark integrating HPLC-ESI-MS/MS 
phytochemical profiling acute oral toxicity and in vivo anti-inflammatory activity. After minor 
revisions, the manuscript will be a good choice to be accepted. 

Response 1: We thank the reviewer for the positive evaluation of our manuscript. The 
suggested minor revisions have been addressed, and the manuscript has been revised. 

Comments 2:  
The language has few awkward phrasing and grammatical issues, so a good proofreading 
pass is recommended. 

Response 2: We thank the reviewer for this suggestion. The manuscript has been revised to 
improve the English language and clarity. 

Comments 3:  
I think author should explain the “CEECP” vs. “extract” consistently.  

Response 3: The terminology has been standardized throughout the manuscript. After the 
initial definition, the extract is consistently referred to as CEECP or its full name to avoid 
ambiguity. 
 

Comments 4:  
Author should avoid the over-repetition of results in discussion section and should strengthen 
the mechanistic links to specific compounds. 

Response 4: Additional information has been added in the Discussion section, as follows: 
“These anti-inflammatory properties of ellagitannins are supported by experi-mental data in 
a variety of models. Corilagin has shown in vivo efficacy in in treating cholestasis at 40 mg/kg, 
lowering oxidative stress indicators and reducing NF-κB acti-vation, evidenced by decreased 
nuclear translocation of the p65 subunit. Chebulanin exhibited anti-arthritic effects at 80 
mg/kg and inhibited NF-κB and MAPK signaling pathways, as demonstrated by reduced 
phosphorylation of IκBα and NF-κB p65, as well as suppression of MAPK components such 
as p38 and JNK. Interestingly, less po-lar and more structurally complex tannins like 
chebulagic acid and pedunculagin demonstrated greater activity at lower concentrations (5
25 µM), inhibiting NF-κB and MAPK activation in cellular models and suppressing pro
inflammatory mediators like NO, PGE2, TNF-α, and IL-6. These effects were associated with 
decreased phos-phorylation of IκBα, reduced nuclear levels of NF-κB subunits (p65 and p50), 
and inhi-bition of MAPK signaling proteins, including p38, JNK, and ERK.” (Lines 489-501). 

Comments 5:  
Figures with high-resolution and error bars are suggested for production. 

Response 5: All figures have been carefully checked and are provided at a resolution of 300 
dpi, ensuring adequate quality for publication. The error bars were also corrected. 

Comments 6:  
It will be great if author can add the retention time in Table 1 

Response 6: The retention time was already included in Table 1. A footnote has been added 
defining ‘R.T.’ as retention time, to improve clarity. 

Comments 7:  
Tables should have uniform formatting and should include all statistical letters. 

Response 7: The tables have been revised to ensure uniform formatting and now include all 
statistical letters. 

Comments 8: 
The author should mention the column temperature, injection volume, and exact ESI 
parameters in methods. 

Response 8: The requested information has been incorporated into the manuscript. All 
parameters are now clearly described in Section “4.2 Extraction and chemical characterization 
of the CEECP by LC–MSⁿ”. The corresponding text is shown below: 
“Column temperature was maintained at room temperature (20 °C)”. (Lines 542-543). 
“For the analysis, a sample of 1 mg of the crude ethanolic extract of C. pluviosum stem bark 
(CEECP) was dissolved in methanol to obtain a 1 mg·mL⁻¹ solution. The solution was filtered 
through a 0.45 µm PVDF membrane, and 10 µL was injected into the chromatographic 
system.” (Lines 549-553). 

Comments 9: 
I suggest the author to mention the animal ethics approval number, housing conditions and 
statistical tests.  

Response 9: The animal ethics approval number and housing conditions are described in 
Section 4.3.1, while the statistical analyses are detailed in Section 4.6 (Statistical Analysis). 
 

Comments 10:  
The author should explain why the (50/100/200 mg/kg) doses were selected? Either on 
preliminary data or based on previous literature?  

Response 10: The justification for the use of the doses in the experiment is now present in the 
paper, as described below: 
“Doses of 50, 100, and 200 mg/kg were selected for the anti-inflammatory as-says based on 
previous studies evaluating the pharmacological effects of species from the genus Cenostigma, 
in which this range is commonly employed in in vivo models. Additionally, the selection of 
these doses was supported by the results of the acute oral toxicity test. Therefore, the chosen 
doses fall within a safe range for pharmacological evaluation while allowing the assessment 
of dose-dependent effects.” (Lines 636-641). 

Comments 11:  
I think the author should mention the kit manufacturer and validation details. 
details. 

Response 11: The manuscript has been revised to include the kit manufacturer and relevant 
validation 
The corresponding “4.3.3. Biochemical and hematological analysis text is shown below: 

"At the end of toxicity assays, blood was collected from the animals and the fol-lowing 
biochemical parameters were evaluated: total protein, albumin, alanine aminotransferase 
(ALT), aspartate aminotransferase (AST), alkaline phosphatase, gamma-glutamyl transferase 
(GGT), urea and creatinine, using specific kits (Labtest Diagnóstica, Lagoa Santa, Brazil) and 
a COBAS Mira Plus analyzer (Roche Diagnostics Systems, Basel, Switzerland). Hematological 
analysis was performed using an automatic ana-lyzer (Counter-ABC Vet Animal Blood, 
Montpellier, France) and optical microscopy; the parameters evaluated were: erythrocytes, 
hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin 
(MCH), mean corpuscular hemoglo-bin concentration (MCHC) and total and differentiated 
leukocyte analysis.” (Lines 597-608). 
“Cytokine levels in the peritoneal fluid were quantified using a bead-based multiplex 
immunoassay (Milliplex MAP 7-plex kit, Millipore, Merck, Darmstadt, Germany), ac-cording 
to the manufacturer’s instructions”. (Lines 670-673). 

Comments 12:  
I suggest to expand the discussion slightly on structure–activity relationships for key 
compounds and their comparison with related species.  

Response 12: The Discussion section has been expanded to include considerations on 
structure–activity relationships of key compounds and their comparison with related species. 
The corresponding text is shown below: 
“These anti-inflammatory properties of ellagitannins are supported by experimental data in a 
variety of models. Corilagin has shown in vivo efficacy in treating cholestasis at 40 mg/kg, 
lowering oxidative stress indicators and reducing NF-κB activation, evidenced by decreased 
nuclear translocation of the p65 subunit. Chebulanin exhibited anti-arthritic effects at 80 
mg/kg and inhibited NF-κB and MAPK signaling pathways, as demonstrated by reduced 
phosphorylation of IκBα and NF-κB p65, as well as suppression of MAPK components such 
as p38 and JNK. Interestingly, less polar and more structurally complex tannins like 
chebulagic acid and pedunculagin demonstrated greater activity at lower concentrations (5-25 µM), inhibiting NF-κB and MAPK activation in cellular models and suppressing pro-inflammatory mediators like NO, PGE2, TNF-α, and IL-6. These effects were associated with 
decreased phosphorylation of IκBα, reduced nuclear levels of NF-κB subunits (p65 and p50), 
and inhibition of MAPK signaling proteins, including p38, JNK, and ERK”. (Lines 489-501). 

Comments 13:  
Add a limitation statement for single-dose toxicity testing and need for mechanistic 
validation.  

Response 13: The statement regarding the recommended limit dose (2000 mg/kg) is present 
in the Discussion section. An additional statement has now been incorporated to explicitly 
acknowledge the limitations of acute exposure and the need for further studies. The revised 
text reads as follows: 
“Although no signs of acute toxicity were observed following a single oral administration at 
the limit dose over a 14-day observation period, our study is limited to acute exposure and 
does not provide information on potential subchronic or chronic effects. Therefore, additional 
studies involving repeated-dose toxicity and mechanistic approaches are necessary to better 
characterize the toxicological profile of the CEECP.” (Lines 356-360). 

Comments 14:  
Make sure that all references have DOIs and complete any placeholders. 

Response 14: We thank the reviewer for this suggestion. All references have been checked, 
and any placeholders have been completed. 

Comments 15:  
Cite (https://www.tandfonline.com/doi/full/10.2147/DDDT.S494417) in Introduction or in 
discussion when discussing ethnopharmacological validation and the need for integrated 
toxicity of traditionally used plants.  
(https://www.sciencedirect.com/science/article/pii/S0753332222002165) 
Cite for chemical characterization of extracts of leaves of Kadsura coccinea by UHPLC and assessment of their 
antioxidant and anti-inflammatory activities. 
(https://www.sciencedirect.com/science/article/abs/pii/S002228602401785X) 
Cite for anti inflammatory activity evaluation and molecular docking analysis compounds isolated and 
potential NF-κB modulation mechanisms.  

Response 15: The recommended reference has been incorporated into the manuscript in the 
Introduction and Discussion sections to improve the text quality. 

4. Response to Comments on the Quality of English Language 
Point 1: The English is fine and does not require any improvement. 
Response: We thank the reviewer for this suggestion. The manuscript has been revised to 
improve the English language.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I agree that the manuscript I reviewed is suitable for publication.