Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the current study, the authors have investigated Murraya exotica roots for their chemical composition. Twenty compounds were isolated and chemically identified using different spectroscopic analyses including extensive NMR measurements. The isolated compounds were tested for their anti-inflammatory activities. The manuscript is interesting and well written. I have only a few remarks to be addressed

• The authors should mention the family of the plant.
• A flowchart for the isolation process (scheme of isolation) will be beneficial.
• The double bond equivalents (degree of unsaturation) of each compound should be calculated and added to your spectral data interpretation.
• It is better to make the double arrows indicating the NOESY correlation with different colors to those of the HMBC
• In the supplementary part, add partial expansion for the NMR spectra highlighting the key 2 D NMR correlations.
• In the supplementary part (if possible), provide the semi prep. HPLC chromatograms for each isolated compound.

Author Response

Q1.       The authors should mention the family of the plant.

Response: Thank you for this suggestion. We have added the family name (Rutaceae) to the text where the plant is first introduced to ensure botanical accuracy.

Q2.       A flowchart for the isolation process (scheme of isolation) will be beneficial.

Response: We agree that a flowchart would improve clarity. We have created a scheme illustrating the extraction and isolation workflow and included it in the Supplementary Information as Figure S1.3.

Q3.        The double bond equivalents (degree of unsaturation) of each compound should be calculated and added to your spectral data interpretation.

Response: We have calculated the degrees of unsaturation for all isolated compounds and incorporated these values into the characterization data in section 2.1 to assist readers in understanding the structural features.

Q4.       It is better to make the double arrows indicating the NOESY correlation with different colors to those of the HMBC

Response: We apologize for the confusion caused by the arrow style in Figure 2. We wish to clarify that no NOESY correlations were depicted in this figure. The double-headed arrows were intended to represent mutual HMBC correlations between the proton and carbon signals. To prevent future misunderstanding by readers, we have added an explanatory note in the legend of Figure 2 stating: "Double-headed arrows indicate mutual HMBC correlations."

Q5.       In the supplementary part, add partial expansion for the NMR spectra highlighting the key 2D NMR correlations.

Response: We have adjusted the layout and enlarged the size of the 2D NMR spectra in the Supplementary Information. We believe that these adjustments ensure the key correlations are distinguishable.

Q6.       In the supplementary part (if possible), provide the semi prep. HPLC chromatograms for each isolated compound.

Response: We sincerely appreciate the reviewer’s suggestion to include the semi-preparative HPLC chromatograms to demonstrate the isolation process and purity. Regrettably, we are unable to provide these specific chromatograms as the raw data files were not archived at the time of the isolation experiments. However, we wish to assure the reviewer that the purity of all isolated compounds was strictly verified prior to structural elucidation and biological assays.

The high purity of the compounds (>95%) is clearly evidenced by the 1D and 2D NMR spectra provided in the Supplementary Information, which show sharp signals and clean baselines without discernible impurities. We hope this alternative evidence suffices to demonstrate the quality of the isolated compounds.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present Phytochemical investigation of the roots of Murraya exotica. They isolated 20 coumarins, 12 of them are new compounds. The structures and absolute configurations of the new compounds were elucidated by a combination of 1D and 2D NMR spectra, HRESIMS analyses, single-crystal X-ray diffraction and ECD exciton coupling. The authors have competently analyzed the spectral data. They have compared the experimental and calculated ECD spectra and have applied chemical hydrolysis to determine the absolute configurations. Anti-inflammatory evaluation of the new compounds was carried out.
The manuscript is well written. The English is sufficiently good. 

The manuscript's title, abstract, scheme, tables and figures are adequate to the content.

I have no corrections to the reviewed manuscript.

It will be a plus to include in the text the IUPAC names of the new compounds.

Author Response

Q1.       It will be a plus to include in the text the IUPAC names of the new compounds.

Response: We appreciate this suggestion. We have added the IUPAC names for all 12 new compounds in Section 4.4.

4.4.1. Exoticoumarin A (1) [8-((S)-2-hydroxy-3-(((1S,2S)-1-hydroxy-1-(7-methoxy-2-oxo-2H-chromen-8-yl)-3-methylbut-3-en-2-yl)oxy)-3-methylbutyl)-5,7-dimethoxy-2H-chromen-2-one]

4.2. Exoticoumarin B (2) [8-((R)-2-(((S)-4-(5,7-dimethoxy-2-oxo-2H-chromen-8-yl)-3-hydroxy-2-methylbutan-2-yl)oxy)-3-hydroxy-3-methylbutyl)-5,7-dimethoxy-2H-chromen-2-one]

4.4.3. Exoticoumarin C (3) [(S)-1-(5,7-dimethoxy-2-oxo-2H-chromen-8-yl)-3-hydroxy-3-methylbutan-2-yl (E)-3-(3-((S)-2,3-dihydroxy-3-methylbutyl)-2-hydroxy-4,6-dimethoxyphenyl)acrylate]

4.4.4. Exoticoumarin D (4) [(S)-1-(5,7-dimethoxy-2-oxo-2H-chromen-8-yl)-3-hydroxy-3-methylbutan-2-yl (E)-3-(3-((1R,2R)-1,2-dihydroxy-3-methylbut-3-en-1-yl)-2-hydroxy-4-methoxyphenyl)acrylate]

4.4.5. Exoticoumarin E (5) [(S)-1-(5,7-dimethoxy-2-oxo-2H-chromen-8-yl)-3-hydroxy-3-methylbutan-2-yl (E)-3-(3-((1S,2S)-1,2-dihydroxy-3-methylbut-3-en-1-yl)-2-hydroxy-4-methoxyphenyl)acrylate]

4.4.6. Exoticoumarin F (6) [(S)-1-(5,7-dimethoxy-2-oxo-2H-chromen-8-yl)-3-hydroxy-3-methylbutan-2-yl (E)-3-(2-hydroxy-3-((1R,2R)-2-hydroxy-1-methoxy-3-methylbut-3-en-1-yl)-4-methoxyphenyl)acrylate]

4.4.7. Exoticoumarin G (7) [(S)-1-(5,7-dimethoxy-2-oxo-2H-chromen-8-yl)-3-hydroxy-3-methylbutan-2-yl (E)-3-(2-hydroxy-3-((1S,2S)-2-hydroxy-1-methoxy-3-methylbut-3-en-1-yl)-4-methoxyphenyl)acrylate]

4.4.8. Exoticoumarin H (8) [(S)-1-(6-hydroxy-5,7-dimethoxy-2-oxo-2H-chromen-8-yl)-3-methylbut-3-en-2-yl 3-methylbutanoate]

4.4.9. Exoticoumarin I (9) [7-methoxy-8-((1R,2R)-1,2,3,4-tetrahydroxy-3-methylbutyl)-2H-chromen-2-one]

4.4.10. Exoticoumarin J (10) [(1R,2R)-2-hydroxy-1-(7-methoxy-2-oxo-2H-chromen-8-yl)-3-methylbut-3-en-1-yl (8Z,11Z)-octadeca-8,11-dienoate]

4.4.11. Exoticoumarin K (11) [(1R,2R)-1-hydroxy-1-(7-methoxy-2-oxo-2H-chromen-8-yl)-3-methylbut-3-en-2-yl (10E,12Z)-9-hydroxyoctadeca-10,12-dienoate]

4.4.12. Exoticoumarin L (12) [(7-methoxy-2-oxo-2H-chromen-8-yl)methyl 3-methylbutanoate]

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Introduction

I suggest adding the part of the plant that has been used to the sentence where the pharmacological research is mentioned (line 47).

Line 55- I suggest change “aqueous EtOH” by “ethanolic”.

Materials and methods

Line 64- I suggest change “absorptions” by “absorption bands”

Line 550- Add the range of evaluated concentrations of the compounds, and indicate the solvent used to resuspend the compounds (negative control).

Results

Line 276- In the results for anti-inflammatory activity, why wasn't the positive control included? And why wasn't an analysis of variance and mean comparisons performed?

Discussion

I suggest strengthening the discussion by comparing their results with similar previous studies.

Line 372- They mention that the identification of anti-inflammatory agent 1 scientifically validates the traditional use of this plant for pain treatment, but was the concentration of agent 1 in the extract determined?

Author Response

Q1.       I suggest adding the part of the plant that has been used to the sentence where the pharmacological research is mentioned (line 47).

Response: We have revised the sentence in line 47 to explicitly state that the aerial parts of Murraya exotica (leaves and twigs) were the subject of the pharmacological research mentioned.

Q2.       Line 55-I suggest change “aqueous EtOH” by “ethanolic”.

Response: We have corrected "aqueous EtOH" to "ethanolic" as suggested.

Materials and methods

Q3.       Line 64- I suggest change “absorptions” by “absorption bands”

Response: We have changed "absorptions" to "absorption bands" in the Materials and Methods section.

Q4.       Line 550- Add the range of evaluated concentrations of the compounds, and indicate the solvent used to resuspend the compounds (negative control).

Response: We thank the reviewer for the valuable suggestion. As requested, we have added the concentration range and solvent information at line 550. The revised sentence now reads: "The tested compounds were evaluated at concentrations of 40, 20, 10, 5, 2.5, and 1.25 μM, with DMSO (dimethyl sulfoxide) used as the solvent and negative control." This addition provides a clearer description of the experimental conditions.

Q5.       Line 276- In the results for anti-inflammatory activity, why wasn't the positive control included? And why wasn't an analysis of variance and mean comparisons performed?

Response: We apologize for this oversight. The positive control Quercetin (IC₅₀ = 16.37 ± 0.96 μM) has been added to the revised figures and tables to facilitate comparison. Relevant statistical notations have also been updated in the figure legends.

Discussion

Q6.       I suggest strengthening the discussion by comparing their results with similar previous studies.

Response: We have expanded the Discussion (the last paragraph) to include comparisons with similar previous studies. The specific content added to the main text is presented as follows:

In the broader context of Traditional Chinese Medicine, the identification of potent anti-inflammatory agents in this study, particularly exoticoumarin A (1), supports the phytochemical rationale for the historical use of M. exotica in treating pain and rheumatic disorders. Distinct from previous investigations, which primarily characterized complex heterodimers [10] and phenylproenols [11] in the roots, or cyclopropane-bearing derivatives in the aerial parts [37], the current study reveals a rich diversity of C-8 substitued coumarins. Furthermore, unlike these earlier studies that were limited to preliminary phenotypic screening of NO inhibition, the present work advances our understanding by elucidating the underlying molecular mechanism at the protein level. This finding not only enriches the species' chemical diversity but also provides mechanistic evidence for its active constituents. Nevertheless, the current study is subjected to certain limitations. Specifically, as the anti-inflammatory effects and the proposed signaling model were verified exclusively in in vitro cellular models, future in vivo investigations are needed to evaluate the bioavailability, safety profile, and therapeutic efficacy of 1 in complex physiological systems.

 

Q7.       Line 372- They mention that the identification of anti-inflammatory agent 1 scientifically validates the traditional use of this plant for pain treatment, but was the concentration of agent 1 in the extract determined?

Response: Thank you for the correction. Accordingly, we have rephrased the Discussion to be more objective. We now cautiously state that the presence of compound 1 supports the traditional use, rather than definitively validating it.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

This interesting manuscript describes the isolation and identification of 12 new coumarin hybrids from the roots of Murraya exotica. Two of them presented significant antiinflammatory activity. There are some remarks specified below:

• Page 1, Abstract line 1; page 2 line 50; dimers: These compounds are not exactly dimers, but coumarin hybrids. Only, compounds 1 and 2 are biscoumarins.
• - Page 2, line 46 ... gastrointestinal disorders [6]: In ref. [6] antiinflammatory activities are presented.
• Page 3, line 95 planar structure of 1: According to ORTEP of 1, the molecule is not planar. The two coumarin moieties are planar, but the molecule isn't planar.
• Page 10, line 214, omphamurin isovalerate [20]: In this reference there isn't any omphamurin, which is presentd in Phytochemistry 1981, 20, 178-179.
• Page 15, line 317, seven biscoumarins (1-7): two biscoumarins (1,2).
• Page 15, line 331, biscoumarins (1-7): biscoumarins (1,2) and coumarin hybrids (3-7).
• Page 15, line 333, biscoumarins: coumarin hybrids.
• Pages 18-19, Exoticoumarin B (2) Exoticoumarin H (8), Exoticoumarin J (10): There is not any HO- absorption in the IR spectra.
• Page 23, ref. [8]: The authors must be checked; the names are the same with ref. [7].
• Page 23, ref. [21] and [27]: the main names of the authors must be checked.

Author Response

Q1．    Page 1, Abstract line 1; page 2 line 50; dimers: These compounds are not exactly dimers, but coumarin hybrids. Only, compounds 1 and 2 are biscoumarins.

Response: We agree with the reviewer's precise classification. We have revised the terminology throughout the manuscript to refer to compounds 3-7 as "coumarin hybrids" and restricted the term "biscoumarins" to compounds 1 and 2.

Q2．    Page 2, line 46 ... gastrointestinal disorders [6]: In ref. [6] anti-inflammatory activities are presented.

Response: We have replaced the appropriate reference for gastrointestinal disorders.

Q3．    Page 3, line 95 planar structure of 1: According to ORTEP of 1, the molecule is not planar. The two coumarin moieties are planar, but the molecule isn't planar.

Response: Thank you for the correction. We have rephrased this to state that the "2D structure" was established, rather than implying the 3D molecule is planar.

Q4．    Page 10, line 214, omphamurin isovalerate [20]: In this reference there isn't any omphamurin, which is presented in Phytochemistry 1981, 20, 178-179.

Response: We apologize for the citation error. We have corrected reference [20] to the correct paper Phytochemistry 1981, 20, 178-179.

Q5．    Page 15, line 317, seven biscoumarins (1-7): two biscoumarins (1,2).

Response: We have corrected this line as "two biscoumarins (1 and 2), five coumarin hybrids (3-7), and two rare coumarin fatty acid esters (10 and 11)".

Q6．    Page 15, line 331, biscoumarins (1-7): biscoumarins (1,2) and coumarin hybrids (3-7).

Response: We have made the suggested correction to distinguish the chemical classes accurately.

Q7．    Page 15, line 333, biscoumarins: coumarin hybrids.

Response: We have replaced "biscoumarins" with "coumarin hybrids".

Q8．    Pages 18-19, Exoticoumarin B (2) Exoticoumarin H (8), Exoticoumarin J (10): There is not any HO- absorption in the IR spectra.

Response: We appreciate your keen observation regarding this inconsistency. The absence of clear hydroxyl signals in the initial data was attributed to the residual solvent interference (MeCN). We have since re-measured the IR spectra for compounds 2, 8, and 10 using fully dried samples, and the original data have been replaced with these corrected values in the revised manuscript and Supporting Information.

Q9．    Page 23, ref. [8]: The authors must be checked; the names are the same with ref. [7].

Response: Thank you for your reminder. We have corrected the author list for Reference [8] in the revised manuscript. We have also conducted a thorough check of the reference list to ensure all citations are accurate and complete.

Q10． Page 23, ref. [21] and [27]: the main names of the authors must be checked.

Response: Thank you for your reminder. We have verified and corrected the author names for references [21] and [27] in the bibliography.

Author Response File: Author Response.docx

Reviewer 5 Report

Comments and Suggestions for Authors

This manuscript reports the isolation and structure elucidation of twenty coumarins (twelve new) from Murraya exotica roots and evaluates their anti-inflammatory activity in LPS-stimulated RAW264.7 macrophages. The chemical work is thorough and the biological screening identifies two active compounds, with exoticoumarin A (1) further investigated at the mechanistic level (NO inhibition, iNOS mRNA/protein, MAPK phosphorylation). Overall, the study is interesting and potentially suitable for publication, but the biological framing and mechanistic claims currently exceed the experimental support. A modest set of additions and careful rewording would significantly strengthen the paper.

 In my opinion, the paper is carefully written and has enough scientific quality to be published in International Journal of Molecular Sciences after minor revision.

• The Introduction is largely chemistry- and ethnopharmacology-oriented and does not adequately contextualize inflammation biology or justify the specific readouts used (NO/iNOS, MAPK). Since the manuscript later proposes a defined signaling mechanism (JNK → iNOS), the Introduction should be expanded by ~1–2 paragraphs to include: The relevance of the LPS/TLR4-RAW264.7 model. Why NO and iNOS are widely used inflammatory readouts and what they represent (and their limitations). A brief overview of canonical pathways downstream of LPS that regulate iNOS (e.g., MAPKs and NF-κB/AP-1) and how coumarins have been reported to modulate these pathways.
• Positive control is required (at minimum in the NO inhibition assay)The NO inhibition data would be substantially more interpretable if benchmarked against a well-established positive control under the same experimental conditions (LPS dose, incubation time, cell density). I strongly recommend including a positive control at least for the NO release assay (Figure 8A/C) and reporting it clearly in both Methods and Figure legends.
  Suggested positive controls: Diclofenac (widely used anti-inflammatory reference compound in LPS-stimulated macrophage assays; please include a concentration range appropriate for RAW264.7 and report the resulting NO inhibition and cell viability). L-NAME (NO synthase inhibitor; commonly used in NO/Griess assays; e.g., 50–200 µM). Aminoguanidine (iNOS-selective inhibitor; often 25–100 µM). Alternatively, dexamethasone can be used as a broad anti-inflammatory control.
  At minimum, please add one positive control condition in the NO assay and provide the corresponding inhibition level (and ideally an IC₅₀ if tested across concentrations), alongside a viability readout to exclude cytotoxicity. This will confirm assay performance and will help contextualize the potency of compounds 1 and 11.

• Mechanistic claims are overstated; rewording is necessary unless additional pathway data are added.

• The current Abstract and Conclusions use causal language (e.g., "directly mediated") and imply broad MAPK selectivity ("no observed activity against other MAPK subfamilies"). Based on the presented excerpt, only JNK and ERK1/2 appear to have been assessed. Without additional data, the following statements should be revised:

  Replace causal language ("directly mediated by attenuation of JNK phosphorylation") with associative language ("associated with", "correlated with", "consistent with involvement of").

  Avoid general claims about "other MAPK subfamilies" unless p38 (and ideally additional nodes) is tested. If only ERK is tested, state only that ERK1/2 phosphorylation was not significantly altered under the tested conditions.

  If the authors do not intend to add further experiments, the mechanism should be presented explicitly as a partial/working model. It is reasonable to mention p38 and NF-κB as plausible contributors in the proposed mechanism, but they must be labeled as hypothesized/not tested and the claims should be appropriately bounded.

  Supplementary Information

• Proposed biosynthetic pathway  of 1: 

  Eherification    -----------  should be  etherification

• Proposed biosynthetic route  of 2.Nucleophilic opening of the epoxide occurs in this case at the most substituted carbon. therefore, there can be no configuration inversion since the most substituted carbon is not chiral.

Author Response

Q1.       The Introduction is largely chemistry- and ethnopharmacology-oriented and does not adequately contextualize inflammation biology or justify the specific readouts used (NO/iNOS, MAPK). Since the manuscript later proposes a defined signaling mechanism (JNK → iNOS), the Introduction should be expanded by ~1–2 paragraphs to include: The relevance of the LPS/TLR4-RAW264.7 model. Why NO and iNOS are widely used inflammatory readouts and what they represent (and their limitations). A brief overview of canonical pathways downstream of LPS that regulate iNOS (e.g., MAPKs and NF-κB/AP-1) and how coumarins have been reported to modulate these pathways.

Response: We appreciate your guidance. In the revised manuscript, we have added a paragraph detailing the roles of macrophages, the LPS model, inflammatory mediators, and the MAPK signaling pathway. This provides a stronger biological rationale for our subsequent bioactivity assay. The specific content added to the main text is presented as follows:

Inflammation constitutes a complex defense response in living tissues, underpinning a broad spectrum of both physiological maintenance and pathological progression [12]. The activation of innate immune cells, such as macrophages, is central to this process. Upon activation, macrophages release a storm of pro-inflammatory cytokines (e.g., IL-1b, TNF-α, and IL-6) and reactive oxygen species (ROS), which are critical mediators in the exacerbation of inflammatory diseases [13]. The lipopolysaccharide (LPS)-induced model is the standard paradigm for simulating this response [14]. LPS acts as a potent immunostimulant, triggering the upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)—the key enzymes responsible for the synthesis of nitric oxide (NO) and prostaglandin E2 (PGE2), respectively [15,16]. Furthermore, the mitogen-activated protein kinase (MAPK) signaling pathways play a pivotal role in orchestrating this inflammatory cascade. Major MAPK subfamilies, including ERK1/2, p38, and JNK, function as essential signal transducers that relay environmental stimuli to the nucleus [17]. Therefore, the modulation of MAPK signaling pathways represents a promising therapeutic strategy for the development of anti-inflammatory agents.

Q2.       Positive control is required (at minimum in the NO inhibition assay) The NO inhibition data would be substantially more interpretable if benchmarked against a well-established positive control under the same experimental conditions (LPS dose, incubation time, cell density). I strongly recommend including a positive control at least for the NO release assay (Figure 8A/C) and reporting it clearly in both Methods and Figure legends.

Suggested positive controls: Diclofenac (widely used anti-inflammatory reference compound in LPS-stimulated macrophage assays; please include a concentration range appropriate for RAW264.7 and report the resulting NO inhibition and cell viability). L-NAME (NO synthase inhibitor; commonly used in NO/Griess assays; e.g., 50–200 µM). Aminoguanidine (iNOS-selective inhibitor; often 25–100 µM). Alternatively, dexamethasone can be used as a broad anti-inflammatory control.

At minimum, please add one positive control condition in the NO assay and provide the corresponding inhibition level (and ideally an IC₅₀ if tested across concentrations), alongside a viability readout to exclude cytotoxicity. This will confirm assay performance and will help contextualize the potency of compounds 1 and 11.

Response: We thank the reviewer for this valuable suggestion. Accordingly, we have included Quercetin—a widely reported natural product with established NO inhibitory activity—as a positive control in our NO inhibition assays. Under the same experimental conditions, quercetin exhibited an IC₅₀ value of 16.37 ± 0.96 μM and showed no cytotoxicity at the concentrations tested. These data have been added to the Methods, Results, and Figure 8 legend, providing a benchmark for the potency of compounds 1 and 11 and confirming assay validity.

Q3.       Mechanistic claims are overstated; rewording is necessary unless additional pathway data are added.

The current Abstract and Conclusions use causal language (e.g., "directly mediated") and imply broad MAPK selectivity ("no observed activity against other MAPK subfamilies"). Based on the presented excerpt, only JNK and ERK1/2 appear to have been assessed. Without additional data, the following statements should be revised:

Replace causal language ("directly mediated by attenuation of JNK phosphorylation") with associative language ("associated with", "correlated with", "consistent with involvement of").

Avoid general claims about "other MAPK subfamilies" unless p38 (and ideally additional nodes) is tested. If only ERK is tested, state only that ERK1/2 phosphorylation was not significantly altered under the tested conditions.

If the authors do not intend to add further experiments, the mechanism should be presented explicitly as a partial/working model. It is reasonable to mention p38 and NF-κB as plausible contributors in the proposed mechanism, but they must be labeled as hypothesized/not tested and the claims should be appropriately bounded.

Response: We agree that our claims should strictly reflect our data.

We have revised the Abstract, Results, and Conclusions to use associative language.

We have removed broad claims about other MAPK subfamilies We now specifically state that "ERK1/2 phosphorylation was not significantly altered and avoid referring to p38 or other pathways we did not test.

In the Discussion, we now present our proposed mechanism as a working model, acknowledging that other pathways like p38 or NF-κB may also be involved but were not assessed in this study.

 

Supplementary Information

Q3.       Proposed biosynthetic pathway of 1:

Eherification-----------should be etherification

Response: We have corrected the typo "Eherification" to "Etherification" in the Supplementary Information.

Q3.       Proposed biosynthetic route of 2. Nucleophilic opening of the epoxide occurs in this case at the most substituted carbon. therefore, there can be no configuration inversion since the most substituted carbon is not chiral.

Response: Thank you for this astute chemical observation. We have revised the proposed biosynthetic scheme and the accompanying text for compound 2.

Author Response File: Author Response.docx