LC-MS Profiling and Biological Activity of Unexplored Leucas nubica Benth. (Lamiaceae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a comprehensive LC-HRMS–based phytochemical profiling of Leucas nubica aerial parts combined with in vitro antioxidant and enzyme inhibitory assays. The study provides the first detailed metabolite inventory for this species, revealing a rich composition dominated by phenylethanoid glycosides and flavonoids. The analytical workflow is robust, the MS/MS interpretation is carefully executed, and the biological assays are performed using established protocols. Overall, the work is well aligned with the scope of Plants and represents a valuable contribution to the phytochemical and pharmacognostic literature. The manuscript is generally well prepared; however, several clarifications and refinements. I therefore recommend minor revision.

（1）While the LC-HRMS profiling is thorough, many compounds are annotated at tentative confidence levels. It would be helpful to clearly distinguish between confirmed identifications (using reference standards) and tentatively annotated metabolites, for example by summarizing the number of compounds in each confidence category. This clarification would improve transparency without diminishing the value of the dataset.

（2）The MS/MS-based dereplication is technically sound but occasionally overly detailed, particularly for structurally related phenylethanoid glycosides and flavonoids. Condensing repetitive fragmentation descriptions and emphasizing broader chemical patterns would improve readability and flow.

（3）The antioxidant and enzyme inhibitory activities are clearly presented; however, statements attributing these effects to specific metabolite classes should be framed more cautiously. Emphasizing that the observed activities reflect extract-level effects, rather than direct actions of individual compounds, would strengthen scientific accuracy.

（4）Since verbascoside is reported as a dominant constituent, a brief qualitative discussion linking the abundance of major compound classes with the observed bioactivities would enhance coherence between the chemical and biological sections, even without quantitative correlation analysis.

（5）References to potential applications in health-promoting or functional products should be slightly tempered, making clear that such applications remain prospective and require further validation.

（6）A careful proofreading is recommended to correct minor grammatical inconsistencies and ensure consistent use of terminology (e.g., metabolite confidence levels, enzyme assay units).

Author Response

Responses to Reviewer 1

This manuscript presents a comprehensive LC-HRMS–based phytochemical profiling of Leucas nubica aerial parts combined with in vitro antioxidant and enzyme inhibitory assays. The study provides the first detailed metabolite inventory for this species, revealing a rich composition dominated by phenylethanoid glycosides and flavonoids. The analytical workflow is robust, the MS/MS interpretation is carefully executed, and the biological assays are performed using established protocols. Overall, the work is well aligned with the scope of Plants and represents a valuable contribution to the phytochemical and pharmacognostic literature. The manuscript is generally well prepared; however, several clarifications and refinements. I therefore recommend minor revision.

（1）While the LC-HRMS profiling is thorough, many compounds are annotated at tentative confidence levels. It would be helpful to clearly distinguish between confirmed identifications (using reference standards) and tentatively annotated metabolites, for example by summarizing the number of compounds in each confidence category. This clarification would improve transparency without diminishing the value of the dataset.

Response: Thank you for the valuable recommendation. The following sentence was added in the Results and discussion: “In general, in the studied L. indica extract 15 compounds were identified with level B, 1 with level C, 41 with level D1, and 21 with level D2 (Table 1).”

（2）The MS/MS-based dereplication is technically sound but occasionally overly detailed, particularly for structurally related phenylethanoid glycosides and flavonoids. Condensing repetitive fragmentation descriptions and emphasizing broader chemical patterns would improve readability and flow.

Response: Thank you for the comment. The text was corrected according the recommendation (See Results and discussion).

（3）The antioxidant and enzyme inhibitory activities are clearly presented; however, statements attributing these effects to specific metabolite classes should be framed more cautiously. Emphasizing that the observed activities reflect extract-level effects, rather than direct actions of individual compounds, would strengthen scientific accuracy.

Response: Thank you for the comment. The text was corrected accordingly (See Results and Discussion).

（4）Since verbascoside is reported as a dominant constituent, a brief qualitative discussion linking the abundance of major compound classes with the observed bioactivities would enhance coherence between the chemical and biological sections, even without quantitative correlation analysis.

Response: Thank you for the valuable comment. A discussion was added (See Results and Discussion).

（5）References to potential applications in health-promoting or functional products should be slightly tempered, making clear that such applications remain prospective and require further validation.

Response: Thank you for the comment. The text was corrected accordingly.

（6）A careful proofreading is recommended to correct minor grammatical inconsistencies and ensure consistent use of terminology (e.g., metabolite confidence levels, enzyme assay units).

Response: Thank you for the comment. All text was corrected.

Reviewer 2 Report

Comments and Suggestions for Authors

Studying the composition of metabolites synthesized by various members of the plant kingdom is a rapidly developing area of ​​scientific research. In this article, the authors focused on Leucas nubica, a pharmaceutically valuable member of the genus Leucas. They analyzed the composition of predominantly phenolic metabolites extracted with methanol from its aerial parts, as well as the biological activity of these extracts. The methodological approach of the study is adequate to the objectives and is modern.

Regarding the presentation of the material in the article, a number of comments are necessary:
1. Abstract: Clarify L. nubica aerial parts (are these leaves?), and more accurately formulate the concluding sentence, as this is only a hypothesis regarding the activity of the phenylethanoid glycosides and flavonoids present in the extract.

2. The Introduction requires significant revision. This concerns the first paragraph, which contains a botanical description of Leucas nubica leaves and other plant parameters that were not the subject of the study and discussion (lines 34-41).
The second paragraph presents data on the metabolites of another representative of the genus Leucas, L. aspera, identified in various organs. How does this relate to the subject of study, Leucas nubica? The same comment applies to the pharmacological information (lines 65-100). These comments should be taken into account when revising the introduction so that the reader is clear about the purpose of studying Leucas nubica, rather than presenting information about the already well-studied plant L. aspera.
3. The Results section contains Table 1, which presents data primarily on the phenolic metabolome of methanol extracts of Leucas nubica. It takes up almost 5 pages. This material could easily be placed in an appendix, and the article could present these data in a table by the main classes of these metabolites, referring to the formatting used in articles in the journal Plants. The headings in Section 2, as well as the contents of the subsections, are unclear. Why are phenylethanoid glycosides highlighted, but no details on flavonoids are provided? The determination of the total phenolic content includes all classes of phenolic compounds, data on which is presented in Section 2.1.2.
The lack of an analysis of the correlations between the main parameters studied (polyphenols, antioxidant and enzymatic activity) is regrettable; this would significantly improve the article.
4. The Materials and Methods section requires revision. Detailed information on the collection and storage of plant material is needed. How were the leaves collected (before flowering), at what stage of ontogenesis, and their age? How were they dried? The biochemical methods are presented very briefly, requiring the reader to search for the original sources, which significantly limits the significance of the data presented.

Conclusion: The presented version of the article requires significant revision.

Comments on the Quality of English Language

The text requires minor stylistic revisions.e text requires minor stylistic revisions.

Author Response

Responses to Reviewer 2

Studying the composition of metabolites synthesized by various members of the plant kingdom is a rapidly developing area of ​​scientific research. In this article, the authors focused on Leucas nubica, a pharmaceutically valuable member of the genus Leucas. They analyzed the composition of predominantly phenolic metabolites extracted with methanol from its aerial parts, as well as the biological activity of these extracts. The methodological approach of the study is adequate to the objectives and is modern.

Regarding the presentation of the material in the article, a number of comments are necessary:
1. Abstract: Clarify L. nubica aerial parts (are these leaves?), and more accurately formulate the concluding sentence, as this is only a hypothesis regarding the activity of the phenylethanoid glycosides and flavonoids present in the extract.

Response: Thank you for the valuable comment. The aerial parts were used for the study. The abstract was corrected according to the recommendation. 

2. The Introduction requires significant revision. This concerns the first paragraph, which contains a botanical description of Leucas nubica leaves and other plant parameters that were not the subject of the study and discussion (lines 34-41).
   The second paragraph presents data on the metabolites of another representative of the genus Leucas, L. aspera, identified in various organs. How does this relate to the subject of study, Leucas nubica? The same comment applies to the pharmacological information (lines 65-100). These comments should be taken into account when revising the introduction so that the reader is clear about the purpose of studying Leucas nubica, rather than presenting information about the already well-studied plant L. aspera.

Response: The first paragraph was revised and becomes now: L. nubica Benth. is an annual herb characterized by cymes with white or pale-colored flowers and trigonous-oblong nutlets.

The phytochemical data on Leucas species has chemophenetic significance. The previous phytopharmacological studies delineated the trends and could be helpful in the exploration of L. nibica activity. The aforementioned paragraphs were revised in more consistent way:

To the best of our knowledge, there are not data on the phytochemical composition of the species. On the other hand, L. aspera (Willd.) Link is the most valuable representative of the genus [3]. A series of lignans from 2, 5-bis-aryl-3, 4-dimethyl tetrahydrofuran, dibenzylbutane and 8, 4'-oxyneolignan type alongside diterpenes from isopimarane-8(14)-15-diene type were isolated from L. aspera whole plant (4, 6). Numerous long-chain compounds and flavonoids have been characterized in the species [5, 10–12]. The ethnomedicinal tradition, pharmacological properties and bioactive compounds of the L. aspera are the subject of a review by Prajapati et al. (2010) and Hajare et al. (2025) highlighting the antioxidant capacity of the plant extracts [3,13]. In Africa and Asia, the plant is used in the ethnopharmacological approach as anti-inflammatory, antipyretic, analgesic, anti-rheumatic and anti-bacterial agent.

Significant advances have been achieved in exploring the anti-inflammatory activity of Leucas species underlying the inhibition of prostaglandins alongside the inflammation-related enzymes (lipoxygenase and proteinase) (4,6,16-18). Indeed, the lignans, flavonoids, and isopimarane and spiro-labdane diterpenoids hold significance for the anti-inflammatory activity. In addition to evoking an anti-inflammatory response, some Leucas species display cytotoxicity against cancer cell lines [13, 20, 21].

 

3. The Results section contains Table 1, which presents data primarily on the phenolic metabolome of methanol extracts of Leucas nubica. It takes up almost 5 pages. This material could easily be placed in an appendix, and the article could present these data in a table by the main classes of these metabolites, referring to the formatting used in articles in the journal Plants. The headings in Section 2, as well as the contents of the subsections, are unclear. Why are phenylethanoid glycosides highlighted, but no details on flavonoids are provided? The determination of the total phenolic content includes all classes of phenolic compounds, data on which is presented in Section 2.1.2.

Response: Thank you for the valuable comment. The Results section was revised accordingly. Table 1 was placed in Supplemental material. The discussion was revised according to the recommendation.

The lack of an analysis of the correlations between the main parameters studied (polyphenols, antioxidant and enzymatic activity) is regrettable; this would significantly improve the article.

Response: We thank the reviewer for this constructive suggestion. However, in the present study, all biological and chemical evaluations were performed on a single Leucas nubica extract. Therefore, statistical correlation analysis (e.g. Pearson or Spearman's ) is not applicable here, as correlation requires variability across multiple independent samples (n ≥ 3–5) in order to generate meaningful coefficients and significance values. With only one sample, any calculated correlation would be artificial and statistically invalid.

In order to address the reviewer’s concern in a scientifically appropriate manner, we have strengthened the qualitative interpretation of the relationships between the chemical composition and the bioactivities in the Discussion section. Based on the literature, we can infer that the observed abilities are explained by the presence of certain phenolic acids and flavonoids, such as caffeic acid derivatives, chlorogenic acid, luteolin, apigenin and cirsiliol.

4. 
   The Materials and Methods section requires revision. Detailed information on the collection and storage of plant material is needed. How were the leaves collected (before flowering), at what stage of ontogenesis, and their age? How were they dried? The biochemical methods are presented very briefly, requiring the reader to search for the original sources, which significantly limits the significance of the data presented.

Response: Thank you for the comment. Leucas nubica aerial parts were collected from city Erkowit (18° 42' 0" North, 37° 0' 0" East), Eastern Sudan, during the full flowering stage, in December, 2024. The collected plant material was dried at room temperature (20-22˚C) and 50% of relative humidity. The aerial parts were dried until a constant weight of the plant material.

For the sake of similarity, we have not included all details of the protocols as these have been previously published. However, all experimental details have been given in the supplemental materials

 

Conclusion: The presented version of the article requires significant revision.

Response: The article was revised according all reviewer recommendations.

The text requires minor stylistic revisions.e text requires minor stylistic revisions.

Response: All text was revised.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors paid close attention to the recommendations and made corrections and additions to the text of the article. All this improved its presentation.
However, the introduction fails to reflect the value and relevance of studying Leucas nubica. Only the first paragraph is devoted to it, while the rest of the text is information about the already well-studied L. aspera.
The addition about flavonoids (lines 270-279) is inconsistent with the text of the paragraph above, which is devoted to examining the activity of one representative phenolic compound, verbescoside. The presentation of the material in the article needs to be coordinated.
Furthermore, these are personal reflections, as the biological activity of extracts containing a mixture of different substances was analyzed.
The conclusion contains information about the regulation of enzyme activity, which was not the subject of the study and should be taken into account.
Conclusion: revision of the article.

Comments on the Quality of English Language

The text requires minor stylistic revisions.e text requires minor stylistic revisions.

Author Response

Reviewer 2

The authors paid close attention to the recommendations and made corrections and additions to the text of the article. All this improved its presentation.
However, the introduction fails to reflect the value and relevance of studying Leucas nubica. Only the first paragraph is devoted to it, while the rest of the text is information about the already well-studied L. aspera.

Response: Thank you for the comment. The Introduction was modified as follows:

“The whole plant is renowned for the treatment of jaundice in the traditional medicine of eastern Soudan [3]. In the previous study of the L. nubica whole plant extracts, the total phenolic content ranged between 0.216 gallic acid/g (dichloromethane extract) and 1.015 mg gallic acid/g (methanol extract), while total flavonoids varied from 0.400 mg quercetin/g to 0.580 mg quercetin/g, respectively [3]. In the antioxidant potential assessment assays, methanol and ethyl acetate extracts scavenged DPPH radicals (47%) and DMPD (48%), respectively, whereas reducing power was estimated at 1.117 (absorbance in FRAP) and 0.361 (absorbance in PRAP), respectively. Metal chelating capacity reached 48% (ethyl acetate extract). L. nubica extracts (at 10 mg/mL) have not been cytotoxic towards human CCRF-CEM leukemia cells. On the other hand, in Africa and Asia the Leucas species including L. aspera (Willd.) Link., L. ciliata Benth. and L. lavandulifolia Sm. are used in the ethnopharmacological approach as anti-inflammatory, antipyretic, analgesic and anti-bacterial agents [4–6]. Indeed, the lignans, flavonoids, and isopimarane and spiro-labdane diterpenoids hold significance for the anti-inflammatory and antioxidant activity.

To the best of our knowledge, there is not data on the phytochemical composition of L. nubica. Taken together, the cited studies generate further interest in the species and prompted us to undertake in-depth profiling of secondary metabolites in L. nubica aerial parts by means of ultra-high-performance liquid chromatography coupled with hybrid quadrupole–Orbitrap high-resolution mass spectrometry.”

The addition about flavonoids (lines 270-279) is inconsistent with the text of the paragraph above, which is devoted to examining the activity of one representative phenolic compound, verbescoside. The presentation of the material in the article needs to be coordinated.
Furthermore, these are personal reflections, as the biological activity of extracts containing a mixture of different substances was analyzed.

Response: Thank you for the valuable comment.

The additional text was modified as follows:

“Besides verbascoside, cirsiliol and apigenin 7-O-glucoside being among predominant flavonoids in the extract, could also contribute to the antioxidant activity of the extract. It have been proved that these flavonoids neutralize reactive oxygen species and protecting cells from oxidative damage, by influencing antioxidant pathways (like Nrf-2/NF-κB) [41,42]. “

 

All sentences were supported by the relevant references.

The conclusion contains information about the regulation of enzyme activity, which was not the subject of the study and should be taken into account.

Response: Thank you for the comment. The Conclusion was modified as follows:

“Furthermore, an inhibitory activity was proven against the key enzyme in melanin biosynthesis tyrosinase.  In the context of carbohydrate metabolism, the studied extract possesses promising ɑ-glucosidase inhibitory activity along with moderate ɑ-amylase inhibition.”   

Conclusion: revision of the article.

The text requires minor stylistic revisions.e text requires minor stylistic revisions.

Response: The text was revised according to the reviewer recommendation.