F1000ResearchF1000Research2046-1402F1000 Research LimitedLondon, UK10.12688/f1000research.164618.2Brief ReportArticlesGas Chromatography-Mass spectrometry (GC-MS) Revealed Bioactive Constituents of
Brassica oleracea var.
viridis (Collard Greens) Used to Treat Male Infertility in Uganda.
[version 2; peer review: 1 approved, 2 approved with reservations]
IkuomolaEmmanuel OrireConceptualizationInvestigationMethodologyValidationhttps://orcid.org/0009-0004-4167-6717a1OwuDaniel UdofiaMethodologySupervisionWriting – Review & Editinghttps://orcid.org/0000-0002-8264-91311OkaVictor OtuMethodologySupervision1BisongSunday AgbaInvestigationSupervisionValidation2Paul-ChimaUgwu OkechukwuMethodologySupervisionValidation3AjaPatrick MaduabuchiConceptualizationMethodologySupervisionWriting – Review & Editinghttps://orcid.org/0009-0006-2450-94604
Physiology, Faculty of Biomedical Sciences,, Kampala International University - Western Campus, Bushenyi, Western Region, 20000, Uganda
Department of Physiology, University of Calabar, College of Medical Sciences, Calabar, Cross River, Nigeria
Publication and Extension, Kampala International University - Western Campus, Bushenyi, Western Region, 20000, Uganda
Biochemistry, Faculty of Biomedical Sciences, Kampala International University - Western Campus, Bushenyi, Western Region, 20000, UgandaIkuomolaemmanuelorire@kiu.ac.ug
Medicinal plants play a crucial role in pharmacological research, as many pharmaceutical companies rely on them for raw materials. Collard greens, also known as “Sukuma wiki,” are a member of the
Brassica oleracea var. viridis family and are known for their medicinal properties. The aim of this study was to identify phytochemicals in the ethanol extract of collard green leaves that has traditional use in treating male infertility in Uganda.
Method
The leaves were dried and crushed into fine powder and the powdered leaves (800 g) A was cold macerated in 5 liters of 99% ethanol (1:5 w/v) for 72 hours in sterile glass container. The ethanol extract (5g) was subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis.
Results
Phytochemical analysis of the Ethanol extract from
Brassica oleracea var. viridis (Collard green) leaves revealed the presence of various compounds, including flavonoids, alkaloids, phenolic compounds, fatty acids, and terpenoids. Gas Chromatography-Mass Spectrometry (GC-MS) analysis identified 77 bioactive compounds, such as 2-Methoxy-4-vinylphenol, 2, 7-Dimethyl-1, 7-octadien-3-amine, Octadecanoic acid, 9, 12, 15-Octadecatrienoic acid, Pentadecanoic acid, and several phenolic derivatives.
Conclusion
In conclusion, the phytochemicals identified in
Brassica oleracea var. viridis (Collard greens), including Phytol, Omega-3 fatty acids, phenols, flavonoids, and sterols, demonstrate potential benefits for enhancing male fertility and reproductive health. These phytochemicals exert their beneficial action through their antioxidant, anti-inflammatory, and possibly neuroprotective properties. There is need for further research to establish the effectiveness of these phytochemicals in clinical settings and provide concrete guidelines for their use in treating male infertility.
Collard greensGC-MSSukuma wikiPhytochemicalsPhytolThe author(s) declared that no grants were involved in supporting this work.Amendments from Version 1
This revised version incorporates substantial improvements and clarifications compared with the previously published article. The introduction has been expanded to provide stronger scientific context, particularly highlighting the ethnomedicinal relevance of
Brassica oleracea var. viridis in Uganda and linking its traditional use to current evidence on phytochemicals with fertility-enhancing potential. The methodology section has been revised to include more precise details on plant collection, authentication, voucher specimen deposition, extraction procedures, and GC-MS operating conditions, thereby enhancing reproducibility and transparency. In the results section, a comprehensive GC-MS profile of 77 identified compounds is now presented with a clearer explanation of inclusion thresholds (≥1.90% area percentage), retention times, molecular formulae, and structural data. Several key bioactive constituents, including phytol, omega-3 fatty acids, tocopherols, sterols, and phenolic derivatives, are now explicitly discussed with respect to their pharmacological and reproductive health implications. The discussion has been strengthened by integrating recent literature on antioxidant, anti-inflammatory, and fertility-related mechanisms of the identified compounds, while also acknowledging contrasting findings on antioxidant supplementation in male infertility. Additional improvements include: Expanded referencing with up-to-date citations in Vancouver style. Clearer conclusions emphasizing the multi-targeted therapeutic potential of the identified phytochemicals. Explicit recommendations for future research and clinical validation studies. Inclusion of declarations (ethics, consent, data availability, reporting guidelines, competing interests, funding, and authors’ contributions) for compliance with journal standards. Enhanced figures, tables, and legends to improve readability and interpretation. Overall, the revised version provides greater methodological rigor, expanded data presentation, and stronger integration of findings with existing knowledge, thereby improving the scientific quality and clarity of the article.
List of abbreviationsDHA
Docosahexaenoic acid
GCMS
Gas Chromatography-Mass Spectrometry
IOE
Ikuomola Orire Emmanuel
Introduction
For centuries, medicinal plants have played a vital role in traditional healing systems across cultures, primarily due to their rich content of bioactive compounds.
1 These phytochemicals such as flavonoids, alkaloids, tannins, terpenoids, and saponins have demonstrated significant therapeutic potential.
2 Acting individually or synergistically, they exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-diabetic, anti-cancer, and reproductive health-enhancing effects.
3 Their contribution to drug discovery continues to be profound, as the identification of novel therapeutic agents often begins with the screening of natural products from diverse plant sources.
4
Brassica oleracea var.
viridis (commonly known as collard greens or “Sukuma wiki” in Swahili) is a nutrient-rich leafy vegetable widely cultivated and consumed in Uganda and other parts of East Africa as shown in
Figure 1. Characterized by large, broad leaves ranging in color from deep green to bluish-green, collard greens thrive in a variety of climates and are valued both as a food source and in ethnomedicine. Traditionally, this plant has been used to manage various health conditions, including male reproductive issues, though its phytochemical basis remains underexplored.
Diagram of
<italic toggle="yes">Brassica oleracea</italic> var. viridis (collard green).
<sup>
<xref ref-type="bibr" rid="ref20">20</xref>
</sup>
The health-promoting properties of collard greens are largely attributed to their high content of vitamins C, E, and K, dietary fiber, calcium, and a diverse range of bioactive compounds. These constituents support immune function, bone health, digestion, and oxidative balance.
5 Additionally, preliminary evidence suggests potential anti-hyperglycemic and fertility-enhancing effects, reinforcing its traditional use in managing male infertility.
6,
7
Gas Chromatography-Mass Spectrometry (GC-MS) is a robust and precise analytical technique employed in phytochemical investigations to identify and quantify chemical constituents in complex plant matrices.
8,
9 This method is especially effective in uncovering bioactive compounds that may explain the therapeutic value of traditionally used plants.
This study aims to analyze the bioactive constituents of
Brassica oleracea var.
viridis collected in Uganda using GC-MS. By identifying these compounds, the research seeks to provide scientific validation for the ethnomedicinal use of collard greens in the treatment of male infertility and contribute to the growing body of knowledge on plant-based fertility therapies.
MethodologyChemicals
Only analytical-grade chemicals and reagents were used for the study.
Plant collection and identification
Fresh leaves of
Brassica oleracea var.
viridis (collard greens) were collected from Bwejuragye in Ishaka Town, Bushenyi, Igara County, Western Uganda with GPS coordinates of approximately 0°32′42.0″S 30°08′18.0″Ephyt. The plant was identified and authenticated by Dr. Eunice Olet of the Department of Botany, Faculty of Biological Sciences, Mbarara University of Science and Technology. A voucher specimen was deposited in the university’s herbarium under the reference number IOE-24-001. Details of the plant collection and identification are presented in
Table 1.
Process of plant collection and identification.
PLANT COLLECTION DETAILS
ITEMS
PROPERTIES
DATE OF COLLECTION
03-09-2024
COLLECTION NUMBER
IOE-24-001
LOCAL NAME:
Sukuma Wiki
FAMILY NAME:
Cruciferous Family/Acephala group
GENERIC NAME:
Chepkilumnda
SCIENTIFIC NAME:
Brassica oleracea var. viridis
GROWTH FORM:
Rosette Growth
COLOR:
Dark Green
TYPE OF INFLORESCENCE:
Raceme
LEAF SHAPE:
Ovate Shape
TYPE OF MARGIN:
Smooth or Slightly Undulating Margin
LEAF VENATION:
Palmate Venation Pattern
PUBESCENCE:
Glabrous (Smooth And Hairless) Leaves
LEAF ARRANGEMENT:
Alternate Leaf Arrangement
COMMON PLANTS AROUND:
Tomatoes, Onions, Garlic, Beans, Carrots
TYPE OF ECO SYSTEM:
Subtropical And Tropical Gardens
LOCALITY:
Bwejuragye, ishaka town
HABITAT:
Subtropical And Tropical Areas
Preparation of plant material
Fresh leaves (2 kg) of
Brassica oleracea var.
viridis (collard greens) were collected from fields located in Bwejuragye, Bushenyi Local Government Area, Uganda. The samples were subsequently transported to the herbarium unit at Mbarara University for proper botanical identification and authentication. The leaves were thoroughly washed with distilled water to remove surface contaminants such as dirt and sand, and then shed-dried at ambient temperature of 26°C. After complete drying, the leaves were finely ground into a powder and stored in airtight containers in a dry environment for future extraction.
The extraction process was performed using a modified version of the method described by Abdulrahman et al.
10 A total of 800 g of the powdered leaves was cold macerated in 5 liter of 99% ethanol (1:5 w/v) for 72 hours in sterile glass container. The mixture was kept in the dark to prevent degradation at room temperature (26
± 2°C) with intermittent shaking every 6-12 hours to enhance solute diffusion and extraction efficiency. The resulting ethanolic mixture was then filtered through sterile Whatman No. 1 filter paper. The filtrate was concentrated to dryness reduced pressure using a rotary evaporator below 40°C having a constant weight. The dried crude extracts were stored at 4°C in an amber glass bottle that was tightly sealed until further analysis. Approximately 5 g of the ethanol extract was subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis, and the remaining extract was stored in a refrigerator for subsequent use in further analyses.
Gas Chromatography-Mass Spectrometry (GC-MS) analysis
GC-MS analysis was performed using a Shimadzu gas chromatograph-mass spectrometer.
11,
12 Chromatographic separation was achieved with a DB-5 fused-silica capillary column in inert MSD mode, equipped with a Triple-Axis detector. High-purity helium (99.999%) served as the carrier gas at a constant flow rate of 1.0 mL/min. The system was operated under the following conditions: ion source temperature at 250 °C, interface temperature at 300 °C, pressure at 16.2 psi, and out time of 1.8 mm. A 1 μL sample was injected in split mode (1:50) at 300 °C.
The column temperature program began at 36 °C (held for 5 min), ramped to 150 °C at 4 °C/min, then to 250 °C at 20 °C/min, and was held for 5 min, resulting in a total run time of 47.5 minutes. Relative percentage abundances were determined by comparing individual peak areas to the total chromatogram area. Data acquisition and instrument control were managed using MS Solution software, and compound identification was based on mass spectral comparisons with the NIST20 library.
Results
Phytochemical screening of the Ethanol extraction of
Brassica oleracea var. viridis (Collard Greens) leaves revealed the presence of the phytochemical constituent’s flavonoids, alkaloids, phenolic compounds, fatty acids, terpenoids, and other compounds as detailed in
Table 2.
Compounds present selected ethanol extract of
<italic toggle="yes">Brassica oleracea</italic> var. viridis (collard greens) and their phytochemical properties.
Gas Chromatography-Mass Spectrometry (GC-MS) profiling of ethanol extract of Brassica oleracea var. viridis (Collard Greens)
Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the ethanol extract obtained from the leaves of
Brassica oleracea var.
viridis (collard greens) identified a total of 77 distinct phytochemical constituents exhibiting a broad spectrum of bioactive properties. The resulting chromatogram is presented in
Figure 2, while
Table 3 details the identified compounds, including those meeting the inclusion threshold of ≥1.90% area percentage. Compounds with an area percentage below this threshold were excluded from further analysis. The table also provides information on each compound’s retention time (RT), molecular formula, molecular weight (MW), and chemical structure.
Showing the chromatograph of ethanol extract of Brassica oleracea var. viridis.
Selected by %age area (1.90 upward for inclusion criteria, exclusion is 1.90 downwards) of ethanol extract of
<italic toggle="yes">Brassica Oleracea</italic> var viridis (collard greens).
Among the identified compounds are several notable bioactive constituents, including Dimethyl trisulfide, 2-Piperidinemethanamine, 2,7-Dimethyl-1,7-octadien-3-amine, Oxalic acid cyclohexyl propyl ester, 2-Pyrrolidinone, 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, and 2,2-Difluoroethylbenzene. Others include 3,4-Anhydro-d-galactosan, dl-trans-Chrysanthemic acid, Phenol derivatives, Indole, 2-Methoxy-4-vinylphenol, and various pyrrolidine and cyclohexane derivatives.
Further constituents of pharmacological interest include 7,10,13-Hexadecatrienoic acid (Z,Z,Z), 9,12,15-Octadecatrienoic acid methyl ester, phytol, γ-tocopherol, α-tocospiro A and B, stigmasterol, ergost-5-en-3-ol (3β), and γ-sitosterol. Many of these compounds have been previously reported to exhibit antioxidant, anti-inflammatory, antimicrobial, and reproductive health-supportive properties, which may explain the traditional use of collard greens in managing male infertility.
The identified bioactive molecules span multiple chemical classes, including fatty acid esters, phenolics, alkaloids, terpenoids, sterols, and tocopherols, underscoring the pharmacological potential of
Brassica oleracea var.
viridis. Selected compounds and their proposed biological pathways relevant to male reproductive health are illustrated in
Figure 3.
Showing the selected potential compounds and their biosynthesis pathways.
Discussion
Phytochemical screening of the ethanol extract from
Brassica oleracea var.
viridis (Collard Greens) has revealed a diverse array of phytoconstituents with potential health benefits, particularly in relation to reproductive health, as summarized in
Table 4. Among the identified compounds, Phytol, a terpenoid, stands out due to its notable antioxidant properties. Previous studies have demonstrated that Phytol exhibits significant antioxidant activity in vitro, including scavenging hydroxyl radicals and nitric oxide, as well as inhibiting the formation of thiobarbituric acid reactive substances (TBARS).
13 These findings are consistent with a systematic review by,
14 which linked antioxidants to the restoration of oxidative stress-induced sperm abnormalities. However, it is important to acknowledge the contrasting results presented by,
15 who showed that antioxidant supplementation might not enhance semen parameters or DNA integrity in men with male factor infertility. This highlights the complexity of antioxidant supplementation in fertility therapies and suggests that a more nuanced approach may be necessary.
Showing the selected potential compounds of ethanol extract of
<italic toggle="yes">Brassica oleracea</italic> var. viridis (collard greens) predicted for male infertility treatment.
S/N
Compounds
Phytochemical constituents
Potential use for male infertility treatment
1
Phytol
Terpenoids
It supports sperm cell function by enhancing overall cellular health. (Steiner
et al. 2020)
Omega-3 fatty acid, known for improving sperm quality, motility and overall reproductive health.(Safarinejad
et al. 2012)
3
Phenol, 4-ethenyl-2,6-dimethoxy-
Phenolic
Known for antioxidant properties that may protect sperm from oxidative stress. (Gulcin, 2020)
4
Gamma-Sitosterol
Steroids
Have antioxidant and anti-inflammatory properties, potentially benefiting male fertility. (Reddy
et al. 2022)
Additionally, the screening identified several omega-3 fatty acids, including 9,12,15-octadecatrienoic acid (Z,Z,Z), pentadecanoic acid, and 7,10,13-hexadecatrienoic acid (Z,Z,Z). These compounds are well-documented for their beneficial effects on sperm quality, motility, and overall reproductive health. A study indicated that omega-3 supplementation could enhance antioxidant activity in human seminal fluid, leading to improvements in sperm count, motility, and morphology.
16 A study reported that omega-3 supplementation significantly improved sperm motility, increased docosahexaenoic acid (DHA) levels in seminal plasma, and enhanced total sperm count and cell density.
17
The presence of phenolic compounds and flavonoids in the
B. oleracea var.
viridis extract also warrants attention, given their potent antioxidant properties. In particular, 4-ethenyl-2,6-dimethoxyphenol has been noted for its potential neuroprotective effects, which may have implications for psychogenic male infertility.
18 Furthermore, Gamma-Sitosterol, a plant sterol, exhibits both antioxidant and anti-inflammatory activities, potentially promoting male fertility through modulation of oxidative stress and inflammatory pathways. Research by
19 on β-sitosterol further supports its role in inhibiting the growth and spread of prostate cancer cells, which could indirectly enhance reproductive health by preserving prostate function.
Conclusion
In conclusion, the
Brassica oleracea var.
viridis (Collard Greens) ethanol extract contains diverse phytochemicals such as phytol, omega-3 fatty acids, phenolic compounds, flavonoids, and sterols. These phytochemicals play significant potential benefits for enhancing male fertility and reproductive health through antioxidant, anti-inflammatory, and potentially neuroprotective pathways.
Recommendation
Future research should focus on elucidating the specific mechanisms by which these compounds interact and contribute to male fertility, as well as assessing their clinical efficacy and safety for therapeutic use in reproductive health. These trials should specifically assess the effect of these phytochemicals such as phytol, omega-3 fatty acids, phenols, flavonoids, and sterols on sperm quality, motility, and overall fertility outcomes to establish a clearer understanding of their clinical potential of these phytochemicals in treatment of male infertility.
DeclarationsEthics approval
The authors confirm that all guidelines set by the University’s research ethics for plant collection, characterization, and documentation was duly followed. The plant specimen was identified by the Department of Botany, Faculty of Science, Mbarara University of Science and Technology, Uganda. The experimental protocols received approval from the Kampala International University Research Ethics Committee. The plant collection process adhered to local guidelines and does not require further confirmation.
Data availability statement
Open Science Framework: Gas Chromatography-Mass spectrometric (GC-MS) Revealed Bioactive Constituents of
Brassica Oleracea var.
Viridis (Collard Greens) Used as Ethnomedicine to Treat Male Infertility in Uganda. (
https://doi.org/10.17605/OSF.IO/QND7X)
21
This project contains the following extended data
ETHANOLIC EXTRACT sukuma wikki file.pdf
Data are available under the terms of the
Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).
Acknowledgements
The technical assistance of Mrs. Ikuomola Odunayo Ibukun and Akintoye Peace is greatly acknowledged. We confirm that all individuals named in the Acknowledgments and Methods sections have agreed to the inclusion of their names and institutional affiliations in this manuscript.
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10.17605/OSF.IO/QND7X10.5256/f1000research.188560.r417218Reviewer response for version 2MorenikeAdeoye-Isijola1Refereehttps://orcid.org/0000-0002-3186-5432
Babcock University, Ilishan-Remo, Ogun State, Nigeria
Competing interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
10.5256/f1000research.188560.r418701Reviewer response for version 2KhanAmanulla1Refereehttps://orcid.org/0000-0001-7205-0267
Anjuman Islam Janjira Degree College of Science, Murud Janjira, Raigad, Maharashtra, India
Competing interests: No competing interests were disclosed.
Gas Chromatography-Mass Spectrometry (GC-MS) Revealed Bioactive Constituents of
Brassica oleracea var.
viridis (Collard Greens) Used to Treat Male Infertility in Uganda
Grammar / Style:Minor corrections:
“These phytochemicals such as flavonoids, alkaloids…” →
“These phytochemicals, including flavonoids, alkaloids…”
Replace
“as shown in Figure 1” with a reference to the updated figure.
Scientific Clarity:
Strengths: Clear ethnomedicinal context.
Improvements:Cite recent studies (last 5–7 years) on
Brassica oleracea bioactive compounds and male fertility.Avoid redundancy; streamline phrases like
“These constituents support immune function, bone health, digestion, and oxidative balance” to maintain focus on reproductive health.
Plant Collection & Identification: Detailed and well-documented. Minor grammar correction:
“Fresh leaves (2 kg) … were subsequently transported to the herbarium unit at Mbarara University for proper botanical identification and authentication” →
“…were transported to the herbarium at Mbarara University for botanical authentication.”
Extraction Procedure:
Clearly mention the ratio (w/v) for ethanol. extraction. Standardize units:
“26 2°C” →
“26 ± 2 °C”.Use past tense consistently.
GC-MS Analysis: Clarify split ratio and sample injection mode. Include column specifications (length, diameter, film thickness) for reproducibility. Tables 2–4: Ensure consistent formatting and capitalization for chemical names.
Figures: Figure 1: Replace with a high-resolution, original photograph of collard greens collected from the field. Figure 2
(GC-MS chromatogram): Replace with a high-resolution chromatogram; label peaks and retention times clearly. Figure 3
(biosynthetic pathway): Replace with original/redrawn diagrams relevant to the identified compounds; avoid screenshots from other sources.
Strengths: Detailed plant authentication and voucher specimen deposition. Comprehensive GC-MS analysis. Effective linkage of phytochemicals to male fertility mechanisms.
Limitations / Recommendations: Include quantitative analysis (concentration of compounds). Discuss potential toxicity or adverse effects. Provide experimental reproducibility details (replicates, GC-MS calibration). Ensure figures and tables are high-quality and original.
The manuscript is scientifically sound and demonstrates a comprehensive GC-MS analysis of
Brassica oleracea var. viridis. With improvements to figure quality, grammar, formatting, citation updates, and clearer discussion of biological relevance and limitations, this manuscript would meet high standards suitable for indexing.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
Partly
Is the study design appropriate and is the work technically sound?
Partly
Are the conclusions drawn adequately supported by the results?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
Reviewer Expertise:
Phytochemistry and Medicinal Botany
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
IkuomolaEmmanuelDepartment of Physiology, Kampala International University - Western Campus, Bushenyi, Western Region, Uganda
Competing interests: The Authors declares no competing interests
23102025
Reviewer 3
Comments:
Grammar / Style:Minor corrections:
“These phytochemicals such as flavonoids, alkaloids…” →
“These phytochemicals, including flavonoids, alkaloids…”
Authors’ Response: Thank you for the correction. The necessary changes have been made in the introductory section of the manuscript.
Replace
“as shown in Figure 1” with a reference to the updated figure.
Authors response: We thank the reviewer for pointing this out. The phrase has been updated as requested to correctly reference the figure.
Scientific Clarity:
Strengths: Clear ethnomedicinal context.
Authors response: We sincerely thank the reviewer for this positive feedback. We are pleased that the clear presentation of the ethnomedicinal context, which grounds this laboratory investigation in the traditional use of
Brassica oleracea var.
viridis for treating male infertility in Uganda, was recognized as a strength of our work.
Improvements: Cite recent studies (last 5–7 years) on
Brassica oleracea bioactive compounds and male fertility.
Authors response: We thank the reviewer for this valuable suggestion. We have now integrated recent studies (published within the last 5-7 years) into the
manuscript sections which support the role of
Brassica oleracea bioactive compounds in promoting male reproductive health. This update strengthens the scientific context of our findings.
Avoid redundancy; streamline phrases like
“These constituents support immune function, bone health, digestion, and oxidative balance” to maintain focus on reproductive health.
AUTHORS RESPONSE: We thank the reviewer for this valuable suggestion. We have streamlined the text as recommended, removing the broad list of general health benefits and refocusing the narrative specifically on the compounds' relevance to male reproductive health. The changes have been made in the
Introduction section.
Plant Collection & Identification: Detailed and well-documented. Minor grammar correction:
“Fresh leaves (2 kg) … were subsequently transported to the herbarium unit at Mbarara University for proper botanical identification and authentication” →
“…were transported to the herbarium at Mbarara University for botanical authentication.”
Authors response: We thank the reviewer for this positive feedback and for the suggested improvement. The sentence has been revised as recommended to be more concise.
Extraction Procedure:
Clearly mention the ratio (w/v) for ethanol. extraction. Standardize units:
“26 2°C” →
“26 ± 2 °C”.Use past tense consistently.
Authors response: We thank the reviewer for these precise observations. We have clarified the ethanol ratio, corrected the temperature unit, and ensured the consistent use of past tense in the described procedure.
Ratio Clarified: Changed "(1:5 w/v)" to "(at a ratio of 1:5 w/v)" for absolute clarity.
Units Standardized: Corrected "26 2°C" to "26 ± 2 °C" and added a space before "°C" for "40 °C" to ensure consistency.
Past Tense & Grammar: Corrected "was concentrated to dryness reduced pressure" to "was concentrated to dryness under reduced pressure" and "having a constant weight" to "until a constant weight was achieved" for grammatical correctness and consistent use of the past tense.
GC-MS Analysis: Clarify split ratio and sample injection mode. Include column specifications (length, diameter, film thickness) for reproducibility.
Authors response: We thank the reviewer for this critical suggestion to enhance the reproducibility of our methods. We have clarified the split ratio and injection mode and have added the standard column specifications (length, internal diameter, and film thickness) to the GC-MS analysis section.
Tables 2–4: Ensure consistent formatting and capitalization for chemical names.
Authors response: We thank the reviewer for highlighting this lack of consistency. We have carefully revised all three tables {2,3 and 4} to ensure chemical names follow standard IUPAC capitalization rules (sentence case for common names, proper capitalization for systematic names) and that formatting is uniform throughout.
Figures: Figure 1: Replace with a high-resolution, original photograph of collard greens collected from the field. Figure 2
(GC-MS chromatogram): Replace with a high-resolution chromatogram; label peaks and retention times clearly. Figure 3
(biosynthetic pathway): Replace with original/redrawn diagrams relevant to the identified compounds; avoid screenshots from other sources.
Authors response: We thank the reviewer for these critical recommendations to enhance the quality and originality of our figures. We have taken the following actions:
Figure 1: The original diagram has been replaced with a new, high-resolution, original photograph of
Brassica oleracea var.
viridis (collard greens) collected from the field in Bwejuragye, Bushenyi, Uganda, as specified in the methodology.
Figure 2: The GC-MS chromatogram has been replaced with a high-resolution version. Key peaks corresponding to the major bioactive compounds discussed in the manuscript are now clearly labeled with their respective retention times.
Figure 3: The previous pathway diagram has been removed. It has been replaced with an original, self-created schematic that illustrates the proposed mechanistic pathways.
Strengths: Detailed plant authentication and voucher specimen deposition. Comprehensive GC-MS analysis. Effective linkage of phytochemicals to male fertility mechanisms.
Authors response: We sincerely thank the reviewer for their positive assessment and for recognizing the strengths of our work, particularly the rigorous plant authentication, comprehensive phytochemical profiling, and the functional interpretation of the results in the context of male fertility.
Limitations / Recommendations: Include quantitative analysis (concentration of compounds). Discuss potential toxicity or adverse effects. Provide experimental reproducibility details (replicates, GC-MS calibration). Ensure figures and tables are high-quality and original.
Authors response: We thank the reviewer for these critical and constructive points. We have addressed them by adding a dedicated "Limitations and Future Perspectives" section to the manuscript. The key additions are summarized below.
Key additions
Added a New Section: A "Limitations and Future Perspectives" section has been inserted before the conclusion.
Addressed Quantification: Explicitly stated the qualitative/semi-quantitative nature of the study and recommended quantitative analysis for future work.
Addressed Toxicity: Acknowledged the lack of toxicity data and recommended future toxicological studies.
Addressed Reproducibility: Specified that the analysis was done in triplicate and mentioned instrument calibration.
Confirmed Figure Quality: Reiterated that all figures have been replaced with high-quality, original versions as per the reviewer's previous comment.
10.5256/f1000research.181158.r390229Reviewer response for version 1MorenikeAdeoye-Isijola1Refereehttps://orcid.org/0000-0002-3186-5432
Babcock University, Ilishan-Remo, Ogun State, Nigeria
Competing interests: No competing interests were disclosed.
The topic needs to be revised if possible and, should read: " Gas Chromatography - Mass spectrometry.......(not "spectrometric").
The revision of the topic is suggested because ethnomidicine is the basis for suggesting that the
Brassica oleracea var. Viridis is used to treat male infertility in Uganda and no previous scientific evidence is referenced or alluded to. Also, reports of the ethnobotanical survey should be included, if available.
Please note that:
Scientific names are always italicized
The genus is always capitalized
The species is never capitalized
4. The method in the abstract says the "point of this study was to find out...."
What bioactive compounds might be in the ethanol extract of collard green leaves
how well they work as antioxidants.
.....may be responsible for their traditional use in treating male fertility
Note that every objective (as shown above) must have clearly defined materials and methods but the only materials and methods seen is that of the GC-MS which addresses only the first objective which is to "find out what bioactive compounds might be in the ethanol extract of collard green leaves"
The second objective mentioned, which was to "find out how well they work as antioxidants" was not attended to by any materials and method carrying out the antioxidant capacity assays such as DPPH, ABTS, FRAP, NO or any other of the tests for radical scavenging activity, lipid peroxidation, reducing power; TEAC and TRAP are assays with mechanisms specifically for antioxidant activity
5. In the introduction, the statement below, must be punctuated with an appropriate reference compulsorily because the later part of the sentence is the crutch on which the justification for the research work is resting on:
"Additionally preliminary evidence suggests potential anti-hyperglycemic and fertility-enhancing effects, reinforcing its traditional use in managing male infertility" (reference not included)
Is the work clearly and accurately presented and does it cite the current literature?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
IkuomolaEmmanuelDepartment of Physiology, Kampala International University - Western Campus, Bushenyi, Western Region, Uganda
Competing interests: “I confirm that there are no competing interests that could be perceived as influencing the validity or importance of the peer review reports.”
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“We sincerely thank the reviewer for their constructive feedback and valuable recommendations. The authors’ detailed responses to the comments are provided below.”
Reviewer Comments:
The topic needs to be revised if possible and, should read: " Gas Chromatography - Mass spectrometry.......(not "spectrometric").
Author’s
Response: The topic has been revised to read “Gas chromatography-Mass spectrometry” as suggested
2. The revision of the topic is suggested because ethnomedicine is the basis for suggesting that the
Brassica oleracea var. Viridis is used to treat male infertility in Uganda and no previous scientific evidence is referenced or alluded to. Also, reports of the ethnobotanical survey should be included, if available. Author’s
Response: The topic has been revised the word ethnomedicine has been deleted to read “Gas Chromatography-Mass spectrometry (GC-MS) Revealed Bioactive Constituents of
Brassica Oleracea var.
Viridis (Collard Greens) Used to Treat Male Infertility in Uganda. However, the report of the ethnobotanical survey is not available.
3. Please note that:
Scientific names are always italicized
The genus is always capitalized
The species is never capitalized
Author’s
Response: The botanical name has been corrected to show the genus and species in small letters.
4. The method in the abstract says the "point of this study was to find out...."
What bioactive compounds might be in the ethanol extract of collard green leaves
how well they work as antioxidants.
.....may be responsible for their traditional use in treating male fertility
Note that every objective (as shown above) must have clearly defined materials and methods but the only materials and methods seen is that of the GC-MS which addresses only the first objective which is to "find out what bioactive compounds might be in the ethanol extract of collard green leaves"
The second objective mentioned, which was to "find out how well they work as antioxidants" was not attended to by any materials and method carrying out the antioxidant capacity assays such as DPPH, ABTS, FRAP, NO or any other of the tests for radical scavenging activity, lipid peroxidation, reducing power; TEAC and TRAP are assays with mechanisms specifically for antioxidant activity.
Author’s
Response: The objective has been modified to focus on only the GC-MS. The sentence now reads “The aim of this study was to identify bioactive compounds in the ethanol extract of collard green leaves that has traditional use in treating male infertility in Uganda”.
5. In the introduction, the statement below, must be punctuated with an appropriate reference compulsorily because the later part of the sentence is the crutch on which the justification for the research work is resting on:
"Additionally preliminary evidence suggests potential anti-hyperglycemic and fertility-enhancing effects, reinforcing its traditional use in managing male infertility" (reference not included).
Author’s
Response: The supporting references have been included in the introduction as suggested. The reference numbers are 6 and 7.
10.5256/f1000research.181158.r388513Reviewer response for version 1ThamerFaten Hameed1Refereehttps://orcid.org/0000-0002-6808-2917
Sana'a University, Sana'a, Yemen
Competing interests: No competing interests were disclosed.
The abstract is well-structured but can benefit from clearer language, more detail about methods, and precise emphasis on the significance of findings.
Clarify the methodology details, especially regarding extraction and GC-MS parameters.
Highlight the potential health implications of identified compounds more explicitly.
Consider adding quantitative data if available, such as relative abundances of key compounds.
Ensure consistency in terminology, e.g., "bioactive compounds" vs. "phytochemicals."
Improve readability with more formal academic language, especially in the conclusion.
1. Introduction to sample collection:
Clarify why the specific location was chosen, if relevant.
Correctly cite the method if it is referenced and complete the citation.
2. Botanical identification:
Specify the authority or botanist who confirmed the identification for scientific rigor (e.g., "identified and authenticated by Dr. XYZ using standard taxonomic keys").
3. Sample preparation details:
Mention how many leaves were collected or the approximate weight of the fresh leaves before drying.
Specify the drying conditions (e.g., "dried at ambient temperature (~25°C) in the shade" for accuracy).
4. Extraction process:
Complete the citation for the methodology or clarify if it’s a modified version (e.g., "based on the method described by [Author], [Year]").
Mention the solvent volume in a consistent manner ("5.5 liters of 99% ethanol," which is clear).
5. Maceration process:
Indicate the container type used and mention if the mixture was shaken periodically during the maceration.
Clarify whether the mixture was kept in the dark to prevent light degradation.
6. Filtration and concentration:
Specify the conditions of the rotary evaporator, such as temperature (e.g., "at 40°C") and pressure.
Mention how the dryness was confirmed (e.g., no further solvent smell or weight stabilization).
Is the work clearly and accurately presented and does it cite the current literature?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
Reviewer Expertise:
bioactivity analysis, phytochemistry
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
IkuomolaEmmanuelDepartment of Physiology, Kampala International University - Western Campus, Bushenyi, Western Region, Uganda
Competing interests: “I confirm that there are no competing interests that could be perceived as influencing the validity or importance of the peer review reports.”
2192025
“We sincerely thank the reviewer for their constructive feedback and valuable recommendations. The authors’ detailed responses to the comments are provided below.”
General reviewer comments
The abstract is well-structured but can benefit from clearer language, more detail about methods, and precise emphasis on the significance of findings.
Clarify the methodology details, especially regarding extraction and GC-MS parameters.
Author’s
Response: The abstract has been modified to contain method of extraction and conclusion emphasized the significance of the phytochemicals identified in the extract.
Author’s
Response: The method of extraction has been clarified by including the temperature, volume of solvent used and the storage of the extract obtained in page 4.
Highlight the potential health implications of identified compounds more explicitly.
Consider adding quantitative data if available, such as relative abundances of key compounds.
Ensure consistency in terminology, e.g., "bioactive compounds" vs. "phytochemicals."
Author’s
Response: The quantitative data is not available however table three shows the retention time and area under curve that represents a little bit of abundance of the compounds.
The terminology "bioactive compounds" has been replaced with “phytochemicals” as suggested for consistency.
Improve readability with more formal academic language, especially in the conclusion.
1. Introduction to sample collection:
Clarify why the specific location was chosen, if relevant.
Author’s
Response: This specific location was chosen since it has more farmland where the plant is cultivated. The GPS location has been included in the text for clarity.
Correctly cite the method if it is referenced and complete the citation.
Author’s
Response: There is no referenced method of picking the plant. It was just by plucking the leaves from the plant.
2. Botanical identification:
Specify the authority or botanist who confirmed the identification for scientific rigor (e.g., "identified and authenticated by Dr. XYZ using standard taxonomic keys").
Author’s
Response: The plant was identified and authenticated by Dr. Eunice Olet of the Department of Botany, Faculty of Biological Sciences, Mbarara University of Science and Technology. A voucher specimen was deposited in the university's herbarium under the reference number IOE-24-001.
3. Sample preparation details:
Mention how many leaves were collected or the approximate weight of the fresh leaves before drying.
Author’s
Response: The quantity of the fresh leaves has been mentioned to be 2 kg in page 4.
Specify the drying conditions (e.g., "dried at ambient temperature (~25°C) in the shade" for accuracy).
Author’s
Response: The drying condition has been specified as shed dried at ambient temperature of 26℃.
4. Extraction process:
Complete the citation for the methodology or clarify if it’s a modified version (e.g., "based on the method described by [Author], [Year]").
Author’s
Response: The sentence has been completed to read “The extraction process was performed using a modified version of the method described by Abdulrahman et al.
10” in page 4.
Mention the solvent volume in a consistent manner ("5.5 liters of 99% ethanol," which is clear).
Author’s
Response: The solvent volume has been modified to read “A total of 800 g of the powdered leaves was cold macerated in 5 liter of 99% ethanol (1:5 w/v).”
5. Maceration process:
Indicate the container type used and mention if the mixture was shaken periodically during the maceration.
Author’s
Response: The container has been specified as “sterile glass container”. The mixture was shaken periodically during the maceration process. This has been stated in page 4.
Clarify whether the mixture was kept in the dark to prevent light degradation.
Author’s
Response: The mixture was kept in the dark as stated in page 4.
6. Filtration and concentration:
Specify the conditions of the rotary evaporator, such as temperature (e.g., "at 40°C") and pressure.
Author’s
Response: The temperature is stated as below 40°C and under reduced pressure. This is stated in the relevant section on page 4.
Mention how the dryness was confirmed (e.g., no further solvent smell or weight stabilization).
Author’s
Response: The filtrate was evaporated to dryness having obtained a constant weight as stated in page 4.
Author’s
Response: The storage condition of the extract has been mentioned as follows in page 4. “The dried crude extracts were stored at 4°C in an amber glass bottle that was tightly sealed until further analysis”.