Isolation and Characterization of 5-(1-Hydroxyethyl)-Dihydro-2-Furanone from Angiopteris evecta with Potent Anti-Inflammatory and Anti-Leukemic Activities

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates bioactive compounds isolated from A. evecta ethyl acetate extract and evaluates their cytotoxic and anti-inflammatory effects in leukemic cell lines and macrophages. The study combines phytochemical purification, cytotoxicity assays, and spectroscopic characterization, addressing a topic of interest for natural product–based anticancer discovery. The work is generally well organized, and the identification of a lead compound with selective cytotoxicity is promising. However, several conceptual, methodological, and presentation issues should be addressed to strengthen the biological relevance, mechanistic insight, and reproducibility of the study.

Comments

1. While purified compound 4 shows promising cytotoxicity and selectivity toward leukemic cell lines, could the authors clarify what biological pathways or cellular processes are likely responsible for this effect, beyond descriptive cytotoxicity? Include basic mechanistic readouts such as apoptosis vs. necrosis, cell-cycle arrest, or inflammatory signaling markers in RAW264.7 cells to better contextualize the observed IC₅₀ values.
2. The selectivity index (SI) is used to highlight preferential toxicity toward cancer cells, clarify how clinically meaningful these SI values are, particularly for compound 4?
3. PBMCs are used as a “normal” control, discuss limitations of PBMCs as a surrogate for systemic toxicity, and whether additional normal cell types might alter the interpretation.
4. Given the relatively moderate IC₅₀ values (µg/mL range), explain the translational potential of these compounds compared with existing natural-product-derived anticancer leads?
5. The manuscript relies heavily on NMR-based characterization. Clarify whether high-resolution MS or additional orthogonal methods were used to confirm molecular formula and purity? In the supplementary NMR figures, some peak assignments are implied but not explicitly labelled, adding annotated spectra or a table summarizing key correlations (¹H–¹³C, COSY, HMBC) to strengthen confidence in compound identification.
6. The molecular formula reported in Supplementary Figure S2 appears inconsistent with the number of carbons listed could the authors reconcile and clarify this point.
7. For RAW264.7 assays, how was cytotoxicity distinguished from anti-inflammatory or immunomodulatory effects, particularly at concentrations near IC₂₀?
8. Some methodological details (e.g., extraction yield normalization, compound dosing rationale, and solvent controls) are either briefly described or scattered. Kindly streamline and expand the methods section to ensure full reproducibility.
9. The manuscript occasionally shifts between descriptive phytochemistry and biological interpretation. Consider tightening the narrative flow to maintain a clear biological focus.
10. Please ensure consistent use of units (µg/mL vs. µM, if applicable) throughout the text.
11. Abbreviations such as AE EtOAc No. 003 should be defined once and used consistently.
12. Consider briefly summarizing the key findings of the supplementary tables in the main Results section to guide readers.

Author Response

Reviewer 1

This manuscript investigates bioactive compounds isolated from A. evecta ethyl acetate extract and evaluates their cytotoxic and anti-inflammatory effects in leukemic cell lines and macrophages. The study combines phytochemical purification, cytotoxicity assays, and spectroscopic characterization, addressing a topic of interest for natural product–based anticancer discovery. The work is generally well organized, and the identification of a lead compound with selective cytotoxicity is promising. However, several conceptual, methodological, and presentation issues should be addressed to strengthen the biological relevance, mechanistic insight, and reproducibility of the study.

Comments

1. While purified compound 4 shows promising cytotoxicity and selectivity toward leukemic cell lines, could the authors clarify what biological pathways or cellular processes are likely responsible for this effect, beyond descriptive cytotoxicity? Include basic mechanistic readouts such as apoptosis vs. necrosis, cell-cycle arrest, or inflammatory signaling markers in RAW264.7 cells to better contextualize the observed IC₅₀ values.

Response: In response to the reviewer’s comment, we investigated the biological mechanisms underlying the cytotoxic activity of purified compound 4, which is now clearly defined as a ternary mixture (Page 6, Lines 184-185), beyond descriptive IC₅₀ measurements. Specifically, mechanistic studies focusing on cell cycle distribution and apoptosis were conducted and are presented in Sections 2.9. (Page 15, Lines 499-522) and 2.10. (Pages 18-19, Lines 557-576), respectively. Flow cytometric analysis revealed that the ternary mixture induced a pronounced accumulation of KG-1a cells in G2/M phase, accompanied by apoptotic cell death. In contrast, EoL-1 cells exhibited a dose-dependent S phase arrest together with a significant increase in apoptosis following treatment. These findings indicated that the cytotoxic effects of the ternary mixture are mediated primarily through cell line-specific cell cycle arrest and the induction of apoptotic cell death, rather than nonspecific cytotoxicity. In addition, to address the reviewer’s suggestion regarding inflammatory signaling, the effects of the ternary mixture on pro-inflammatory cytokine production were evaluated in LPS-stimulated RAW264.7 macrophages (Section 2.8 Page 14). The ternary mixture significantly suppressed TNF-α and IL-6 production without inducing cytotoxicity, further supporting its biological activity beyond simple growth inhibition.  

2. The selectivity index (SI) is used to highlight preferential toxicity toward cancer cells, clarify how clinically meaningful these SI values are, particularly for compound 4?

Response: The selectivity index (SI) is commonly used as a quantitative indicator of preferential cytotoxicity toward cancer cells relative to normal cells and is defined as the ratio of the IC₅₀ value in normal cells to that in cancer cells [58,59]. In general, an SI value greater than 3 is considered to reflect desirable anticancer selectivity. This definition has been added to the Materials and Methods section 4.7 (Page 27, Lines 937–943). In the present study, compound 4 (defined as the ternary mixture) exhibited SI values of 5.28 for KG-1a cells and 6.34 for EoL-1 cells, indicating a clear preferential toxicity toward leukemic cells over normal PBMCs. These SI values exceed the commonly accepted threshold for selective anticancer activity and suggest a favorable therapeutic window at the in vitro level. Notably, the selectivity of the ternary mixture toward KG-1a cells was higher than that of the reference chemotherapeutic agent doxorubicin under the same experimental conditions, further supporting its potential biological relevance. While these findings are derived from in vitro assays and do not directly predict clinical efficacy, they provide an important preclinical rationale for further mechanism and in vivo evaluation of compound 4 as a potential antileukemic candidate.

3. PBMCs are used as a “normal” control, discuss limitations of PBMCs as a surrogate for systemic toxicity, and whether additional normal cell types might alter the interpretation.

Response: Peripheral blood mononuclear cells (PBMCs) were selected as normal control because they are primary human cells derived from the hematopoietic system, which is the same biological origin as the leukemic cell lines used in this study. PBMCs therefore provide a relevant and widely accepted model for assessing preferential cytotoxicity toward malignant versus normal blood cells in leukemia-related research. However, we acknowledge that PBMCs have inherent limitations as a surrogate for systemic toxicity. PBMCs are non-transformed, slowly proliferating, and heterogeneous primary cells, and they do not represent the cellular complexity or metabolic characteristics of solid organs such as liver, kidney, or epithelial tissues, which are often critical targets of drug-induced toxicity. Consequently, cytotoxicity results obtained using PBMCs primarily reflect hematological selectivity rather than whole-body safety. The use of additional normal cell types, such as fibroblasts or epithelial cell lines, could potentially alter the interpretation of cytotoxic selectivity by revealing off-target effects in non-hematopoietic tissues. Nevertheless, PBMC-based selectivity assays are commonly employed as an initial screening approach in antileukemic studies and provide valuable preliminary information on therapeutic window and blood cell specificity, as reported previously [1,2].

Taken together, while PBMCs offer biologically relevant normal control for leukemia-focused studies, further evaluation using additional normal cell types and in vivo models will be required to comprehensively assess the systemic safety profile of the extracts and compound 4.

References:

1. Han JM, Kim HL, Jung HJ. Ampelopsin Inhibits Cell Proliferation and Induces Apoptosis in HL60 and K562 Leukemia Cells by Downregulating AKT and NF-κB Signaling Pathways. International Journal of Molecular Sciences. 2021; 22(8):4265. https://doi.org/10.3390/ijms22084265
2. de Freitas Gomes A, Batalha ADdSJ, de Castro Alves CE, Galvão de Azevedo R, Rodriguez Amado JR, Pereira de Souza T, Koolen HHF, da Silva FMA, Chaves FCM, Florentino Neto S, et al. Immunomodulatory and Anticancer Effects of Fridericia chica Extract-Loaded Nanocapsules in Myeloid Leukemia. Pharmaceutics. 2024; 16(6):828. https://doi.org/10.3390/pharmaceutics16060828
3. Given the relatively moderate IC₅₀ values (µg/mL range), explain the translational potential of these compounds compared with existing natural-product-derived anticancer leads?

Response: The IC₅₀ values of the crude fractional extract and the ternary mixture reported in this study fall within the microgram-per-milliliter range and were obtained from in vitro cell line–based assays. While these values are moderate compared with highly optimized chemotherapeutic agents, they are comparable to those reported for many natural-product-derived anticancer leads at the early discovery stage. Importantly, the translational potential of these compounds is not defined solely by cytotoxic potency but also by their biological selectivity and mechanistic relevance. In this study, the ternary mixture exhibited favorable selectivity indices toward leukemic cells over normal PBMCs, induced apoptosis and cell cycle arrest, and modulated disease-relevant pathways, including WT1 expression and MAPK-related signaling. In addition, the compound demonstrated anti-inflammatory activity without overt cytotoxicity in RAW264.7 macrophages, suggesting a potentially broader therapeutic profile.

Taken together, these features support the value of the ternary mixture as a mechanistically informed natural-product lead rather than a final drug candidate. Similar to other natural-product-derived anticancer agents, further optimization, in vivo evaluation, and pharmacokinetic studies will be required to fully assess its translational applicability in leukemia treatment.

5. The manuscript relies heavily on NMR-based characterization. Clarify whether high-resolution MS or additional orthogonal methods were used to confirm molecular formula and purity? In the supplementary NMR figures, some peak assignments are implied but not explicitly labelled, adding annotated spectra or a table summarizing key correlations (¹H–¹³C, COSY, HMBC) to strengthen confidence in compound identification.

Response: Purified compound 4 was characterized using multiple orthogonal analytical techniques, including IR spectroscopy, GC–MS, and NMR (¹H, ¹³C, COSY, HMBC). GC–MS analysis revealed that purified compound 4 is a ternary mixture of closely related furanone derivatives. To improve clarity and reproducibility, we have added the following: IR spectral data supporting hydroxyl and lactone carbonyl functionalities (Supplementary Data S1); MS supporting the existence of three compounds (Supplementary Table S2); annotated ¹H and ¹³C NMR spectra (Supplementary Table S3–S4 and Supplementary Figures S1–S2).

6. The molecular formula reported in Supplementary Figure S2 appears inconsistent with the number of carbons listed could the authors reconcile and clarify this point.

Response: The confusion has been addressed with the additional data from IR, GC-MS, and individual components from the ternary mixtures ¹H and ¹³C NMR annotations as shown in the Supplementary data.

7. For RAW264.7 assays, how was cytotoxicity distinguished from anti-inflammatory or immunomodulatory effects, particularly at concentrations near IC₂₀?

Response: To distinguish anti-inflammatory or immunomodulatory effects from cytotoxicity in RAW264.7 macrophages, cytokine production was evaluated in parallel with cell viability under identical treatment conditions. RAW264.7 cells were treated with AE EtOAc No. 003 or the ternary mixture at concentrations close to their IC₂₀ values, which were intentionally selected to minimize cytotoxic effects while allowing biological activity to be assessed. Under LPS-stimulated inflammatory conditions, treatment with AE EtOAc No. 003 or the ternary mixture significantly reduced the production of pro-inflammatory cytokines TNF-α and IL-6 (Results 2.8, Figure 7). Importantly, cell viability analysis performed concurrently demonstrated that RAW264.7 viability remained above 80% compared with the vehicle control (Results 2.8, Figure 8), indicating that the observed cytokine suppression was not attributable to cell death or overt cytotoxicity.

These findings support the conclusion that the reduced TNF-α and IL-6 levels reflect genuine anti-inflammatory or immunomodulatory activity rather than a secondary consequence of reduced cell viability at concentrations near IC₂₀.

8. Some methodological details (e.g., extraction yield normalization, compound dosing rationale, and solvent controls) are either briefly described or scattered. Kindly streamline and expand the methods section to ensure full reproducibility.

Response: We thank the reviewer for highlighting the need to improve clarity and reproducibility of the Methods section. The Materials and Methods have been carefully revised, expanded, and streamlined to consolidate key experimental details that were previously brief or dispersed across sections. Specifically, Section 4.1 (Plant Materials and Extraction) has been expanded to provide a step-by-step description of crude fractional extract preparation, including solvent sequence, extraction duration, drying intervals, filtration, solvent removal, and stock solution preparation (Page 25, Lines 826–833). Extraction yields were normalized and reported as percentage yield (w/w) relative to dried plant material to facilitate reproducibility and comparison across samples.

In addition, the rationale for compound dosing has been clarified across relevant assay sections, with concentrations selected based on IC₁₀, IC₂₀, IC₃₀, or IC₅₀ values derived from preliminary cytotoxicity screening to balance biological activity and cytotoxicity. Solvent controls were standardized throughout all in vitro experiments by maintaining the final DMSO concentration below cytotoxic thresholds and keeping it constant across treatment and control groups.

Collectively, these revisions were implemented to ensure methodological transparency, internal consistency, and full reproducibility of the experimental procedures.

9. The manuscript occasionally shifts between descriptive phytochemistry and biological interpretation. Consider tightening the narrative flow to maintain a clear biological focus.

Response: We thank the reviewer for this constructive comment and agree that maintaining a clear biological focus is essential. To address this concern, the manuscript has been revised to improve narrative flow and better integrate phytochemical description with biological interpretation.

Specifically, the Results (Page 6, lines 187–189 and page 7, lines 211–213) and Discussion sections (page 21, lines 632–638 and pages 21-22, lines 660-678) were reorganized to follow biologically driven progression. Initial phytochemical screening and compound isolation are now presented concisely as enabling steps, while emphasis is placed on how each stage informs subsequent biological investigation. Descriptive phytochemical details were streamlined, and interpretative transitions were added to clearly link compound identification with functional outcomes, including cytotoxicity, WT1 modulation, molecular docking, network pharmacology, and downstream cellular responses such as apoptosis, cell cycle arrest, and anti-inflammatory activity. These revisions were implemented to ensure that phytochemical characterization supports, rather than interrupts, the central biological narrative of the study.

10. Please ensure consistent use of units (µg/mL vs. µM, if applicable) throughout the text.

Response: We have already rechecked for the units in the manuscripts.

11. Abbreviations such as AE EtOAc No. 003 should be defined once and used consistently.

Response: AE EtOAc No. 003 has been first defined as the EtOAc crude fractional extract in Abstract (Page 1, Lines 37-38) and Results 2.2 (Page 4, Lines 144-145).

12. Consider briefly summarizing the key findings of the supplementary tables in the main Results section to guide readers.

Response: To guide readers and highlight the key findings of the supplementary data, concise summaries have been incorporated into the main Results section. Specifically, the cytotoxic screening results of the four purified compounds are now briefly summarized in the main text. Among the isolated compounds, purified compound 4 exhibited the highest yield (30.46%) and the most potent cytotoxic activity against both KG-1a and EoL-1 leukemic cells, while maintaining lower toxicity toward normal PBMCs (Page 6, Lines 162–168; Supplementary Table S1).

In addition, the rationale for defining purified compound 4 as a ternary mixture has been clarified by summarizing the key outcomes of further HPLC purification and NMR characterization in the main text, with detailed spectral data provided in supplementary Data S1, Table S2–S4, and Figures S1–S4 (Page 6, Lines 173–181).

Furthermore, the cytotoxicity assessment in RAW264.7 macrophages has been briefly summarized to emphasize that AE EtOAc No. 003 and the ternary mixture did not markedly affect cell viability at the concentrations used for anti-inflammatory assays, with detailed data presented in Supplementary Table S5 and Figure S5 (Page 14, Lines 465–470).

Reviewer 2 Report

Comments and Suggestions for Authors

 

Review Article o1-06.2026: Isolation and Characterization of (5R,6R)-5-(1-Hydroxyethyl)-Dihydro -2-Furanone from Angiopteris evecta with Potent Anti- Inflammatory and Anti-Leukemic Activities. By Lapamas Rueankham, Natsima Viriyaadhammaa, Wenxian Yin, Yuanzhi Liu, Sawitree Chiampanichayakul, Methee Rungrojsakul, Trinnakorn Katekunlaphan, Siriporn Okonogi Aroonchai Saiai, Arihiro Iwasaki, Christian Nanga Chick, Toyonobu Usuki, and Songyot Anuchapreeda.

Acute myeloid leukemia (AML) is the subtype of leukemia with an increasing prevalence and persistently low survival rates, accompanied by a yearly rise in mortality [1]. AML characterized by the accumulation of undifferentiated blast cells and disruption of the number and function of normal blood cells, resulting from abnormal proliferation and impaired differentiation of hematopoietic stem and progenitor cells (HSPCs) [2,3]. Genetic alterations, including chromosomal aberrations and single-nucleotide variants (SNVs), can transform hematopoietic stem cells or progenitor cells into leukemic stem cells (LSCs) through mutations in genes such as DNMT3A, FLT3, NPM1, CEBPA, or WT1 [4,5]. LSCs possess self-renewal capacity, remain dormant in the G0 phase, and lack differentiation potential, enabling them to evade chemotherapeutic eradication and contribute to AML relapse [6]. Angiopteris evecta known as giant fern or king fern and locally referred to as “Wan Keab Rad” in Thailand, belonging to the family Marattiaceae. In Thai traditional medicine, A. evecta rhizome is an ingredient of the remedy “Kheaw-Hom” and traditionally used as a diuretic, antipyretic, analgesic, and anti-diarrheal agent [18,19]. Nevertheless, the antileukemic effects of A. evecta extracts and their active constituent’s haven´t elucidated. The aim of the authors was to collected dried rhizomes of A. evecta from 12 different locations in Thailand, they extracted with n-Hexane (n-Hex), ethyl acetate (EtOAc), and ethanol (EtOH). These extracts screened and compared for cytotoxic effects against leukemic cell lines (KG-1a and EoL-1 cells). Among thirty-six crude fractional extracts, the ethyl acetate extract from source No. 003 (AE EtOAc No. 003) exhibited the strongest cytotoxic activity against KG-1a and EoL-1 leukemic cell lines, with low toxicity toward normal peripheral blood mononuclear cells. Bioactivity-guided fractionation yielded purified compound 4, a furanone-rich mixture (ternary mixture) dominated by (5R,6R)-5-(1-hydroxyethyl)-dihydro-2-furanone. Purified compound 4 inhibited leukemic cell proliferation by inducing apoptosis, causing cell cycle arrest, and downregulating WT1 expression in EoL-1 cells. Network pharmacology and molecular docking analyses implicated AKT1, MAPK signaling, apoptosis-related pathways, and WT1 as key molecular targets. In addition, AE EtOAc No. 003 and purified compound 4 suppressed TNF-α and IL-6 production in LPS-stimulated macrophages. The most active compound of A. evecta-derived furanone compounds represent promising lead candidates for anti-leukemic drug development.

Results

A total of 36 Crude fractional extracts of A. evecta screened for cytotoxic activity against leukemic cell lines, including KG-1a and EoL-1, using the MTT assay. Based on its potent anti-leukemic activity and comparatively low toxicity to ward normal cells, the crude fractional extract of A. evecta AE EtOAc No. 003 selected as the candidate for further investigation. The fraction AE EtOAc No. 003 (3 g) purified in a column chromatography, obtained four pure compounds, and screened for the cytotoxicity against KG-1a and EoL-1 cells, as well as normal PBMCs, using the MTT assay. The compound 4 characterized as a light-yellow viscous oil, with the highest yield (30.46%). In addition, it exhibited the greatest cytotoxic activity against both leukemic cell lines, with IC50 values of 9.73 ± 0.24 for KG-1a cells and 8.10 ± 0.73 for EoL-1 cells. Notably, compound 4 showed low cytotoxicity toward PBMCs.

-In Line 165 The phrase “the selectivity index (SI) of purified compound 4 indicated a higher efficacy against leukemic cells compared with normal PBMCs,” change by The selectivity index (SI) of compound 4 indicated a higher efficacy against leukemic cells than normal PBMCs,

-In Line 168 to 176 change by “To know if compound 4 was pure, further purification by high-performance liquid chromatography (HPLC) and characterized to determine its chemical properties and molecular structure using nuclear magnetic resonance (NMR) spectroscopy (supplementary Figure S1S4). The NMR spectra compared with previously reported data by Chen Y., 2010 [29] showed a single peak on the HPLC chromatogram, however, revealed a mixture of three coeluting chemical compound (ternary mixture) in a 2 : 2 : 1 ratio: (–)-epi-osmundalactone ((5R,6R)-5,6-dihydro-5-hydroxy-6-methyl-2H-pyran-2-one), (5R,6R)-5-(1-hydroxyethyl)- dihydro-2-furanone, and (5R,6R)-5-(1-hydroxyethyl)-2(5H)-furanone.”

-Based on the docking scores, the compound (5R,6R)-5-(1-hydroxyethyl)-dihydro-2-furanone exhibited the strongest binding affinity toward WT1 (−6.3 kcal/mol). However, (5R,6R)-5-(1-hydroxyethyl)-2(5H)- furanone and epi-osmundalactone yielded scores of −6.2 kcal/mol and −5.3 kcal/mol, respectively. In addition all three compounds showed significant affinities with ERK1 and ERK2 (all ≥ −5.6 kcal/mol), suggesting a potential multi-target inhibitory effect on the MAPK pathway. Furthermore (5R,6R)-5-(1-hydroxyethyl)-dihydro-2-furanone established strong conventional hydrogen bonding interactions with three key sites at the protein-DNA interface: amino acid residue ARG87 (Chain A) and nucleotides DG129 (Chain B) and DC151 (Chain C) (Figure 4AC). These results indicate that the compound stabilized within the WT1 binding pocket, potentially interfering with its biological activity in AML cells.

 

These results indicated that AE EtOAc No. 003 and purified compound 4 inhibited leukemic cell proliferation by inducing cell cycle arrest at G2/M of KG-1a cells and S phase of EoL-1 cells.

In the effects of AE EtOAc No. 003 and purified Compound 4 on Leukemic Cell apoptosis,  indicated that AE EtOAc No. 003 and purified compound 4 suppress leukemic cell growth by inducing apoptosis.

-In Disscusion Line 676 to 682, I suggest deleting the phrases “traditionally used as an ingredient in the Thai herbal remedy Kheaw-Hom. A. evecta, commonly known as giant fern or king fern and locally referred to as Wan Keab Rad in Thailand, has been traditionally used for the treatment of fever, inflammation, and diarrhea [18,19], since is described in Introducction and in Results Rhizomes of A. evecta were collected from 12 different locations in Thailand and extracted using n-Hex, EtOAc, and EtOH, yielding a total of 36 crude fractional extracts for evaluation of antileukemic activity. Initial cytotoxicity screening against KG-1a and EoL-1 leukemic cell lines revealed the most EtOAc crude fractional extracts exhibited pronounced cytotoxic effects.

-In References section only reference 29 has doi information.

Title, Abstract, Introduction, Materials and Methods, and conclusion are adequate for the content of the article.

I suggest publishing at the Journal after minor correction.

Comments on the Quality of English Language

I indicated at the review

Author Response

Reviewer 2

Review Article o1-06.2026: Isolation and Characterization of (5R,6R)-5-(1-Hydroxyethyl)-Dihydro -2-Furanone from Angiopteris evecta with Potent Anti- Inflammatory and Anti-Leukemic Activities. By Lapamas Rueankham, Natsima Viriyaadhammaa, Wenxian Yin, Yuanzhi Liu, Sawitree Chiampanichayakul, Methee Rungrojsakul, Trinnakorn Katekunlaphan, Siriporn Okonogi Aroonchai Saiai, Arihiro Iwasaki, Christian Nanga Chick, Toyonobu Usuki, and Songyot Anuchapreeda.

Acute myeloid leukemia (AML) is the subtype of leukemia with an increasing prevalence and persistently low survival rates, accompanied by a yearly rise in mortality [1]. AML characterized by the accumulation of undifferentiated blast cells and disruption of the number and function of normal blood cells, resulting from abnormal proliferation and impaired differentiation of hematopoietic stem and progenitor cells (HSPCs) [2,3]. Genetic alterations, including chromosomal aberrations and single-nucleotide variants (SNVs), can transform hematopoietic stem cells or progenitor cells into leukemic stem cells (LSCs) through mutations in genes such as DNMT3A, FLT3, NPM1, CEBPA, or WT1 [4,5]. LSCs possess self-renewal capacity, remain dormant in the G0 phase, and lack differentiation potential, enabling them to evade chemotherapeutic eradication and contribute to AML relapse [6]. Angiopteris evecta known as giant fern or king fern and locally referred to as “Wan Keab Rad” in Thailand, belonging to the family Marattiaceae. In Thai traditional medicine, A. evecta rhizome is an ingredient of the remedy “Kheaw-Hom” and traditionally used as a diuretic, antipyretic, analgesic, and anti-diarrheal agent [18,19]. Nevertheless, the antileukemic effects of A. evecta extracts and their active constituent’s haven´t elucidated. The aim of the authors was to collected dried rhizomes of A. evecta from 12 different locations in Thailand, they extracted with n-Hexane (n-Hex), ethyl acetate (EtOAc), and ethanol (EtOH). These extracts screened and compared for cytotoxic effects against leukemic cell lines (KG-1a and EoL-1 cells). Among thirty-six crude fractional extracts, the ethyl acetate extract from source No. 003 (AE EtOAc No. 003) exhibited the strongest cytotoxic activity against KG-1a and EoL-1 leukemic cell lines, with low toxicity toward normal peripheral blood mononuclear cells. Bioactivity-guided fractionation yielded purified compound 4, a furanone-rich mixture (ternary mixture) dominated by (5R,6R)-5-(1-hydroxyethyl)-dihydro-2-furanone. Purified compound 4 inhibited leukemic cell proliferation by inducing apoptosis, causing cell cycle arrest, and downregulating WT1 expression in EoL-1 cells. Network pharmacology and molecular docking analyses implicated AKT1, MAPK signaling, apoptosis-related pathways, and WT1 as key molecular targets. In addition, AE EtOAc No. 003 and purified compound 4 suppressed TNF-α and IL-6 production in LPS-stimulated macrophages. The most active compound of A. evecta-derived furanone compounds represent promising lead candidates for anti-leukemic drug development.

Results

A total of 36 Crude fractional extracts of A. evecta screened for cytotoxic activity against leukemic cell lines, including KG-1a and EoL-1, using the MTT assay. Based on its potent anti-leukemic activity and comparatively low toxicity to ward normal cells, the crude fractional extract of A. evecta AE EtOAc No. 003 selected as the candidate for further investigation. The fraction AE EtOAc No. 003 (3 g) purified in a column chromatography, obtained four pure compounds, and screened for the cytotoxicity against KG-1a and EoL-1 cells, as well as normal PBMCs, using the MTT assay. The compound 4 characterized as a light-yellow viscous oil, with the highest yield (30.46%). In addition, it exhibited the greatest cytotoxic activity against both leukemic cell lines, with IC50 values of 9.73 ± 0.24 for KG-1a cells and 8.10 ± 0.73 for EoL-1 cells. Notably, compound 4 showed low cytotoxicity toward PBMCs.

-In Line 165 The phrase “the selectivity index (SI) of purified compound 4 indicated a higher efficacy against leukemic cells compared with normal PBMCs,” change by The selectivity index (SI) of compound 4 indicated a higher efficacy against leukemic cells than normal PBMCs,

-In Line 168 to 176 change by “To know if compound 4 was pure, further purification by high-performance liquid chromatography (HPLC) and characterized to determine its chemical properties and molecular structure using nuclear magnetic resonance (NMR) spectroscopy (supplementary Figure S1-S4). The NMR spectra compared with previously reported data by Chen Y., 2010 [29] showed a single peak on the HPLC chromatogram, however, revealed a mixture of three coeluting chemical compound (ternary mixture) in a 2 : 2 : 1 ratio: (–)-epi-osmundalactone ((5R,6R)-5,6-dihydro-5-hydroxy-6-methyl-2H-pyran-2-one), (5R,6R)-5-(1-hydroxyethyl)- dihydro-2-furanone, and (5R,6R)-5-(1-hydroxyethyl)-2(5H)-furanone.”

-Line 354-365: Based on the docking scores, the compound (5R,6R)-5-(1-hydroxyethyl)-dihydro-2-furanone exhibited the strongest binding affinity toward WT1 (−6.3 kcal/mol). However, (5R,6R)-5-(1-hydroxyethyl)-2(5H)- furanone and epi-osmundalactone yielded scores of −6.2 kcal/mol and −5.3 kcal/mol, respectively. In addition all three compounds showed significant affinities with ERK1 and ERK2 (all ≥ −5.6 kcal/mol), suggesting a potential multi-target inhibitory effect on the MAPK pathway. Furthermore (5R,6R)-5-(1-hydroxyethyl)-dihydro-2-furanone established strong conventional hydrogen bonding interactions with three key sites at the protein-DNA interface: amino acid residue ARG87 (Chain A) and nucleotides DG129 (Chain B) and DC151 (Chain C) (Figure 4A-C). These results indicate that the compound stabilized within the WT1 binding pocket, potentially interfering with its biological activity in AML cells.

            These results indicated that AE EtOAc No. 003 and purified compound 4 inhibited leukemic cell proliferation by inducing cell cycle arrest at G2/M of KG-1a cells and S phase of EoL-1 cells.

In the effects of AE EtOAc No. 003 and purified Compound 4 on Leukemic Cell apoptosis, indicated that AE EtOAc No. 003 and purified compound 4 suppress leukemic cell growth by inducing apoptosis.

-In Disscusion Line 676 to 682, I suggest deleting the phrases “traditionally used as an ingredient in the Thai herbal remedy Kheaw-Hom. A. evecta, commonly known as giant fern or king fern and locally referred to as Wan Keab Rad in Thailand, has been traditionally used for the treatment of fever, inflammation, and diarrhea [18,19], since is described in Introducction and in Results Rhizomes of A. evecta were collected from 12 different locations in Thailand and extracted using n-Hex, EtOAc, and EtOH, yielding a total of 36 crude fractional extracts for evaluation of antileukemic activity. Initial cytotoxicity screening against KG-1a and EoL-1 leukemic cell lines revealed the most EtOAc crude fractional extracts exhibited pronounced cytotoxic effects.

Response: The sentences in Lines 165; Lines 168-176; and Lines 354-365 have already edited as shown on page 6, lines 168-169; page 6, lines 173-187; page 10, lines 274-286, and in Discussion Lines 615-622 have been removed.

-In References section only reference 29 has doi information.

Response: Doi information of reference 29 has already been removed.

- Title, Abstract, Introduction, Materials and Methods, and conclusion are adequate for the content of the article.

I suggest publishing at the Journal after minor correction.

Response: Thank you very much for your comments and suggestions.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for submitting this well-structured and comprehensive study on the isolation and characterization of bioactive compounds from Angiopteris erecta with anti-inflammatory and anti-leukemic potential. The work is clearly presented, and the multi-faceted approach combining phytochemistry, network pharmacology, molecular docking, and in vitro assays is commendable.

However, as noted in the manuscript, a significant limitation remains regarding the nature of the primary bioactive fraction, referred to as “purified compound 4.” While described as “purified,” this fraction is in fact a ternary mixture of three co-eluting compounds in a 2:2:1 ratio: (-)-epi-osmundalactone, (5R,6R)-5-(1-hydroxyethyl)-dihydro-2-furanone, and (5R,6R)-5-(1-hydroxyethyl)-2(5H)-furanone. Although the study attributes the observed biological effects to this mixture, it is not possible to determine whether the activity originates from one specific component, results from additive effects, or reflects true synergy among the constituents.

• It is recommended to clarify from the abstract and results that "purified compound 4" is actually a ternary mixture, not a single compound, to prevent misinterpretation. In the discussion, it would be valuable to elaborate on how this limitation affects the attribution of the observed biological effects and to explicitly propose the purification and individual evaluation of each component as a necessary future step. Furthermore, if the molecular docking results allow for any hypothesis regarding which component may be the most active, it would be useful to briefly mention this. Finally, it is suggested to review the title and keywords to accurately reflect the nature of the mixture studied, thereby enhancing the transparency and rigor of the work.
• The study reports inconsistent effects of the treatments on WT1 expression across the two tested cell lines. The extract and the purified compound 4 did not significantly affect WT1 expression in KG-1a cells, but the purified compound 4 did downregulate it in EoL-1 cells.  The discussion suggests this is due to cell-line-specific effects or different underlying mechanisms (WT1-dependent vs. WT1-independent).  While this is a plausible explanation, it also highlights a lack of a unified mechanism of action, which weakens the overall hypothesis regarding WT1 as a primary target. The findings suggest the compound's effect is context-dependent and not universally applicable, even within AML models.
• Table 3 presents a selectivity index (SI) for the standard anti-cancer drug doxorubicin as less than 1 (0.66 for KG-1a and 0.77 for EoL-1), indicating it is more toxic to normal PBMCs than to the cancer cells in this specific assay.  While this highlights the superior selectivity of purified compound 4, using a comparator with such poor performance in the given test system might overstate the relative advantage of the new compound. The result for doxorubicin seems unusual and may depend heavily on the specific experimental conditions, which are not fully detailed for this reference drug.
• A substantial part of the mechanistic discussion is based on predictive tools like network pharmacology and molecular docking.  These in silico methods predict potential targets (like AKT1, MAPK pathway) and binding affinities but do not provide experimental proof of these interactions.  The study presents these predictions as strong support for its hypotheses, but they remain correlational and speculative until experimentally verified. For instance, the predicted involvement of the MAPK signaling pathway is not confirmed with experimental data (e.g., Western blots for phosphorylated ERK or other pathway members).

Author Response

Reviewer 3

Thank you for submitting this well-structured and comprehensive study on the isolation and characterization of bioactive compounds from Angiopteris erecta with anti-inflammatory and anti-leukemic potential. The work is clearly presented, and the multi-faceted approach combining phytochemistry, network pharmacology, molecular docking, and in vitro assays is commendable.

However, as noted in the manuscript, a significant limitation remains regarding the nature of the primary bioactive fraction, referred to as “purified compound 4.” While described as “purified,” this fraction is in fact a ternary mixture of three co-eluting compounds in a 2:2:1 ratio: (-)-epi-osmundalactone, (5R,6R)-5-(1-hydroxyethyl)-dihydro-2-furanone, and (5R,6R)-5-(1-hydroxyethyl)-2(5H)-furanone. Although the study attributes the observed biological effects to this mixture, it is not possible to determine whether the activity originates from one specific component, results from additive effects, or reflects true synergy among the constituents.

1. It is recommended to clarify from the abstract and results that "purified compound 4" is actually a ternary mixture, not a single compound, to prevent misinterpretation. In the discussion, it would be valuable to elaborate on how this limitation affects the attribution of the observed biological effects and to explicitly propose the purification and individual evaluation of each component as a necessary future step. Furthermore, if the molecular docking results allow for any hypothesis regarding which component may be the most active, it would be useful to briefly mention this. Finally, it is suggested to review the title and keywords to accurately reflect the nature of the mixture studied, thereby enhancing the transparency and rigor of the work.

Response: We thank the reviewer for this important and insightful comment and fully agree with the concern regarding the nature of “purified compound 4.” In the revised manuscript, this fraction has been explicitly defined as a ternary mixture rather than a single purified compound. Accordingly, the terminology has been corrected in the Abstract, Results, and throughout the manuscript following Section 2.3 (Page 6, Lines 184-185), to prevent any potential misinterpretation.

In addition, the Discussion has been revised to explicitly acknowledge the limitation that the observed biological activities cannot be attributed to a single constituent within the ternary mixture. We now clarify that the anti-leukemic and anti-inflammatory effects observed may arise from the activity of one dominant component, additive effects, or synergistic interactions among the three co-eluting compounds (Page 22, lines 673–678).

We further emphasize that future studies involving complete purification and individual biological evaluation of epi-osmundalactone, 5-(1-hydroxyethyl)-dihydro-2-furanone, and 5-(1-hydroxyethyl)-2(5H)-furanone will be essential to delineate their respective contributions and potential synergistic effects (Pages 24-25, lines 812-817).

Based on molecular docking analysis, 5-(1-hydroxyethyl)-dihydro-2-furanone exhibited the highest binding affinity toward the WT1–DNA complex and MAPK-related proteins. This observation is now briefly discussed as a hypothesis suggesting that this component may play a prominent role in the observed biological activity, while acknowledging that experimental validation is required (Page 24, lines 798-801).

Finally, the title and keywords have been carefully reviewed and revised where appropriate to accurately reflect the nature of the ternary mixture studied, thereby improving transparency and scientific rigor.

2. The study reports inconsistent effects of the treatments on WT1 expression across the two tested cell lines. The extract and the purified compound 4 did not significantly affect WT1 expression in KG-1a cells, but the purified compound 4 did downregulate it in EoL-1 cells.  The discussion suggests this is due to cell-line-specific effects or different underlying mechanisms (WT1-dependent vs. WT1-independent).  While this is a plausible explanation, it also highlights a lack of a unified mechanism of action, which weakens the overall hypothesis regarding WT1 as a primary target. The findings suggest the compound's effect is context-dependent and not universally applicable, even within AML models.

Response: We thank the reviewer for this important comment and agree that the differential effects on WT1 expression between KG-1a and EoL-1 cells indicate a context-dependent mechanism rather than a unified mode of action. As shown in Results Section 2.5 (Page 8) and discussed on pages 22-23 (Lines 704-717), AE EtOAc No. 003 and the ternary mixture inhibited proliferation of KG-1a cells primarily through the induction of apoptosis and G2/M cell cycle arrest, without significantly altering WT1 expression. These findings suggest that WT1-independent mechanisms contribute substantially to growth inhibition in KG-1a cells, likely reflecting phenotypic and molecular heterogeneity among AML subtypes. In contrast, in EoL-1 cells, the ternary mixture significantly downregulated WT1 expression in parallel with reduced cell proliferation, supporting a WT1-associated mechanism in this cellular context. Taken together, these results indicate that WT1 should not be considered a universal primary target of the extract or ternary mixture across AML models. Rather, WT1 modulation appears to be cell line–specific and context-dependent, while apoptosis induction and cell cycle arrest represent more consistent downstream biological effects observed across both leukemic cell lines. Future studies will therefore focus on elucidating the upstream determinants that govern WT1-dependent versus WT1-independent responses, including cellular transport, signaling network differences, and transcriptional regulation, to better define the mechanistic scope of these compounds. We have edited these details in the discussion on pages 22-23 (Lines 704-717).

3. Table 3 presents a selectivity index (SI) for the standard anti-cancer drug doxorubicin as less than 1 (0.66 for KG-1a and 0.77 for EoL-1), indicating it is more toxic to normal PBMCs than to the cancer cells in this specific assay.  While this highlights the superior selectivity of purified compound 4, using a comparator with such poor performance in the given test system might overstate the relative advantage of the new compound. The result for doxorubicin seems unusual and may depend heavily on the specific experimental conditions, which are not fully detailed for this reference drug.

Response: We thank the reviewer for this careful and constructive comment regarding the selectivity index (SI) of doxorubicin. We would like to clarify that SI values in this study were calculated as the ratio of the IC50 in normal PBMCs to that in leukemic cells. For doxorubicin, the IC50 value in PBMCs was not reached at the highest tested concentration (>1,000 ng/mL). Therefore, the SI values for doxorubicin are reported as lower-bound estimates rather than extract values. Based on the corrected presentation, the SI of doxorubicin is >0.66 ng/mL for KG-1a cells and >771.04 for EoL-1 cells, reflecting the high sensitivity of EoL-1 cells to doxorubicin and the limited interpretability of PBMC-based SI measurements for this broadly cytotoxic chemotherapeutic agent. We acknowledge that the initial formatting of Table 3, particularly the use of inequality symbols and mixed units, may have caused confusion. Accordingly, Table 3 has been revised to clarify display the inequality signs and units, and explanatory footnote has been added to indicate that these SI values represent low-bound estimates. Doxorubicin was included as a reference cytotoxic agent to provide contextual comparison rather than as a benchmark for selectivity. We have also revised the results section to clarify this limitation and to avoid overstating the relative advantage of the ternary mixture based solely on SI values. The revised Table 3 has been shown on Page 6.

Compound and Drug                          IC50 values (mean ± SD)                             Selectivity index (SI)

                                                  KG-1a              EoL-1               PBMCs                    KG-1a             EoL-1

Ternary mixture                     9.73 ± 0.24.    8.10 ± 0.73.      51.36 ± 8.37                 5.28                6.34

(Purified compound 4) (µg/mL)                                            

Doxorubicin (ng/mL)       1,509.43 ± 89.78  1.30 ± 0.04        > 1,000                      > 0.66          > 771.04

 

4. A substantial part of the mechanistic discussion is based on predictive tools like network pharmacology and molecular docking.  These in silico methods predict potential targets (like AKT1, MAPK pathway) and binding affinities but do not provide experimental proof of these interactions.  The study presents these predictions as strong support for its hypotheses, but they remain correlational and speculative until experimentally verified. For instance, the predicted involvement of the MAPK signaling pathway is not confirmed with experimental data (e.g., Western blots for phosphorylated ERK or other pathway members).

Response: We thank the reviewer for this important and thoughtful comment. We fully agree that network pharmacology and molecular docking are predictive in silico approaches that generate hypotheses regarding potential targets and signaling pathways, but do not constitute experimental proof of direct molecular interactions or pathway activation. In the revised manuscript, we have carefully revised the Discussion to moderate the interpretation of these in silico results, explicitly stating that the predicted involvement of targets such as AKT1 and MAPK-related proteins represents putative mechanisms rather than confirmed signaling events. These analyses are now presented as supportive, hypothesis-generating evidence that complements the observed cellular phenotypes, including apoptosis induction and cell cycle arrest. We acknowledge that experimental validation of these predictions, such as Western blot analysis of MAPK/ERK pathway activation (e.g., phosphorylated ERK), AKT signaling components, or pharmacological inhibition studies, was not performed in the current study and therefore represents an important limitation. Accordingly, we have explicitly identified these experiments as necessary future directions to validate the predicted signaling pathways and to establish causal mechanistic links. These revisions were made to ensure a clear distinction between computational predictions and experimentally validated findings, and to avoid overinterpretation of in silico data. We discussed this part in the discussion on page 24, lines 798-801.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have done an excellent job revising the manuscript. They 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.

Reviewer 3 Report

Comments and Suggestions for Authors

Accept in present form