Phytochemical Profiling, Toxicological Safety, and Antihyperglycemic Effects of Fouquieria splendens Engelm. Foliar Extract in Streptozotocin-Induced Diabetic Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The conclusion statement is missing in the abstract.
2. It is advised to include the recent epidemiological data on diabetes mellitus (DM) rather than relying on 2021 statistics.
3. Consider splitting the introduction to enhance readability and attract the reader’s attention to the scientific content of the article.
4. The collection time of the plant material is missing. Moreover, it appears that some existing studies have already discussed the antidiabetic activity of this plant. If so, please clarify the novelty of the present work.
5. Please justify the inclusion of bacterial and micronucleus assays for the toxicity. How are they relevant to the core objectives of the study? If they are not directly related, it is advised to remove them.
6. It is unclear why two different strains of animals were used. Please address the ethical concerns associated with this choice.
7. A single dose of streptozotocin (STZ) is generally sufficient to induce Type 2 Diabetes Mellitus (T2DM). The authors are advised to include an appropriate citation, for instance:
   DOI: 10.1155/2018/4601649.
8. Please clarify the rationale for selecting the different doses used in the study.
9. It is advised to include scale bars in all figures for better clarity and scientific accuracy.
   The authors are also advised to refer to and cite the following manuscripts to enrich the research content of the article:DOI: 10.4103/ijpmr.ijpmr_16_24; DOI: 10.1080/13813455.2025.2483501

Author Response

Thank you very much for taking the time to review this manuscript. I sincerely appreciate your insightful comments and constructive suggestions, which have been carefully considered and incorporated to improve the clarity and overall quality of the work. Detailed responses are provided below, and all corresponding revisions or corrections are highlighted in track changes within the re-submitted files.

1. The conclusion statement is missing in the abstract.

The conclusion statement is included within the abstract “EFS exhibited significant antihyperglycemic activity and a favorable safety profile, supporting its potential as a complementary phytotherapeutic agent for diabetes management. These results highlight the pharmacological value of F. splendens and promote the exploration of native plants as adjuncts for chronic disease therapy.”

No changes were made to this comment, the conclusion can be found int he page 1, line 31.

2. It is advised to include the recent epidemiological data on diabetes mellitus (DM) rather than relying on 2021 statistics.

Thank you for your valuable comment. The reference has been updated according to the most recent report published by the International Diabetes Federation, which estimates that 588.7 million adults worldwide are currently living with diabetes. In North America, the total number of cases is 56.2 million. The global prevalence of diabetes is projected to rise to 853 million by 2050. (Duncan, B.B.; Magliano, D.J.; Boyko, E.J. IDF Diabetes Atlas 11th Edition 2025: Global Prevalence and Projections for 2050. Nephrol. Dial. Transplant. 2025, 0, 1–3. https://doi.org/10.1093/ndt/gfaf177)

[3]. Duncan, B.B.; Magliano, D.J.; Boyko, E.J. IDF Diabetes Atlas 11th Edition 2025: Global Prevalence and Projections for 2050. Nephrol. Dial. Transplant. 2025, 0, 1–3. https://doi.org/10.1093/ndt/gfaf177

The changes can be found on page 2, lines 47–50, where the updated citation [3] has been included. This modification also resulted in an additional change on page 18, lines 676–677, where the previous reference was updated accordingly.

3. Consider splitting the introduction to enhance readability and attract the reader’s attention to the scientific content of the article.

We appreciate your valuable suggestion. In response, the Introduction section has been revised and divided into four subsections to improve clarity and attract the reader’s attention to the scientific content of the article. The new structure includes: (1) general context, (2) problem statement and need, (3) natural therapeutic potential, and (4) justification of the study.

1.1. Background on Diabetes Mellitus

1.2. Public Health Impact and Socioeconomic Burden

1.3. Limitations of Current Therapies and Rationale for Natural Alternatives

1.4. Fouquieria splendens as a Potential Therapeutic Candidate

The changes can be observed on page 2, from line 40 to page 3, line 100. In addition, on page 2, line 82, the author name “Engelm.”—who originally described the plant has been added.

 

4. The collection time of the plant material is missing. Moreover, it appears that some existing studies have already discussed the antidiabetic activity of this plant. If so, please clarify the novelty of the present work.

The collection time of the plant material has been clarified in the revised version, page 3, lines 111-113 "Plant specimens were collected from a relict population of F. splendens (curatorial number 49021) in the locality of Acatita (23°39'42.6″, -104°22'17.1″) within the municipality of Mezquital in the state of Durango during October and November 2022".

Regarding the novelty of the study, this is the first report employing the foliar extract of Fouquieria splendens Engelm. for the treatment of diabetes. The present work demonstrates, through in vivo assays, the antihyperglycemic efficacy of the extract and provides the first toxicological characterization, including acute oral toxicity, mutagenicity, and genotoxicity evaluations. To the best of our knowledge, no previous studies have comprehensively assessed both the biological activity and the safety profile of the F. splendens foliar extract in experimental models of diabetes.

5. Please justify the inclusion of bacterial and micronucleus assays for the toxicity. How are they relevant to the core objectives of the study? If they are not directly related, it is advised to remove them.

The inclusion of the bacterial reverse mutation (Ames) test and the micronucleus assay was intended to provide a comprehensive toxicological profile of the Fouquieria splendens foliar extract. During the drug development process, a series of preclinical studies are required to ensure both the efficacy and safety of candidate compounds. Among these, the evaluation of mutagenic and genotoxic potential is mandatory according to international regulatory guidelines, including those established by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Specifically, the Ames test was performed following the OECD Guideline 471 [OECD, 2020. Test No. 471: Bacterial Reverse Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. https://doi.org/10.1787/9789264071247-en], which recommends the use of Salmonella typhimurium tester strains to identify the mutagenic potential of chemical substances and natural extracts. In parallel, the micronucleus assay was conducted according to the OECD Guideline 474 [OECD, 2016. Test No. 474: Mammalian Erythrocyte Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. https://doi.org/10.1787/9789264264762-en], to assess the potential of the extract to induce chromosomal damage or genotoxic effects in peripheral blood erythrocytes.

In the context of the present study, these assays were incorporated to establish a comprehensive safety profile of the F. splendens extract. Given the promising antihyperglycemic activity demonstrated in the in vivo model, it was essential to confirm that the extract does not pose mutagenic or genotoxic risks prior to advancing to more extensive preclinical or clinical evaluations. Therefore, both tests are directly aligned with the core objective of the study, which is to evaluate the therapeutic potential of F. splendens while ensuring its toxicological safety and regulatory compliance for future pharmacological development.

6. It is unclear why two different strains of animals were used. Please address the ethical concerns associated with this choice.

Two different animal strains were employed in the study in accordance with international regulatory and toxicological testing requirements. The use of distinct strains is justified by the specific sensitivity and suitability of each model for different assay types.

Specifically, BALB/c mice were used for the in vivo genotoxicity (micronucleus) assay because this strain is recognized for its higher sensitivity for said test, which increases the reliability of detecting potential genotoxic effects, as recommended in the OECD Guideline 474.

 To ensure ethical compliance and minimize the number of animals used, the same BALB/c mice were subsequently employed in the acute toxicity assessment. This was feasible because the micronucleus assay involves the collection of peripheral blood samples, which does not require animal sacrifice. Therefore, both evaluations were conducted on the same group of animals, adhering to the principles of the 3Rs (Replacement, Reduction, and Refinement) and in full accordance with institutional and national ethical guidelines for animal experimentation. This approach allowed us to obtain all the necessary safety data while minimizing animal use and distress, ensuring that the study remained scientifically rigorous and ethically responsible.

7. A single dose of streptozotocin (STZ) is generally sufficient to induce Type 2 Diabetes Mellitus (T2DM). The authors are advised to include an appropriate citation, for instance: DOI: 10.1155/2018/4601649.

The dose of streptozotocin (STZ) used in this study is consistent with previously established and widely accepted protocols for the induction of experimental diabetes. Several studies have reported that a single intraperitoneal injection of STZ ranging from 40 to 70 mg/kg is effective in inducing a diabetic state in rodents.

This approach follows the procedures described by Furman (2021), who highlights that this dosage range reliably produces hyperglycemia and is one of the most frequently employed methods for experimental diabetes induction [Furman, B.L. Streptozotocin-Induced Diabetic Models in Mice and Rats. Curr. Protoc. 2021, 1(4), e78. https://doi.org/10.1002/cpz1.78].

Furthermore, according to the same reference, fasting blood glucose levels exceeding 150 mg/dL (8.3 mmol/L) are considered indicative of mild hyperglycemia, confirming the successful induction of diabetes. Therefore, the dosage and induction protocol used in this study align with recognized methodological standards and ensure the reproducibility and validity of the diabetic model.

The suggested reference [Jayachandran M, Vinayagam R, Ambati RR, Xu B, Chung SSM. Guava Leaf Extract Diminishes Hyperglycemia and Oxidative Stress, Prevents β-Cell Death, Inhibits Inflammation, and Regulates NF-kB Signaling Pathway in STZ Induced Diabetic Rats. Biomed. Res. Int. 2018, 2018, 4601649. https://doi.org/10.1155/2018/4601649] has also been reviewed and cited in the revised version to support this methodological approach.

On page 5, lines 216–217, the following text was added: “following previously established protocols for experimental diabetes induction [26,27].”

Additionally, on page 19, lines 734–738, the following references were included:

[26] Furman, B.L. Streptozotocin-Induced Diabetic Models in Mice and Rats. Curr. Protoc. 2021, 1(4), e78. https://doi.org/10.1002/cpz1.78

[27] Jayachandran, M.; Vinayagam, R.; Ambati, R.R.; Xu, B.; Chung, S.S.M. Guava Leaf Extract Diminishes Hyperglycemia and Oxidative Stress, Prevents β-Cell Death, Inhibits Inflammation, and Regulates NF-κB Signaling Pathway in STZ-Induced Diabetic Rats. Biomed. Res. Int. 2018, 2018, 4601649. https://doi.org/10.1155/2018/4601649

8. Please clarify the rationale for selecting the different doses used in the study.

The selection of the different doses used in this study was based on both toxicity data and previous studies on antihyperglycemic plant extracts and standard antidiabetic drugs. For the plant extract, toxicity studies reveal that the maximum safe dose can reach up to 2000 mg/kg. Based on these findings, we selected a dose of 200 mg/kg to ensure safety and allow for evaluation of pharmacological efficacy.

Regarding the reference drugs, the doses were selected based on previously published studies demonstrating their efficacy in diabetic models. The selected dose of 100 mg/kg of metformin has been shown to significantly reduce blood glucose levels in rodent models of diabetes [Aguila-Muñoz, D.G.; Jiménez-Montejo, F.E.; López-López, V.E.; Mendieta-Moctezuma, A.; Rodríguez-Antolín, J.; Cornejo-Garrido, J.; Cruz-López, M.C. Evaluation of α-Glucosidase Inhibition and Antihyperglycemic Activity of Extracts Obtained from Leaves and Flowers of Rumex crispus L. Molecules 2023, 28(15), 5760. https://doi.org/10.3390/molecules28155760; Atanu, F.O.; Avwioroko, O.J.; Ilesanmi, O.B.; Yakubu, O.E. Metformin Potentiates the Antidiabetic Properties of Annona muricata and Tapinanthus globiferus Leaf Extracts in Diabetic Rats. Pharmacogn. J. 2021, 13(3), 614–619. https://doi.org/10.5530/pj.2021.13.77].

 

Similarly, a 10 mg/kg dose of sitagliptin effectively lowers blood glucose levels [Kizilay, G.; Ersoy, O.; Cerkezkayabekir, A.; Topcu-Tarladacalisir, Y. Sitagliptin and Fucoidan Prevent Apoptosis and Reduce ER Stress in Diabetic Rat Testes. Andrologia. 2021, 53(3), e13858. https://doi.org/10.1111/and.13858; Shawky, L.M.; Morsi, A.A.; El Bana, E.; Hanafy, S.M. The Biological Impacts of Sitagliptin on the Pancreas of a Rat Model of Type 2 Diabetes Mellitus: Drug Interactions with Metformin. Biology. 2020, 9, 6. https://doi.org/10.3390/biology9010006; Jadhav, P.B.; Jadhav, S.B.; Zehravi, M.; Mubarak, M.S.; Islam, F.; Jeandet, P.; Khan, S.L.; Hossain, N.; Rashid, S.; Ming, L.C.; Sarker, M.M.R.; Azlina, M.F.N. Virtual Screening, Synthesis, and Biological Evaluation of Some Carbohydrazide Derivatives as Potential DPP-IV Inhibitors. Molecules. 2022, 28(1),149. https://doi.org/10.3390/molecules28010149]

Therefore, the doses used in the present study were carefully selected to ensure safety and pharmacological relevance, thus allowing for a reliable evaluation of the antihyperglycemic efficacy of Fouquieria splendens extract and its comparison with standard antidiabetic drugs.

9. It is advised to include scale bars in all figures for better clarity and scientific accuracy.

As suggested, the objective used for each photograph has been specified in Figure 3. The histological sections were captured at 20× magnification for the kidney, liver, and pancreas, and at 10× for the intestine.

On page 12, lines 417–425, the caption of Figure 3 was revised. Accordingly, the figure itself was also modified by removing redundant information from the side and adding continuous and dashed arrows in black and red to improve clarity.

The authors are also advised to refer to and cite the following manuscripts to enrich the research content of the article: DOI: 10.4103/ijpmr.ijpmr_16_24; DOI: 10.1080/13813455.2025.2483501

DOI: 10.4103/ijpmr.ijpmr_16_24
This study is not directly related, as its objective was to identify areas prone to foot ulcers at early stages of diabetes in India.
However, relevant information could be incorporated as a paragraph in the Introduction discussing the general context: “Diabetes leads to increased morbidity and mortality, which continue to rise worldwide. Among its major complications are neuropathy—the leading cause of limb amputations—and ischemia, both being the principal conditions underlying diabetic foot ulcers, which affect approximately 15% of individuals with diabetes.” Nevertheless, the references cited in this article are outdated (2004 and 2008).

DOI: 10.1080/13813455.2025.2483501
Full access to this manuscript was not available, so figures and tables could not be reviewed. However, it addresses the antioxidant capacity of a methanolic flower extract both in vitro and in vivo using a different experimental model (alloxan-induced diabetic mice).

Similar points:

• Phytochemical screening of the extract (flower, methanolic extract)

• Antioxidant studies

• Evaluation of acute toxicity (in mice)

Additionally, the authors:

• Conducted histopathological analysis of cardiac tissue

In their results, the authors reported the identification of bioactive compounds with antioxidant and anti-inflammatory activity. They further noted that the extract suppressed hyperglycemia by regulating the expression of the glucose transporter GLUT4 and histologically restored tissue alterations in diabetic animals.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting study aimed at assessing the phytochemical profiling, toxicological safety, and antihyperglycemic effect of Fouquieria splendens Engelm. The materials and methods are well described; however, some questions remain unanswered by the authors. 

1. Why they didn't choose to use only female mice and not both sexes, as they did for rats?
2. Based on which criteria they have decided to use 2000mg/kg of body weight?
3. In relation to the use of STZ, as this is a toxic agent, it is recommended to mention if they had any losses of animals
4. How have they performed the behavioral and physiological evaluation of the animals? This was done based on an evaluation sheet, after physical observation of the animals, or by using a camera. 
5. Concerning the statistical analysis, why they have chosen to use SEM instead of SD ?

Author Response

Thank you very much for taking the time to review this manuscript. I sincerely appreciate your insightful comments and constructive suggestions, which have been carefully considered and incorporated to improve the clarity and overall quality of the work. Detailed responses are provided below, and all corresponding revisions or corrections are highlighted in track changes within the re-submitted files.

1. Why they didn't choose to use only female mice and not both sexes, as they did for rats?

Thank you for this important question. Female mice were used exclusively for the peripheral blood micronucleus assay, which evaluates the genotoxic potential of a compound or extract. This decision was based on the fact that the BALB/c female strain is particularly sensitive for detecting toxic and immunogenic substances, making it a preferred model for genotoxicity testing [Zhou, C.; Ludmila, T.; Sun, N.; Wang, C.; Pu, Q.; Huang, K.; Che, H. BALB/c Mice Can Be Used to Evaluate Allergenicity of Different Food Protein Extracts. Food Agric. Immunol. 2016, 27(5), 589–603. https://doi.org/10.1080/09540105.2015.1129600; Salamone, M.F.; Mavournin, K.H. Bone Marrow Micronucleus Assay: A Review of the Mouse Stocks Used and Their Published Mean Spontaneous Micronucleus Frequencies. Environ. Mol. Mutagen. 1994, 23(4), 239–273. https://doi.org/10.1002/em.2850230402].

In this study, the use of only female BALB/c mice allowed us to maximize the sensitivity of the micronucleus assay while adhering to the principle of reduction in animal use. While rats of both sexes were used for other toxicological and pharmacological endpoints to account for possible sex related differences in metabolic response or efficacy.

2. Based on which criteria they have decided to use 2000mg/kg of body weight?

The dose of 2000 mg/kg body weight was employed in the toxicity studies, specifically in the acute oral toxicity assay and the in vivo peripheral blood micronucleus test. This dose was selected in accordance with the guidelines established by the Organization for Economic Co-operation and Development (OECD), which provide internationally recognized standards for preclinical toxicological testing and are widely used in drug development by regulatory agencies such as the FDA and EMA.

 

For the acute oral toxicity study, the dose selection followed the OECD Guideline 423 (Acute Toxic Class Method) [OECD. Test No. 423: Acute Oral Toxicity – Acute Toxic Class Method. In OECD Guidelines for the Testing of Chemicals, Section 4; OECD Publishing: Paris, France, 2002. https://doi.org/10.1787/9789264071001-en].

 

In the case of the micronucleus assay in peripheral blood, the dose was determined based on the results obtained from the acute oral toxicity study and in accordance with OECD Guideline 474 [OECD. Test No. 474: Mammalian Erythrocyte Micronucleus Test. In OECD Guidelines for the Testing of Chemicals, Section 4; OECD Publishing: Paris, France, 2016. https://doi.org/10.1787/9789264264762-en]

3. In relation to the use of STZ, as this is a toxic agent, it is recommended to mention if they had any losses of animals

Although mortality associated with the streptozotocin (STZ)-induced diabetes protocol has been reported, these effects are mainly observed in mice and at higher doses. In our study, the dose employed did not result in the death of any animal, which is consistent with what has been described in standardized protocols for diabetes induction. [Furman, B.L. Streptozotocin-Induced Diabetic Models in Mice and Rats. Curr. Protoc. 2021, 1(4), e78. https://doi.org/10.1002/cpz1.78]

 

4. How have they performed the behavioral and physiological evaluation of the animals? This was done based on an evaluation sheet, after physical observation of the animals, or by using a camera. 

The behavioral and physiological evaluation of the animals was conducted through direct visual observation, following the criteria and procedures established in the OECD Guideline 423 for Acute Oral Toxicity. The evaluation included continuous monitoring of clinical signs such as changes in locomotor activity, posture, piloerection, tremors, convulsions, salivation, and any other behavioral or physiological alterations, as recommended by the guideline [OECD. Test No. 423: Acute Oral Toxicity – Acute Toxic Class Method. In OECD Guidelines for the Testing of Chemicals, Section 4; OECD Publishing: Paris, France, 2002. https://doi.org/10.1787/9789264071001-en].

 

5. Concerning the statistical analysis, why they have chosen to use SEM instead of SD?

We chose to present our data as mean ± SEM because the SEM is more appropriate when the objective is to compare mean values between multiple groups using inferential statistical tests such as t-tests or ANOVA. The SEM quantifies the precision of the estimated group mean and reflects how well each sample mean represents the underlying population mean. In contrast, the SD describes the dispersion of individual observations around the sample mean and serves as a descriptive measure of variability within a sample. Therefore, expressing the data as mean ± SEM better aligns with the statistical basis of our analyses and provides a clearer representation of the precision of the estimated means.

This approach is consistent with the recommendations discussed by Jaykaran (2010), who emphasized that the SEM is appropriate when the intent is to infer population parameters rather than merely describe sample variability (Jaykaran, Indian J. Pharmacol., 2010; 42(5):329. doi:10.4103/0253-7613.70402; PMID: 21206631; PMCID: PMC2959222). Related discussions on the correct interpretation and misuse of SEM in biomedical research can be found in Nagele (2003) and Tom (2004).

• Nagele P. Misuse of standard error of the mean (SEM) when reporting variability of a sample: a critical evaluation of four anaesthesia journals. Br J Anaesth. 2003;90:514–516. doi:10.1093/bja/aeg087.
• Tom L. Twenty statistical errors even YOU can find in biomedical research articles. Croat Med J. 2004;45:361–370.
• Jaykaran. "Mean ± SEM" or "Mean (SD)"? Indian J Pharmacol. 2010;42(5):329. doi:10.4103/0253-7613.70402. PMID: 21206631; PMCID: PMC2959222.

Additional comment:
As references 3, 26, and 27 were newly added, all subsequent references were renumbered accordingly. The total number of references increased from 71 to 74.

Author Response File: Author Response.pdf