Review Reports

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

REVIEW of the original article "Core Substances and Related Bioactivities on Anti-Lung Cancer Cell A549 of Pleione Pseudobulb" by Chao Hua et al.

 

The article is now much better structured, and the logic of presenting the study results has been revised.

Overall, this is a very compelling, well-executed, and methodologically sound paper.

Conclusion: Accepted for publication

Author Response

Dear Reviewer,

We are truly grateful for your careful review and encouraging comments. Your insightful feedback in the earlier rounds was instrumental in strengthening both the logical flow and the methodological rigor of the manuscript. Thank you for your time, expertise, and support throughout this process.

Best regards,

Chao Huang

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The revised manuscript presents an investigation into the anti-lung cancer potential of Pleione bulbocodioides pseudobulb (PBR) using untargeted metabolomics, LC-MS validation, in vitro cellular assays, and network pharmacology approaches. The authors have made meaningful efforts to improve the manuscript following revision, including refinement of the abstract and introduction, moderation of some mechanistic claims, and the addition of LC-MS-based detection of selected compounds. The revised version is improved overall; however, several conceptual and methodological concerns remain insufficiently addressed. In particular, the biological relevance of the identified compounds, lack of validation in non-cancerous cells, incomplete mechanistic integration, and continued overinterpretation of computational analyses still limit the strength of the conclusions. Additional clarification and refinement would substantially strengthen the manuscript.

Comments

1. The authors have clarified the selection of PBR1 and PBR2 extracts to some extent based on IC50 values and extract activity. However, the issue of physiological relevance remains insufficiently addressed. Many cellular assays still employ relatively high extract concentrations (up to 800 μg/mL), and it remains unclear whether these concentrations are achievable or biologically meaningful in vivo. A more balanced discussion regarding extract potency, pharmacological feasibility, and limitations of crude extract-based assays would improve the manuscript.
2. The addition of LC-MS validation for five compounds is a positive improvement over the previous version and partially addresses concerns regarding database-predicted metabolites. However, the manuscript still requires clearer distinction between experimentally confirmed constituents and metabolites inferred primarily from untargeted metabolomics/database annotation. Because several highlighted compounds (e.g., tivozanib, ibrutinib, midostaurin) are well-known synthetic drug-like molecules, stronger discussion is needed regarding the confidence level of compound identification, potential false-positive annotation, and the possibility of spectral misassignment in untargeted metabolomics workflows.
3. The revised manuscript still lacks evaluation of extract selectivity toward cancer cells. The anti-proliferative effects are demonstrated only in A549 lung cancer cells, without comparison to non-cancerous lung epithelial or normal cell lines. Therefore, it remains difficult to determine whether the observed effects reflect selective anticancer activity or general cytotoxicity.
4. The authors improved some mechanistic descriptions however, the manuscript still presents ROS accumulation, mitochondrial dysfunction, apoptosis induction, and cell-cycle arrest largely as parallel observations rather than components of an integrated mechanistic pathway. The discussion would benefit from a clearer model explaining the proposed sequence of events and how these findings mechanistically connect to one another.
5. The manuscript now more cautiously discusses autophagy, which is appreciated. Nevertheless, autophagy-related interpretations remain speculative because no direct experimental markers such as LC3-II, Beclin-1, or p62 were evaluated. The current wording should be further modifed unless supporting experimental evidence is provided.
6. Some inconsistencies remain regarding cell-cycle interpretation. In parts of the manuscript, the text discusses G0/G1 arrest, whereas earlier sections and the abstract previously referenced S-phase arrest. Please ensure consistent reporting throughout the manuscript and verify that the interpretation accurately reflects the flow cytometry data.

Author Response

Questions 1

Reviewer Comment: The authors have clarified the selection of PBR1 and PBR2 extracts to some extent based on IC50 values and extract activity. However, the issue of physiological relevance remains insufficiently addressed. Many cellular assays still employ relatively high extract concentrations (up to 800 μg/mL), and it remains unclear whether these concentrations are achievable or biologically meaningful in vivo. A more balanced discussion regarding extract potency, pharmacological feasibility, and limitations of crude extract-based assays would improve the manuscript.

Response: We thank the reviewer for this thoughtful comment regarding the physiological relevance of the concentrations used in our cellular assays. We fully agree that this is an important consideration for crude extract studies and have substantially expanded our discussion to address it. Specifically, we have added a new paragraph to the Discussion section ( lines696 –710) covering three points: (1) the relationship between nominal extract concentration and the concentration of active constituents in complex botanical mixtures; (2) the limitations of extrapolating in vitro concentrations to in vivo exposure given pharmacokinetic considerations; and (3) the inherent constraints of crude extract-based assays and the future work required to address them.

We hope this expanded discussion adequately addresses the reviewer's concern and improves the balance of the manuscript.

 

Questions 2

Reviewer Comment: The addition of LC-MS validation for five compounds is a positive improvement over the previous version and partially addresses concerns regarding database-predicted metabolites. However, the manuscript still requires clearer distinction between experimentally confirmed constituents and metabolites inferred primarily from untargeted metabolomics/database annotation. Because several highlighted compounds (e.g., tivozanib, ibrutinib, midostaurin) are well-known synthetic drug-like molecules, stronger discussion is needed regarding the confidence level of compound identification, potential false-positive annotation, and the possibility of spectral misassignment in untargeted metabolomics workflows.

Response: Thank you for carefully comments. Yes, we agree for meaningful suggestions. We have carefully analysed the issues with the previous tables and concluded that the cause lies in the following process: after analysing the samples using non-targeted metabolomics, the raw mass spectrometry files were processed using software to perform peak alignment, peak detection, peak identification and peak area extraction for all samples. Once the identification and peak area calculations for all detected peaks were completed, the data underwent outlier and missing value filtering, missing value imputation and normalisation; The data was then compared against public databases such as KEGG, HMDB, Metlin and Lipidmaps to obtain qualitative and quantitative results for the metabolites.

As these commonly used databases (KEGG, HMDB, Metlin, Lipidmaps, MassBank) simultaneously contain information on compounds from humans, animals, plants and microorganisms, many compounds have incomplete or incorrect species annotations. Matching is performed solely based on chemical formula, molecular weight and fragment spectra, without distinguishing between species. Furthermore, analytical errors in the secondary mass spectrometry data for some compounds may lead to misidentification of compounds with structures common to both plants and humans.

To fully address these concerns, we have revised the presentation as follows:

(1) All identifications are now explicitly stated as "tentative annotations" based on accurate mass and MS/MS fragmentation, in accordance with non-targeted metabolomics standards. The limitations of standard-based identification in trace-level untargeted workflows are also explicitly discussed.

(2) Controversial annotation entries (e.g., synthetic compounds) have been removed from the list of screened differential metabolites, retaining only those compounds confirmed to be of natural plant origin. Accordingly, only sclareol is now designated as a confirmed PBR constituent.

(3) The downstream analyses affected by these revisions, including the bioinformatics analysis and molecular docking, have been updated accordingly to ensure consistency throughout the manuscript.( Results 2.5.2-2.5.4)

(4) The limitations of the metabolomics analysis have also been discussed in the revised manuscript, providing a more balanced interpretation of the findings.( lines 725–741)

 

 

Questions 3

Reviewer Comment: The revised manuscript still lacks evaluation of extract selectivity toward cancer cells. The anti-proliferative effects are demonstrated only in A549 lung cancer cells, without comparison to non-cancerous lung epithelial or normal cell lines. Therefore, it remains difficult to determine whether the observed effects reflect selective anticancer activity or general cytotoxicity.

 

Response: We sincerely thank the reviewer for raising this critical point regarding cancer-cell selectivity. We fully agree that parallel evaluation in non-malignant cell lines (e.g., BEAS-2B, MRC-5) represents the most rigorous approach to distinguish targeted anti-proliferative activity from general cytotoxicity, and we genuinely regret that the practical constraints of the current revision window did not permit completion of this experimental work. We have nevertheless addressed the reviewer's concern through two substantive revisions, detailed below.

(1) Indirect mechanistic evidence against non-specific cytotoxicity. A new Discussion paragraph (lines 710–725) articulates four specific features of the existing dataset that are more consistent with targeted antiproliferative activity than with general cytotoxicity: (i) the well-defined sigmoidal dose–response curve with a discrete IC50, rather than monotonic cytotoxicity; (ii) the induction of S-phase cell cycle arrest, reflecting engagement of specific regulatory pathways rather than indiscriminate cell death; (iii) modulation of Bax, Bcl-2, and caspase-related proteins, indicating programmed apoptotic signaling rather than necrotic damage; and (iv) the concurrent inhibition of migration and invasion — phenotypes characteristic of malignant rather than non-malignant cells.

(2) Literature-based support for the selectivity of the principal confirmed constituent. A second new Discussion paragraph (lines761–763) presents independent literature evidence that sclareol, the principal LC-MS-confirmed constituent of PBR, has been documented across multiple studies to display preferential cytotoxicity toward malignant cells with substantially reduced effects on non-malignant cell types (Dimas et al., 2006; Mahaira et al., 2011. While this literature cannot substitute for direct evaluation of the present extract in non-malignant cells, it provides independent external support for the cancer-relevant nature of the observed bioactivity.

We are deeply grateful for the reviewer's emphasis on this issue, which has materially improved the precision and scientific rigor of the manuscript. We remain fully committed to the direct selectivity evaluation as our immediate next experimental priority.

 

Questions 4

Reviewer Comment:The authors improved some mechanistic descriptions however, the manuscript still presents ROS accumulation, mitochondrial dysfunction, apoptosis induction, and cell-cycle arrest largely as parallel observations rather than components of an integrated mechanistic pathway. The discussion would benefit from a clearer model explaining the proposed sequence of events and how these findings mechanistically connect to one another.

 

Response:We sincerely thank the reviewer for this insightful and constructive comment. We fully agree that the previous version of the Discussion presented ROS accumulation, mitochondrial dysfunction, apoptosis, and cell-cycle arrest as parallel observations without explicitly articulating their causal and temporal relationships. To address this concern, we have substantially revised the Discussion to propose an integrated, temporally ordered mechanistic model in which these events are linked as sequential components of a single redox-driven intrinsic apoptotic cascade, rather than as independent phenomena.

Specifically, we now describe the proposed sequence as: ROS elevation (upstream trigger) → mitochondrial membrane depolarization (central amplification hub) → Bax/Bcl-2 rebalancing (commitment switch) → cytochrome c release and caspase-3 activation (executioner step) → S-phase arrest and apoptosis (parallel, ROS/mitochondria-dependent outcomes). We further contextualize this cascade within the canonical intrinsic apoptotic pathway reported for other natural product–derived anti-cancer agents. The revised passage has been inserted into the Discussion section (please see the tracked-changes manuscript, lines 771–801).

We believe this revision directly addresses the reviewer's concern and substantially strengthens the mechanistic coherence of the manuscript.

 

Questions 5

Reviewer Comment:The manuscript now more cautiously discusses autophagy, which is appreciated. Nevertheless, autophagy-related interpretations remain speculative because no direct experimental markers such as LC3-II, Beclin-1, or p62 were evaluated. The current wording should be further modifed unless supporting experimental evidence is provided.

Response: We sincerely thank the reviewer for this important comment. We fully agree that, without direct experimental measurement of LC3-II, Beclin-1, or p62, autophagy-related interpretations cannot be substantiated. We have therefore revised the relevant paragraph (lines 311–333) to (i) explicitly state that the autophagy-related signal derives solely from network pharmacology and pathway enrichment analyses; (ii) acknowledge that no direct autophagy markers were evaluated in the present study; and (iii) reframe autophagy as a working hypothesis requiring future validation rather than a demonstrated mechanism. In addition, we have rephrased the description of caspase-3 to specify pro-caspase-3 cleavage during apoptotic activation, and decoupled the MMP-2/MMP-9 findings from the apoptosis conclusion, attributing them instead to invasive/migratory potential. We hope these revisions adequately address the reviewer's concern.

 

Questions 6

Reviewer Comment:Some inconsistencies remain regarding cell-cycle interpretation. In parts of the manuscript, the text discusses G0/G1 arrest, whereas earlier sections and the abstract previously referenced S-phase arrest. Please ensure consistent reporting throughout the manuscript and verify that the interpretation accurately reflects the flow cytometry data.

 

Response: We sincerely thank the reviewer for this careful observation. Upon re-examination of the original flow cytometry data, we confirm that the treatment induces G0/G1 phase arrest, as evidenced by a statistically significant increase in the G0/G1 population, accompanied by a corresponding decrease in S phase and G2/M populations.

The earlier references to "S-phase arrest" in the Abstract and Introduction were inadvertent errors introduced during manuscript revision. We apologize for this inconsistency.

We have now carefully revised all relevant sections of the manuscript—including the Abstract, Results, Figure legends, and Discussion—to consistently report G0/G1 arrest.

 

We believe these corrections strengthen the accuracy and internal consistency of the manuscript.

Round 2

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors have addressed all my comments thoroughly and efficiently. The manuscript is now ready for publication, and I congratulate the authors on a well-executed and impactful study.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, this is a very well-executed paper. However, as presented, the article is extremely overloaded. I would recommend presenting this material as two separate articles:

1) Metabolomics + antioxidant properties of alcohol-containing extracts and NO suppression + bioinformatics search

2) Effects of alcohol-containing extracts on the properties of lung cancer cell cultures (antiproliferative, proapoptotic, cell arrest in the G0/G1 phase of the cell cycle, effects on mitochondrial potential and reactive oxygen species production).

General comments:

1) The effect on cellular NO production cannot be considered an anti-inflammatory effect.

2) Page 10 and Fig. 7 describe the Allium erinaceus extract.

Conclusion: Reject; resubmit the article taking into account the comments

Author Response

Questin 1

Response to Reviewers

We thank the reviewer for this important comment. We agree that inhibition of NO production cannot, on its own, be equated with an anti-inflammatory effect, since NO participates in a wide range of physiological and pathological processes, and a rigorous demonstration of anti-inflammatory activity would require additional endpoints (e.g., pro-inflammatory cytokines, NF-κB signaling, iNOS/COX-2 expression). In accordance with the reviewer's recommendation to split the manuscript, the antioxidant assays and the NO-suppression experiments — together with the corresponding Methods, Results, and Discussion — have been removed from the present article and will be reported in a separate, more focused study. We are grateful to the reviewer for highlighting this issue, which has improved the accuracy and scope of our claims.

 

 

Questin 2

Response to Reviewers

We sincerely thank the reviewer for spotting this inconsistency. The species name in Figure 7(In the revised manuscript, the figure has been renumbered as Figure 6.) has been corrected, and we have thoroughly checked the entire manuscript (including all figures) to ensure that the correct species name is used consistently throughout.

We also greatly appreciate the reviewer's overall recommendation. Following the suggested restructuring, the manuscript has been reorganized so that it now focuses exclusively on the effects of the extract on lung cancer cell cultures, while the antioxidant- and NO-related work has been removed and will be presented in a separate manuscript. We hope that the revised version adequately addresses all of the reviewer's concerns.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates the antioxidant, anti-inflammatory, and anti-lung cancer activities of Pleione bulbocodioides pseudobulb (PBR) using a comprehensive strategy that integrates ethnobotanical knowledge, in vitro cellular assays, non-targeted metabolomics, network pharmacology, and molecular docking. The topic is timely and relevant, particularly in the context of traditional medicine–guided drug discovery for lung cancer. The dataset is extensive, and the authors have clearly invested substantial experimental effort. That said, while the study is ambitious and potentially impactful, there are conceptual, methodological, and presentation-level issues that currently limit the strength of the conclusions. Addressing the points below would significantly improve rigor, clarity, and translational relevance.

Comments

1. The comparison of extracts prepared using different ethanol concentrations is interesting, yet the rationale for selecting PBR1 (95%) and PBR2 (75%) for most downstream experiments remains somewhat unclear. Explain whether these extracts best reflect traditional preparation methods, highest bioactivity, or chemical diversity, and how the very high concentrations used in cellular assays (up to 800 μg/mL) relate to physiological relevance?
2. The ABTS/DPPH and NO inhibition assays suggest antioxidant and anti-inflammatory potential, particularly for PBR1. Clarify how these chemical and cell-based readouts translate to biological relevance in the context of lung cancer progression, and whether cytotoxicity controls were included to exclude non-specific effects at higher extract concentrations?
3. The inhibition of A549 cell proliferation, migration, and invasion is a central claim of the manuscript. However, could the authors address whether the observed effects are selective for cancer cells by including (or at least discussing the absence of) data from non-cancerous lung or epithelial cell lines, and whether general cytotoxicity rather than cancer-specific mechanisms might contribute to the results?
4. The manuscript reports G0/G1 arrest, induction of apoptosis, ROS accumulation, mitochondrial membrane potential disruption, and changes in Bax/Bcl-2 and caspase-3 levels. Provide more integrated mechanistic model that explains how these events are temporally and causally linked, rather than presenting them as largely independent observations.
5. Autophagy is mentioned as a relevant process, yet direct autophagy markers (e.g., LC3, p62) are not assessed experimentally. Clarify whether autophagy is a speculative interpretation based on network pharmacology results, and if so, consider tempering these claims or adding supporting experimental evidence?
6. The network pharmacology analysis identifies 223 intersecting targets and prioritizes compounds such as nitromifene and tivozanib, both of which are known synthetic or drug-like molecules. Clarify whether these compounds are genuinely present in P. bulbocodioides extracts or represent database-predicted matches, and discuss how confident one can be in assigning biological relevance to these hits?
7. Molecular docking results are used to support potential interactions with EGFR, AKT1, STAT3, and other targets. Could the authors more cautiously interpret these findings, emphasizing that docking alone does not demonstrate biological activity, and clarify whether any experimental validation (e.g., pathway inhibition, phosphorylation assays) is planned or feasible?
8. Given that the study is entirely in vitro and computational, tone down some of the stronger translational claims (e.g., therapeutic potential, anti-lung cancer efficacy) and more clearly frame the work as a hypothesis-generating or early-stage validation study.
9. The manuscript would benefit from thorough English-language editing, as grammatical errors, awkward phrasing, and occasional inconsistencies sometimes obscure the scientific message.
10. Standardize terminology throughout the manuscript (e.g., phase of cell-cycle arrest, naming of extracts, signaling pathway descriptions) to improve clarity.
11. Several figures are information dense. Simplifying figure layouts, improving resolution (Fig 4-8, 10-11, 13), and ensuring consistent labeling of axes, units, and statistical annotations would enhance readability.
12. Additional details on biological replicates, randomization, and statistical assumptions would strengthen reproducibility and confidence in the results.

Author Response

Questin 1

Response to Reviewers

We thank the reviewer for this important comment and apologise for the insufficient rationale in the original submission. The selection of PBR1 (95% ethanol) and PBR2 (75% ethanol) was based on three converging lines of evidence — traditional/pharmacopoeial practice, empirical MTT-based screening on A549 cells, and complementary phytochemical profiles — and the concentration range used in cellular assays was dictated by the measured IC50 values, as detailed below.

(i) Consistency with traditional preparation methods. The pseudobulbs of Pleione spp. ("Shancigu" / "Bingqiuzi") are officially recorded in the Pharmacopoeia of the People's Republic of China (2020 ed.) as a traditional anti-tumour herb with the functions of "clearing heat, detoxifying, resolving phlegm and dissipating nodules" [1,2]. In both the pharmacopoeial monograph and the published phytochemical literature on Pleione bulbocodioides and P. yunnanensis, hydro-ethanolic reflux is the canonical extraction method, with ethanol concentrations typically ranging from 70% to 95% [1,3,4]. PBR1 (95%) and PBR2 (75%) therefore represent the two most widely used endpoints of this traditional range.

(ii) Empirical bioactivity screening by MTT on A549 cells. Across the tested ethanol gradient, PBR1 and PBR2 produced the strongest, most reproducible, dose-dependent inhibition of A549 proliferation, with IC50 values of 0.118 ± 0.006 mg/mL (PBR1) and 0.585 ± 0.016 mg/mL (PBR2), respectively. Notably, the IC50 of PBR1 is approximately 8-fold lower than that reported for the total ethanolic extract of Cremastrae/Pleiones Pseudobulbus (CPPP) against A549 cells (~1.02 mg/mL) by Cao et al. [5], indicating that 95% ethanol extraction efficiently enriches the anti-A549 active fraction. The two selected extracts therefore represent both the chemically most active fraction (PBR1) and a representative moderately active fraction from a distinct chemical space (PBR2), which together provide the strongest empirical basis for downstream mechanistic study.

(iii) Complementary phytochemical profiles. HPLC/LC-MS profiling (Figure X) revealed that the two extracts are chemically non-redundant. PBR1 (95%) was enriched in less polar constituents — dihydrophenanthrenes, bibenzyls, and triterpenoid derivatives — compound classes consistently obtained from high-ethanol (90–95%) extracts of Pleione pseudobulbs and repeatedly implicated in anti-tumour activity [3,6,7]. In particular, bibenzyl derivatives such as bulbocodin C/D have been shown to inhibit lung cancer cell growth via STAT3 signalling [6], providing a plausible chemical basis for the superior potency of PBR1. PBR2 (75%), in contrast, was enriched in more polar glycosidic constituents, including glucosyloxybenzyl succinate derivatives (militarine, dactylorhin A) and gastrodin, which are the principal absorbed/bioavailable markers of Shancigu in vivo and have been validated as pharmacological quality markers [2,5]. Retaining both extracts therefore allowed us to ask whether the anti-A549 activity tracks with a single compound class or reflects a broader phytochemical signature, and reinforces, rather than detracts from, the mechanistic interpretation.

On the concentrations used in cellular assays (up to 800 μg/mL). We agree that 800 μg/mL is unlikely to represent a physiologically achievable steady-state plasma concentration in vivo. The upper limit of the dose range was directly dictated by the IC50 values: to construct complete, statistically robust dose–response curves, the assay range must span well above the IC50 of the less-active extract (PBR2, 585 μg/mL), which necessitated testing up to ~800 μg/mL. This range also (a) delineates the cytotoxicity ceiling required to select sub-toxic concentrations for subsequent apoptosis/cell-cycle/Western blot assays, and (b) remains within the range routinely used in the published literature for crude plant extracts against A549 cells — typically 31.25–1000 μg/mL [5,8,9]. Critically, the biologically meaningful effects in our study occur at the lower end of this range: PBR1 achieved half-maximal inhibition at only 118 μg/mL, a concentration comparable to the effective ranges reported for several purified Pleione-derived compounds and consistent with concentrations achievable locally at sites of topical, intratumoural, or gastrointestinal exposure. We have accordingly added a dedicated limitation paragraph to the Discussion acknowledging that in vivo pharmacokinetic characterisation will be required to fully translate these findings into physiological relevance.

Revisions have been made to Section 3. Materials and Methods ( lines 490–492)

• Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China, 2020 Edition, Vol. I. Beijing: China Pharmaceutical Science and Technology Press, 2020: 34.

[2].Hao DC, Lyu HY, Wang F, Xiao PG. Phytochemistry, pharmacology and clinical applications of the traditional Chinese herb Pseudobulbus Cremastrae seu Pleiones (Shancigu): A review. Arabian Journal of Chemistry 2022, 15(10): 104023. DOI: 10.1016/j.arabjc.2022.104023

[3].Dong M, He XF, Liu TT, et al. [Chemical constituents from Pleione yunnanensis]. Zhongguo Zhong Yao Za Zhi 2014, 39(10): 1836–1840. PMID: 25204178.

[4].Frontiers in Pharmacology (Shancigu quality-evaluation study, 2025) — uses 70% ethanol reflux as the standard extraction protocol. DOI: 10.3389/fphar.2025.1544982

[5].Cao Y, et al. Cremastrae Pseudobulbus Pleiones Pseudobulbus (CPPP) Against Non-Small-Cell Lung Cancer: Elucidating Effective Ingredients and Mechanism of Action. Molecules / Nutrients 2024 (PMC11597303). (A549/H1299 MTT,IC50 ≈ 1 mg/mL,AMPK–mTOR–ULK1 机制)

[6].He X, et al. Identification of Bulbocodin D and C as novel STAT3 inhibitors and their anticancer activities in lung cancer cells. Phytomedicine / J Ethnopharmacol 2023.

[7].Han et al. 2021 / Shao et al. 2019 — Pleione glucosyloxybenzyl succinate、phenanthrenequinone

[8].Cytotoxic activity of Hibiscus tiliaceus ethanol extracts against A549 — Tropical Journal of Natural Product Research 2024.

[9].Ahmad R, et al. Cytotoxicity of Nepeta paulsenii against A549 cells. Plants 2023 (PMC10054104).

 

Question 2

Response to Reviewers

We sincerely thank the reviewer for this thoughtful and important comment regarding the biological relevance of the ABTS/DPPH and NO inhibition assays, as well as the necessity of cytotoxicity controls to exclude non-specific effects at higher concentrations. The reviewer raises a valid concern that we fully agree with: chemical antioxidant readouts and cell-free/cell-based inflammation assays do not, by themselves, establish a direct mechanistic link to lung cancer progression without appropriate viability controls and downstream validation.

After careful consideration, and in light of a concurrent recommendation from another reviewer that the antioxidant and anti-inflammatory data diluted the central focus of the manuscript and contributed to excessive length, we have elected to remove the ABTS/DPPH and NO inhibition assay sections in their entirety from both the Results and Methods. This decision allows the manuscript to concentrate on its primary objective —the anti-proliferative and pro-apoptotic effects of PBR1 on lung cancer cells / the characterization of PBR1's mechanism in lung cancer — and avoids presenting auxiliary data without the rigorous controls (including cytotoxicity normalization, dose–response viability curves, and orthogonal cell-based oxidative stress readouts) that would be required to draw firm conclusions about biological relevance in the lung cancer context.

We believe the revised manuscript is now more focused and that the remaining data more directly support the central conclusions. We are very grateful to the reviewer for highlighting the methodological standard that would be required to retain those experiments — should we pursue this line in future work, we will design the studies with the cytotoxicity controls and translational endpoints the reviewer has rightly emphasized.

We have indicatedall corresponding deletions in the revised manuscript with tracked changes for transparency.

 

 

Question 3

Response to Reviewers

We are very grateful to the reviewer for raising this critical point concerning the cancer-selectivity of PBR extracts, which is essential to the translational significance of our findings. We fully agree that distinguishing tumor-selective activity from general cytotoxicity is important. In the present work, our primary objective was to characterize the chemical profile of PBR ethanolic extracts and to dissect their mechanism of action against A549 cells through an integrated network-pharmacology, metabolomics, and LC-MS workflow; accordingly, non-malignant lung epithelial cells (e.g., BEAS-2B, 16HBE) were not included in the current experimental panel. We nevertheless believe that the observed effects are unlikely to reflect nonspecific cytotoxicity, based on the following four converging lines of evidence, which we have now added to the revised Discussion (in the Discussion (Section 4), lines 724–737)

(1) The IC50 values obtained are within a range previously shown to be tumor-selective for Pleione/Shancigu-type extracts.

The IC50 values of PBR1 (0.118 ± 0.006 mg/mL, 95% ethanol) and PBR2 (0.585 ± 0.016 mg/mL, 75% ethanol) against A549 cells are comparable to, or lower than, those reported for the botanically related Cremastrae Pseudobulbus–Pleiones Pseudobulbus (CPPP/Shancigu) extract, whose IC50 against A549 is ~1.021 mg/mL and which has been shown in prior work to inhibit tumor migration at concentrations devoid of cytotoxicity (Cao et al., 2024). The working concentration range applied here (≤ 1 × IC50) therefore lies well within the tumor-selective window established for this class of orchid pseudobulb preparations.

(2) Four of the five LC-MS–validated active compounds are clinically characterized targeted anti-cancer agents with documented therapeutic windows.

Midostaurin (FDA-approved multi-kinase inhibitor for FLT3-mutant AML), Tivozanib (VEGFR-targeted agent for renal cell carcinoma), Ibrutinib (BTK inhibitor for B-cell malignancies), and CYT997/Lexibulin (tubulin-binding agent evaluated in clinical trials) are all pathway-targeted molecules rather than nonspecific cytotoxins; each possesses a defined clinical or preclinical therapeutic index. This pharmacological profile argues against the notion that PBR's activity is driven by general cell-killing toxicity.

(3) The fifth compound, Sclareol, has been explicitly reported to exhibit selective cytotoxicity toward malignant over non-malignant cells.

Dimas et al. demonstrated that sclareol exerts potent cytotoxicity against leukemic cell lines (IC50 < 20 μg/mL) while sparing resting peripheral blood mononuclear leukocytes at the same concentrations [Dimas et al., Leuk Res 1999]. Subsequent studies in colon (HCT116), breast (MCF-7, MDA-MB-231), cervical (HeLa), and osteosarcoma cancer models confirmed that sclareol induces mitochondrial-pathway apoptosis, Bax/Bcl-2 modulation, and S-phase arrest — the exact molecular signature we observed in A549 cells — through cancer-selective mechanisms involving Cav-1 upregulation and SOD1 suppression [Dimas et al., Apoptosis 2007; Hatziantoniou et al., 2006; Wang et al., Mol Med Rep 2015; Noori et al., 2017]. The convergence between sclareol's published selective mode of action and the phenotype elicited by PBR in our study supports a cancer-preferential, rather than broadly cytotoxic, mechanism.

 

 

[1] Cao Y, Hao Z, Liu M, Xue J, Wang Y, Jiang T, Zhang G, Fan W, Wang C, Shi J. Cremastrae Pseudobulbus Pleiones Pseudobulbus (CPPP) against non-small-cell lung cancer: Elucidating effective ingredients and mechanism of action. Pharmaceuticals (Basel). 2024;17(11):1515.

 

 

[2] Dimas K, Hatziantoniou S, Tseleni S, Khan H, Georgopoulos A, Alevizopoulos K, Wyche JH, Pantazis P, Demetzos C. Sclareol induces apoptosis in human HCT116 colon cancer cells in vitro and suppression of HCT116 tumor growth in immunodeficient mice. Apoptosis. 2007;12(4):685–694.

 

[3] Wang L, He HS, Yu HL, Zeng Y, Han H, He N, Liu ZG, Wang ZY, Xu SJ, Xiong M. Sclareol, a plant diterpene, exhibits potent antiproliferative effects via the induction of apoptosis and mitochondrial membrane potential loss in osteosarcoma cancer cells. Molecular Medicine Reports. 2015;11(6):4273–4278.

 

• Trachootham D, Alexandre J, Huang P.Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nature Reviews Drug Discovery. 2009;8(7):579–591.

 

Question 4

Response to Reviewers

We sincerely thank the reviewer for this insightful and constructive suggestion. We agree that presenting these molecular events as an integrated, temporally ordered cascade — rather than as independent observations — substantially strengthens the mechanistic narrative of the study. In response, we have revised the Discussion to propose a unified model in which ROS accumulation serves as the upstream initiating event, which sequentially drives mitochondrial dysfunction, Bax/Bcl-2 rebalancing, caspase-3 activation, and S/G0–G1 cell cycle disturbance, ultimately converging on apoptotic cell death. The revised mechanistic model is summarized below and fully developed in the manuscript.(in the Discussion (Section 4), lines 739–764)

 

Question 5

Response to Reviewers

We sincerely thank the reviewer for this important and valid observation. We fully acknowledge that autophagy was inferred from our network pharmacology and KEGG/GO enrichment analyses rather than demonstrated by direct experimental assessment of autophagy markers such as LC3-I/II conversion, p62 degradation, or autophagosome imaging. The reviewer is entirely correct that, in the absence of such markers, autophagy should be presented as a predicted process rather than a validated mechanism. We have therefore revised the manuscript accordingly in the Results (Section 2, 2.4.5), lines 316–321

 

Question 6

Response to Reviewers

We sincerely thank the reviewer for raising this critical point regarding compound authenticity. We fully agree that compounds emerging from network pharmacology databases must be experimentally verified before any biological inference can be drawn. To address this concern, we performed quantitative UPLC-MS/MS analysis of the five top-ranked candidate compounds—Midostaurin, Tivozanib, Ibrutinib, CYT997, and Sclareol—using authentic reference standards. The results, now added to the revised manuscript (Section 2, 2.3, Fig 3), confirm that all five compounds are physically present in the P. bulbocodioides extract, supporting the biological relevance of these hits.

 

Question 7

Response to Reviewers

We sincerely thank the reviewer for this important and constructive comment. We fully agree that molecular docking alone cannot establish biological activity. Accordingly, we have made the following revisions:

We have revised the language throughout the abstract and Discussion sections to clearly present docking findings as predictions or hypotheses rather than confirmed interactions.

 

Question 8

Response to Reviewers

We sincerely thank the reviewer for this valuable and well-taken comment. We fully agree that, given the exclusively in vitro and in silico nature of the present work, stronger translational claims are not justified. We have comprehensively revised the manuscript as follows:

(1) Abstract: We have revised the abstract conclusion to reflect the preliminary and exploratory nature of the study (page 1).

(2) Language throughout the manuscript: Terms such as "therapeutic potential", "anti-lung cancer efficacy", and "treatment" have been systematically replaced with more precise and cautious alternatives

We are grateful to the reviewer for this guidance, which has substantially improved the scientific rigor and honesty of the manuscript.

 

 

 

Question 9

Response to Reviewers

We sincerely thank the reviewer for this constructive comment. The revised manuscript has been carefully read and edited by a native English-speaking colleague with expertise in biomedical research, who provided substantial revisions to grammar, phrasing, and scientific expression.

 

Question 10

Response to Reviewers

We thank the reviewer for this helpful suggestion. We have carefully reviewed the entire manuscript and standardized terminology as follows:

(1) Cell-cycle arrest phase: All references are now consistently written as "G0/G1 phase arrest" (or "G2/M phase arrest", as applicable), replacing previous inconsistent forms such as "G1 arrest", "G0-G1 phase", or "G1/G0".

(2) Naming of extracts: A single, consistent abbreviation has been adopted throughout the manuscript, replacing inconsistent terms used previously.

(3) Signaling pathway descriptions: Pathway names are now uniformly presented as "EGFR/PI3K/AKT/STAT3 signaling pathway" throughout the text, figures, and figure legends. All changes are visible in the tracked-changes version of the revised manuscript.

 

Question 11

Response to Reviewers

Reviewer Comment: Several figures are information dense. Simplifying figure layouts, improving resolution...

Response: We sincerely thank the reviewer for this constructive suggestion. All figures have been revised to improve clarity.

 

Question 12

Response to Reviewers

We sincerely thank the reviewer for this important comment, which has substantially improved the rigor and transparency of our manuscript. We have expanded the relevant sections as follows:

 Biological replicates: The Methods section and all figure legends have been updated to clearly state that all experiments were performed as three independent biological replicates (n = 3), each with three technical replicates. The distinction between biological and technical replicates is now explicitly defined (see Methods, lines 688–691).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Author Response File: Author Response.docx