Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript explores the preventive effects of Polygonatum multiflorum tuber polysaccharides (PMTL) on diphenoxylate-induced constipation in rats. The study integrates biochemical, histological, molecular, and microbiota analyses to build a mechanistic model. The work is original, methodologically sound, and potentially valuable for the development of functional foods or nutraceuticals targeting constipation. However, several aspects require clarification, deeper analysis, and more cautious interpretation.

1. The abstract should better emphasize the novelty (why PMTL vs. other pectin-rich fibers).

1. Effect sizes (e.g., % changes in neurotransmitters or SCFAs) could be included briefly to highlight magnitude of effect.
2. The introduction could better differentiate PMTL from other prebiotic fibers (e.g., inulin, resistant starch). What makes PMTL particularly promising?
3. References are sometimes dated—newer microbiota-focused constipation research (2022–2024) should be included.
4. Lack of sample size justification (power analysis). This weakens statistical robustness.
5. Methods for polysaccharide characterization are too superficial—structural detail (molecular weight, monosaccharide composition by HPLC/GC) would strengthen reproducibility.
6. SCFA quantification should present absolute concentrations (µmol/g) rather than relative differences.
7. Microbiota sequencing: missing information about sequencing depth, normalization, and correction for multiple testing.
8. PMTL is rich in soluble pectin (460 mg/g). Fecal water retention significantly improved with 10% PMTL.Dose-response not linear (10% > 20%). Needs mechanistic explanation (e.g., excessive fiber causing slower fermentation).
9. PMTL increased carmine propulsion rate. Lack of direct measurement of intestinal transit time weakens clinical translatability.
10. Constipation reduced goblet cells and crypt depth, reversed by PMTL. PMTL reduced colonic apoptosis. Quantification of goblet cells per field/crypt would add rigor.
11. PMTL restored excitatory neurotransmitters (MTL, ACh, SP) and reduced inhibitory ones (SS, NO). Very large increase in SP (over 200%) may be biologically inflated. Authors should discuss methodological bias.
12. PMTL downregulated AQP3/AQP4 and reduced IL-1β/iNOS expression. Mechanism should be linked more explicitly to pectin fermentation and SCFA signaling.
13. PMTL restored microbial diversity and reduced F/B ratio. Beneficial bacteria correlated with SCFAs and neurotransmitters. Functional predictions (PICRUSt2, KEGG pathways) are missing. This limits mechanistic interpretation.
14. PMTL increased acetic acid and butyrate. Absolute values missing; important to compare with physiological ranges in humans.
15. Discussion often repeats results rather than critically analyzing them.
16. The non-linear dose response (10% > 20%) is acknowledged but not explained. Possible hypotheses (excessive fiber fermentation leading to gas, osmotic imbalance) should be considered.
17. The role of polyphenols in PMTL is neglected. These compounds may also contribute to microbiota modulation and anti-inflammatory effects.
18. Clinical translation is not fully addressed: How does rat dosage translate to human intake?
19. Conclusions are too strong given the preclinical (rat) model. Must highlight limitations before extrapolating to humans.
20. Future directions should be proposed: clinical trials, synergistic use with probiotics, structural optimization of PMTL.

Final Recommendation:
The manuscript presents innovative and promising findings, but major revisions are needed before publication, particularly in the methodological detail, critical discussion, and cautious interpretation of translational relevance.

Author Response

Dear reviewer,

We are very pleased to receive your letter with the reviewer’s comments for our manuscript “foods-3847498”. We thank the reviewers for thoroughly reviewing our manuscript and making thoughtful comments. The manuscript has been revised carefully, and the detailed corrections requested by the reviewers have been done and listed below point by point.

 

1. 1. The abstract should better emphasize the novelty (why PMTL vs. other pectin-rich fibers).

Response: Thanks for the reviewer’s patience and detailed comments which have enabled us to improve our manuscript, and according to the reviewer’s guide, the sentence was revised to follows:

lines 13-14: “This study was designed to explore the preventive effects of pectin-enriched Premna microphylla turczleaves (PMTL) consumption on experimental constipation.” was revised as: “This study for the first time explored the preventive effects of a novel pectic polysaccharide from Premna microphylla turcz leaves (PMTL) on experimental constipation.”

 

2. Effect sizes (e.g., % changes in neurotransmitters or SCFAs) could be included briefly to highlight magnitude of effect.

Responses: We thank the reviewer for the insightful comments. According to the reviewer’s guide, this sentence has been revised as follow:

lines 361-363: “As shown in Figure 6A, the colonic levels of total SCFAs in the CM rats were decreased significantly as compared with the rats in NC group (p < 0.01).” was revised as: “As shown in Figure 6A, the total SCFAs levels in the colon of rats in the NC group and the CM group were 234.6 ± 60.9 μM/g and 106.9 ± 26.3 μM/g, respectively. It can be found that the total SCFAs level in the colon of rats in the CM group decreased by 54.4% compared with the NC group (p < 0.01).”

lines 364-366: “As shown in Figure 6B--F, the levels of butyric acid, isobutyric acid and valeric acid of the constipated rats were markedly lower than that of the NC rat (p < 0.05).” was revised as: “As shown in Figure 6B-F, the levels of butyric acid, isobutyric acid and valeric acid in constipated rats were 65.9%, 26.6% and 43.1% lower than those in NC rats (p < 0.05).”

lines 366-368：“However, ingestion of the CM rats with 10% or 20% PMTL significantly increased the colonic levels of butyric acid (p < 0.05)” was revised as: “The levels of acetic acid and butyric acid in CM rats given 10%PMTL were 28.7 ± 1.9 μg/g and 130.5 ± 15.3 ng/g, respectively, which increased by 142.7% and 100.4% compared with the model group (p < 0.05).”

 

3. The introduction could better differentiate PMTL from other prebiotic fibers (e.g., inulin, resistant starch). What makes PMTL particularly promising?

Responses: We thank the reviewer for the insightful comments. According to the reviewer’s guide, this sentence has been revised as follow:

lines 51-54: “To be specific, pectin is the most abundant nutrients in PMTL, which has extensive healthy effects including prebiotics properties [23-26], and thus we speculate that consumption of dietary PMTL might exert the potential to regulate the structure of gut microbiome for relieving constipation.” was revised as: “Pectin possesses a broader spectrum of health benefits relative to other prebiotics, including pronounced prebiotic properties. Notably, pectin constitutes the most abundant nutrient in PMTL, therefore we hypothesize that dietary intake of PMTL may modulate the gut microbiota structure, thereby alleviating constipation [23-26].”

 

4. References are sometimes dated—newer microbiota-focused constipation research (2022–2024) should be included.

Responses: We sincerely appreciate your valuable suggestion. In accordance with your comment, the relevant references have been revised as follows:

lines 508-510: “1. Reichardt, F.; Chassaing, B.; Nezami, B.G.; Li, G.; Tabatabavakili, S.; Mwangi, S.; Srinivasan, S. Western diet induces colonic nitrergic myenteric neuropathy and dysmotility in mice via saturated fatty acid- and lipopolysaccharide-induced TLR4 sig-nalling. J. Physiol. 2017, 595, 1831–1846.” was revised as: “1. Tran, D.L.; Sintusek, P. Functional Constipation in Children: What Physicians Should Know. World J. Gastroenterol. 2023, 29, 1261–1288.”

 

lines 512: “3. Hayat, U.; Dugum, M.; Garg, S. Chronic constipation: Update on management. Cleve. Clin. J. Med. 2017, 84, 397–408.” was revised as: “3. Salvi, F.; Petrino, R.; Conroy, S.P.; Liperoti, R.; Paoletti, L.; Beccacece, A.; dell'Aquila, G.; Fedecostante, M.; Cherubini, A. Constipation: A Neglected Condition in Older Emergency Department Patients. Intern. Emerg. Med. 2024, 19, 1977–1986.

 

lines 533: “13. Rodriguez, R.W. Off-label uses of alvimopan and methylnaltrexone. Am. J. Health Syst. Pharm. 2014, 71, 1450–1455.” was revised as: “13. Chang, L.; Chey, W.D.; Imdad, A.; Almario, C.V.; Bharucha, A.E.; Diem, S.; Greer, K.B.; Hanson, B.; Harris, L.A.; Ko, C.; Murad, M.H.; Patel, A.; Shah, E.D.; Lembo, A.J.; Sultan, S. American Gastroenterological Association-American College of Gastroenterology Clinical Practice Guideline: Pharmacological Management of Chronic Idiopathic Constipation. Gastroenterology 2023, 164, 1086–1106.”

 

lines 537-538: “16. Hu, T.G.; Wen, P.; Fu, H.Z.; Lin, G.Y.; Liao, S.T.; Zou, Y.X. Protective effect of mulberry (Morus atropurpurea) fruit against diphenoxylate-induced constipation in mice through the modulation of gut microbiota. Food Funct. 2019, 10, 1513–1528. was revised as: “Huang, P.H.; Jian, C.H.; Lin, Y.W.; Huang, D.W. Impact of Premna microphylla Turcz Leaf Water Extracts on the Properties of Gelatin-Carrageenan Edible Film and Its Application in Cherry Tomatoes Storage Food Chem. X 2025, 25, 102186.

 

lines 565-568: “30. Zhai, X.; Lin, D.; Zhao, Y.; Yang, X. Bacterial Cellulose Relieves Diphenoxylate-Induced Constipation in Rats. J. Agric. Food Chem. 2018, 66, 4106–4117.” was revised as: “32. Yang, C.; Chen, X.; Niu, P.; Yang, X.; Lu, Y. Incremental Effects of Eurotium cristatum Fermentation of Soybean on Its Nutrients, Flavor Profile and Laxative Regulation in Experimental Constipated Rats. Food Funct. 2025, 16, 2363–2377.”

 

lines 580-581: “37. Morrison, D.J.; Preston, T. Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes 2016, 7, 189–200.” was revised as: “40. Mann, E.R.; Lam, Y.K.; Uhlig, H.H. Short-Chain Fatty Acids: Linking Diet, the Microbiome and Immunity. Nat. Rev. Immunol. 2024, 24, 577–595.”

 

lines 591-593: “42. Kim, J.E.; Go, J.; Koh, E.K.; Song, S.H.; Sung, J.E.; Lee, H.A.; Lee, Y.H.; Hong, J.T.; Hwang, D.Y. Gallotannin-Enriched Extract Isolated from Galla Rhois May Be a Functional Candidate with Laxative Effects for Treatment of Loperamide-Induced Constipation of SD Rats. PLoS One 2016, 11, e0161144.” was revised as: “45. Liang, S.; He, Z.; Liang, Z.; Wang, K.; Du, B.; Guo, R.; Li, P. Prunus persica (L.) Batsch Blossom Soluble Dietary Fiber Synergia Polyphenol Improving Loperamide-Induced Constipation in Mice via Regulating Stem Cell Factor/C-kit, NF-κB Signaling Pathway and Gut Microbiota. Food Res. Int. 2024, 192, 114761.”

 

lines 620-622: “56. Marín-Manzano, M.C.; Abecia, L.; Hernández-Hernández, O.; Sanz, M.L.; Montilla, A.; Olano, A.; Rubio, L.A.; Moreno, F.J.; Clemente, A. Galacto-oligosaccharides derived from lactulose exert a selective stimulation on the growth of Bifidobacterium animalis in the large intestine of growing rats. J. Agric. Food Chem. 2013, 61, 7560–7567.” was revised as: “62. Zhang, T.; Liu, W.; Lu, H.; Cheng, T.; Wang, L.; Wang, G.; Zhang, H.; Chen, W. Lactic Acid Bacteria in Relieving Constipation: Mechanism, Clinical Application, Challenge, and Opportunity. Crit. Rev. Food Sci. Nutr. 2025, 65, 551–574.”

 

lines 630-631: “60. Bendali, F.; Madi, N.; Sadoun, D. Beneficial effects of a strain of Lactobacillus paracasei subsp. paracasei in Staphylococcus aureus-induced intestinal and colonic injury. Int. J. Infect. Dis. 2011, 15, e787–e794.” was revised as: “66. Maezawa, Y.; Nagasaki, K. Aerococcus urinae: An Emerging, Gram-Positive Pathogen Causing Urinary Tract Infection. Am. J. Med. 2024, 137, e89–e90.

 

5. 5. Lack of sample size justification (power analysis). This weakens statistical robustness.

Responses: Thank you for this critical comment regarding the sample size justification. We fully agree that an a priori power analysis is essential for ensuring statistical robustness and is a standard practice in high-quality research. As this study was initially conceived as a pilot/exploratory investigation, the sample size was determined primarily based on common practices in similar published studies in our field and practical constraints regarding animal availability. We promise that in all future studies, prior efficacy analysis will be conducted at the planning stage.

 

6. Methods for polysaccharide characterization are too superficial—structural detail (molecular weight, monosaccharide composition by HPLC/GC) would strengthen reproducibility.

Responses: Thank you for your valuable suggestion. We fully agree that detailed polysaccharide structure characterization (e.g., molecular weight, monosaccharide composition by HPLC/GC) is crucial for improving reproducibility. In fact, we have conducted a systematic structural analysis of the PMTL polysaccharides—including molecular weight determination by HPGPC and monosaccharide composition by GC-MS. These results will be presented in a separate, companion manuscript focused specifically on the purification and structural characterization, which aligns with the ongoing focus of our subsequent work.

 

7. SCFA quantification should present absolute concentrations (µmol/g) rather than relative differences.

Responses: Thanks for the reviewer’s patience and detailed comments which have enabled us to improve our manuscript. According to the reviewer’s suggestion, this sentence has been corrected as follow:

lines 361-363: “As shown in Figure 6A, the colonic levels of total SCFAs in the CM rats were decreased significantly as compared with the rats in NC group (p < 0.01).” was revised as: “As shown in Figure 6A, the total SCFAs levels in the colon of rats in the NC group and the CM group were 234.6 ± 60.9 μM/g and 106.9 ± 26.3 μM/g, respectively. It can be found that the total SCFAs level in the colon of rats in the CM group decreased by 54.4% compared with the NC group (p < 0.01).”

 

8. Microbiota sequencing: missing information about sequencing depth, normalization, and correction for multiple testing.

Responses: We thank the reviewer for their comments, the sentence has been revised as suggested:

lines 178-182: “The purified PCR products were quantified by QuantiFluorTM-ST Blue Fluorescence Quantification System and mixed in appropriate proportions according to the needs of each sample. The PCR products were sequenced by Illumina MiSeq platform (Illumina, USA), and the sequenced data was processed by statistical analysis.” was revised as: “High-throughput sequencing of the bacterial 16S rRNA gene was performed on the Illumina MiSeq PE250 platform. Raw paired-end reads were demultiplexed, subjected to quality filtering, and merged based on overlap regions to generate high-quality sequences [30]. Denoising of these sequences was carried out to derive amplicon sequence variants (ASVs) along with their abundance profiles. Subsequent analyses, including taxonomic classification, community diversity assessment, and differential abundance testing, were conducted using these ASV data. For taxonomic assignment, sequences were analyzed with the RDP Classifier algorithm against the SILVA SSU123 16S rRNA database, applying a confidence threshold of 70% to ensure classification accuracy.”

 

Relevant references were added as follows:

30. Wang, Y.; Li, T.; Liu, Y.Y.; Yang, C.C.; Liu, L.; Zhang, X.N.; Yang, X.B. Heimao Tea Polysaccharides Ameliorate Obesity via Enhancing Gut Microbiota-Dependent Adipocytes Thermogenesis in Mice Fed with High Fat Diet. Food Funct. 2022, 13, 13014–13027.

 

9. PMTL is rich in soluble pectin (460 mg/g). Fecal water retention significantly improved with 10% PMTL.Dose-response not linear (10% > 20%). Needs mechanistic explanation (e.g., excessive fiber causing slower fermentation).

Responses: We are grateful for the reviewer's helpful comments, the following statement is added before line 433: “It was noteworthy that PMTL rich in soluble pectin significantly elevated fecal water content in constipated CM rats when supplemented at 5%–20% in chow. Notably, its dose-response was non-linear, with 10% PMTL being more effective than 20% (Figure 1D). We speculate that excessive dietary fiber may slow gastrointestinal fermentation, thereby impairing fecal water content regulation in CM rats and weakening the efficacy of the 20% dose [53].”

 

Relevant references were added as follows:

53. Gill, S.K.; Rossi, M.; Bajka, B.; Whelan, K. Dietary Fibre in Gastrointestinal Health and Disease. Nat. Rev. Gastroenterol. Hepatol. 2021, 18, 101–116.

 

10. PMTL increased carmine propulsion rate. Lack of direct measurement of intestinal transit time weakens clinical translatability.

Responses: Thank you for this insightful comment. We fully agree that direct measurement of intestinal transit time is critical to enhancing the clinical translatability of our findings on PMTL-improved intestinal motility—since the carmine propulsion rate only reflects upper gastrointestinal motility and cannot fully represent overall intestinal transit. In our initial study, we used the carmine method (a widely used indirect indicator of intestinal propulsion in rodent models) to preliminarily assess PMTL’s effect on motility, and we will focus on intestinal transit time measurement methods in our subsequent research.

 

11. Constipation reduced goblet cells and crypt depth, reversed by PMTL. PMTL reduced colonic apoptosis. Quantification of goblet cells per field/crypt would add rigor.

Responses: We sincerely thank the reviewer for this valuable suggestion. We fully agree that quantification is essential for strengthening our conclusions. In response to your comment, we have now added a detailed quantitative analysis of goblet cells per crypt as a new panel (Figure 2A) in the revised manuscript.

Accordingly, we have meticulously updated the figure legend for Figure 2 to describe the new quantitative data and the statistical methods used. Furthermore, we have revised the corresponding descriptive text in the Results section of the manuscript：

lines 270-271: “(A) H&E section staining of the colon: (a) Colonic muscle thickness; (b) Colonic crypt thickness…”was revised as: “(A) H&E section staining of the colon: (a) Colonic muscle thickness; (b) Colonic crypt thickness;(c) Goblet cell per crypt…”

 

lines 241-252: “In comparison with the NC group, the structure of goblet cells of rats in CM group was not clear, as well as the number of goblet cells was very small. Besides, the colonic muscle thickness and crypt thickness of rats in the CM group were significantly reduced as compared with the rats in NC group. However, compared with the CM group, the colonic muscle thickness of the rats in the 10% and 20% PMTL groups was significantly increased (p < 0.01), while the colonic crypt length of the rats in all PMTL consumption groups were dramatically ele-vated (p < 0.05). In addition, the colonic epithelium and smooth muscle layer structure of PMTL-treated rats were intact, and the boundary between crypt structure and goblet cells was clear as compared with the CM group. The results of H&E staining images showed that PMTL could not only effectively increase colonic muscle and crypt thickness, but also improve goblet cell morphology and volume in colons of constipated rats.” was revised as: “In comparison with the NC group, the structure of goblet cells of rats in CM group was not clear, and the number of goblet cells per crypt was significantly decreased (p < 0.01). Besides, the colonic muscle thickness and crypt thickness of rats in the CM group were significantly reduced as compared with the rats in NC group. However, compared with the CM group, the colonic muscle thickness of the rats in the 10% and 20% PMTL groups was significantly increased (p < 0.01), while the colonic crypt length of the rats in all PMTL consumption groups were dramatically elevated (p < 0.05). Furthermore, quantitative analysis revealed that PMTL administration, particularly at the 10% dose, markedly increased the number of goblet cells per crypt compared to the CM group (p < 0.01), demonstrating a dose-dependent restorative effect. In addition, the colonic epithelium and smooth muscle layer structure of PMTL-treated rats were intact, and the boundary between crypt structure and goblet cells was clear as compared with the CM group. The results of H&E staining images showed that PMTL could not only effectively increase colonic muscle and crypt thickness, and restore goblet cell numbers, but also improve goblet cell morphology and volume in colons of constipated rats.”

 

12. PMTL restored excitatory neurotransmitters (MTL, ACh, SP) and reduced inhibitory ones (SS, NO). Very large increase in SP (over 200%) may be biologically inflated. Authors should discuss methodological bias.

Responses: Thank you for this insightful comment and for highlighting the potential concern regarding the greater than 200% increase in SP levels. We agree that such a marked increase warrants careful consideration of methodological limitations. As you suggested, we have now added discussion of this point in the revised manuscript：

lines 427-428: “However, these changes in the inhibitory neurotransmitters and excitatory neuro-transmitters were all dramatically reversed by PMTL ingestion in the CM rats.” was revised as: “However, these alterations in inhibitory and excitatory neurotransmitters were dramatically reversed by PMTL ingestion in CM rats. Concomitantly, SP levels were increased markedly (>200%) following PMTL intervention, indicating potent neuroexcitatory effects. Although the magnitude of this change may be influenced by pre-treatment depletion in the constipation model or potential nonlinearity in quantification at high concentrations, the upward trend remains robust and biologically indicative.”

 

13. PMTL downregulated AQP3/AQP4 and reduced IL-1β/iNOS expression. Mechanism should be linked more explicitly to pectin fermentation and SCFA signaling.

Responses: Thank you for this insightful comment. We agree that providing a more explicit mechanistic link between pectin fermentation, SCFA signaling, and the observed downregulation of AQP3/AQP4 and IL-1β/iNOS is crucial for strengthening our manuscript. In response, we have revised the Discussion section to elaborate on the proposed connecting mechanism：

lines 475-477: “These findings suggested suggesting that PMTL may be metabolized by the intestinal microbiota to produce SCFAs, subsequently reliving constipation caused by diphenoxylate in rats.” was revised as: “Mechanistically, the downregulation of pro-inflammatory factors (IL-1β, iNOS) and aquaporins (AQP3, AQP4) was likely mediated by SCFAs derived from PMTL pectin fermentation. Butyrate, in particular, activates GPCRs (e.g., GPR41/43/109a), suppressing NF-κB signaling and subsequent inflammation. The attenuated inflammatory milieu further normalizes water transport, leading to reduced AQP3/AQP4 expression. These findings suggested that PMTL might alleviate constipation via the microbiota–SCFAs–inflammation axis, restoring intestinal homeostasis.”

 

14. PMTL restored microbial diversity and reduced F/B ratio. Beneficial bacteria correlated with SCFAs and neurotransmitters. Functional predictions (PICRUSt2, KEGG pathways) are missing. This limits mechanistic interpretation.

Responses:Thank you for your valuable comment. We fully agree that functional prediction analyses like PICRUSt2/KEGG pathway annotation would deepen mechanistic understanding of PMTL’s effect on gut microbiota, and we acknowledge this as a limitation of our current work.In this study, we focused on validating core phenotypic and correlational relationships (microbial diversity, F/B ratio, SCFA, and neurotransmitter levels) to confirm PMTL’s regulatory role in the gut-microbiota-metabolite axis. While our existing data (e.g., correlations between beneficial bacteria and SCFAs/neurotransmitters) support our conclusions, we recognize the need for functional profiling.

We plan to address this gap in future work—integrating metagenomic sequencing with PICRUSt2/KEGG analyses to characterize microbial functional shifts—and have added a transparent note on this limitation in the “Conclusions” section of the revised manuscript：“To expand the study’s translational value and mechanistic depth, three future directions are proposed: (1) exploring potential synergistic effects of PMTL with probiotics or other prebiotics to enhance its regulatory impact on gut health; (2) investigating structural optimization of PMTL-derived polysaccharides to improve their bioavailability and functional properties; and (3) integrating metagenomic sequencing with PICRUSt2/KEGG analyses to systematically characterize the functional shifts of gut microbiota induced by PMTL, thereby clarifying the functional links between microbial community remodeling and PMTL’s physiological effects.”

 

15. PMTL increased acetic acid and butyrate. Absolute values missing; important to compare with physiological ranges in humans.

Responses: We thank the reviewer for their comments, the sentence has been revised as suggested:

lines 366-368：“However, ingestion of the CM rats with 10% or 20% PMTL significantly increased the colonic levels of butyric acid (p < 0.05)” was revised as: “The levels of colonic acid and butyric acid in CM rats given 10%PMTL were 28.7 ± 1.9 μg/g and 130.5 ± 15.3 ng/g, respectively, which increased by 142.7% and 100.4% compared with the model group (p < 0.05).”

 

lines 468-471: “SCFAs are important metabolites of intestinal microbiota, which can stimulate intestinal peristalsis, increase intestinal osmotic pressure and promote water absorption, so they play an important role in alleviating constipation.” was revised as: “SCFAs as key metabolites derived from gut microbiota, play a crucial role in alleviating constipation by stimulating intestinal peristalsis, increasing luminal osmotic pressure, and enhancing water absorption [61]. In constipated rats, PMTL treatment significantly elevated fecal SCFA levels—particularly acetic acid and butyrate—restoring them to a physiologically beneficial range. This restoration not only counteracted constipation-associated deficits but also reached concentrations considered optimal for colonic health, including anti-inflammatory effects and the promotion of normal intestinal function.”

 

16. Discussion often repeats results rather than critically analyzing them.

Responses: We thank the reviewer for the insightful comments. According to the reviewer’s guide, this sentence has been revised as follow:

lines 404-407：“Furthermore, we also examined the expression of several inflammatory cytokines in the colon, and the results showed that the levels of TNF-α, IL-1β and iNOS in the constipated rats were significantly elevated, while PMTL treatment reversed this trend (p < 0.05, Figure 4E, F and ) was revised as: “The anti-constipate effect of PMTL appears to be mediated, through the modulation of inflammatory pathways. Treatment with PMTL significantly attenuated the constipation-associated upregulation of key pro-inflammatory mediators, including TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS) (p < 0.05, Figure 4E, F and G). The suppression of iNOS is of particular mechanistic importance, as it likely reduces the excessive production of nitric oxide-derived reactive nitrogen species, thereby alleviating nitrosative stress and its detrimental effects on colonic smooth muscle motility [46]. Concurrently, the reduction in TNF-α and IL-1β may contribute to the restoration of epithelial barrier integrity and normalization of mucosal water transport [47]. These findings suggest that the therapeutic efficacy of PMTL extends beyond mere laxation and includes the resolution of underlying mucosal inflammation, which represents a critical pathophysiological component of chronic constipation.”

lines 448-450：“In line with previous studies, our results indicated that diphenoxylate-induced constipation dramatically disturbed the gut microbiota composition of rats, whereas this dysbiosis could be improved by PMTL treatment [54].” was revised as: “Consistent with the established mechanisms of drug-induced intestinal ecological dysregulation, our research results indicate that diphenoxate disrupts the homeostasis of colonic microbiota, and this interference is significantly reversed by PMTL intervention. This recovery may selectively promote the recovery of symbiotic or beneficial taxa by providing fermentable substrates [54].”

 

Relevant references were added as follows:

46. Gao, X.; Hu, Y.; Tao, Y.; Liu, S.; Chen, H.; Li, J.; Zhao, Y.; Sheng, J.; Tian, Y.; Fan, Y. Cymbopogon citratus (DC.) Stapf Aqueous Extract Ameliorates Loperamide-Induced Constipation in Mice by Promoting Gastrointestinal Motility and Regulating the Gut Microbiota. Front. Microbiol. 2022, 13, 1017804.
47. Li X, Xiang Z, Wang X, He H, Xu M, Tan C, Wu X, Zhang J, Dong W. Metformin attenuates colitis via blocking STAT3 acetylation by reducing acetyl-CoA production. J Adv Res. 2025 Mar 31: S2090-1232(25)00218-8.

 

17. The non-linear dose response (10% > 20%) is acknowledged but not explained. Possible hypotheses (excessive fiber fermentation leading to gas, osmotic imbalance) should be considered.

Responses: Thanks for the reviewer’s patience and detailed comments which have enabled us to improve our manuscript. According to the reviewer’s suggestion, this sentence has been corrected as follow:

lines 471-472: “Our results showed that PMTL consumption could significantly increase the fecal SCFAs content in the constipated rat, especially the acetic acid and butyrate.” was revised as: “The non-linear dose-response relationship was observed, in which the 10% PMTL dose showed better efficacy than the 20% dose. This may result from excessive fermentable fiber intake, which can lead to adverse effects—including gas production, bloating, and microbial changes—that counteract the benefits of SCFAs, highlighting the importance of optimal rather than maximal dosing [68].”

 

Relevant references were added as follows:

68. Sasaki, H.; Masutomi, H.; Yamauchi, Y.; Ishihara, K.; Fukuda, S. Effectiveness of Personalized Granola Tailored to the Gut Microbiota for Improving Gut Environment and Mood States. Front. Microbiol. 2025, 16, 1607918.

 

 

18. The role of polyphenols in PMTL is neglected. These compounds may also contribute to microbiota modulation and anti-inflammatory effects.

Responses: We sincerely appreciate your valuable feedback. In response to your comments, we have expanded and strengthened the relevant section in the Discussion：

lines 407-409: “Based on these results, we speculated that PMTL could improve colonic morphology and physiological function of the constipated rats by reducing the expression of inflammatory cytokines.” was revised as: “Based on these results, we speculate that PMTL may improve colonic morphology and physiological function in constipated rats by reducing the expression of inflammatory cytokines. It should also be noted that polyphenols present in PMTL could contribute to these anti-inflammatory effects, suggesting a potential multi-component mechanism behind the observed therapeutic outcomes.”

 

lines 466-468: The presented results suggest that PMTL may alleviate constipation symptoms by increasing the abundance of beneficial bacteria and decreasing the abundance of pathogenic bacteria in the intestine. was revised as: “The obtained results indicate that PMTL alleviates constipation symptoms, potentially through enhancing the proliferation of beneficial intestinal bacteria while suppressing pathogenic bacterial populations. Additionally, the polyphenolic compounds in PMTL are also likely to participate in the regulation of gut microbiota, further supporting its prebiotic function.

 

19. Clinical translation is not fully addressed: How does rat dosage translate to human intake?

Responses: We appreciate you highlighting this issue. The correction has been made in the subsequent version of the manuscript as suggested：

lines 97-100: “After a week of adaptive feeding, all the rats were randomly divided into five groups (n = 10 per group): normal control (NC) group, diphenoxylate-induced constipation model (CM) group, 5% Premna microphylla turcz leaf(5% PMTL) group, 10% PMTL group and 20% PMTL group.” was revised as: “After a week of adaptive feeding, all rats were randomly assigned to five groups (n = 10 per group): normal control (NC), constipation model (CM) induced by diphenoxylate, and three intervention groups receiving PMTL at doses of 5%, 10%, and 20%. The PMTL doses were determined based on cross-species conversion using body surface area normalization to ensure physiologically relevant exposure in the rat model [31].”

 

Relevant references were added as follows:

31. Hughes, R.K.; Shiwani, H.; Rosmini, S.; Augusto, J.B.; Burke, L.; Jiang, Y.; Pierce, I.; Joy, G.; Castelletti, S.; Orini, M.; Kellman, P.; Xue, H.; Lopes, L.R.; Mohiddin, S.; Treibel, T.; Manisty, C.; Captur, G.; Davies, R.; Moon, J.C. Improved Diagnostic Criteria for Apical Hypertrophic Cardiomyopathy. JACC Cardiovasc Imaging 2024, 17, 501–512.

 

20. Conclusions are too strong given the preclinical (rat) model. Must highlight limitations before extrapolating to humans.

Responses: We thank the reviewer for their comments, the sentence has been revised as suggested:

lines 484-486: “These findings suggest that dietary PMTL has the potential to be developed as a functional food to alleviate constipation symptoms in the future.” was revised as: “These preclinical findings suggest that PMTL may hold promise as a functional dietary ingredient for the relief of constipation; however, further clinical studies are necessary to confirm its efficacy and safety in humans.”

 

21. Future directions should be proposed: clinical trials, synergistic use with probiotics, structural optimization of PMTL.

Responses: We extend our sincere gratitude to the reviewers for their insightful and constructive comments, which are greatly appreciated. We are in full agreement that the proposed directions represent essential next steps for advancing this research. In response to these recommendations, we have supplemented the conclusion section of the manuscript with the following statements: “To expand the study’s translational value and mechanistic depth, three future directions are proposed: (1) exploring potential synergistic effects of PMTL with probiotics or other prebiotics to enhance its regulatory impact on gut health; (2) investigating structural optimization of PMTL-derived polysaccharides to improve their bioavailability and functional properties; and (3) integrating metagenomic sequencing with PICRUSt2/KEGG analyses to systematically characterize the functional shifts of gut microbiota induced by PMTL, thereby clarifying the functional links between microbial community remodeling and PMTL’s physiological effects (e.g., SCFA production, neurotransmitter regulation).”

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript foods-3847498 reported that dietary Premna Microphylla Turcz leaf consumption alleviates functional constipation via regulating gut microbiota and the aquaporin transport system in rats. 
Previous investigations have reported that Premna Microphylla Turcz leaf components, such as polysaccharides and phenolics, relieve inflammation-related symptoms. In particular, the protective effect of pectin extracted from Premna microphylla Turcz leaves has been investigated against gut microbiota dysbiosis. 
Some previous publications examples:
Guanglei Song, Fangyuan Chen, Shubo Chen, Shuhui Ye, Polysaccharides from Premna microphylla turcz ameliorate inflammation via the enhancement of intestinal resistance in host, Journal of Ethnopharmacology, Volume 276, 2021, 114208, ISSN 0378-8741, https://doi.org/10.1016/j.jep.2021.114208. 
Xiao Li, Zeliang Wei, Xingyue Wang, Feixia Duan, Lidan Xiong, Jingwen Li, Jing Tian, Lirong Jia, Hong Gao, Premna microphylla Turcz leaf pectin exhibited antioxidant and anti-inflammatory activities in LPS-stimulated RAW 264.7 macrophages, Food Chemistry, Volume 349, 2021, 129164, ISSN 0308-8146, https://doi.org/10.1016/j.foodchem.2021.129164.
Gao J, Zhang M, Zhang L, Wang N, Zhao Y, Ren D, Yang X. Dietary Pectin from Premna microphylla Turcz Leaves Prevents Obesity by Regulating Gut Microbiota and Lipid Metabolism in Mice Fed High-Fat Diet. Foods. 2024 Jul 17;13(14):2248. doi: 10.3390/foods13142248. PMID: 39063332; PMCID: PMC11275460.
In this regard, the present manuscript does not offer new essential comprehension into this research subject.
 In addition, the manuscript presents low-quality figures and a high percent match (40%) in iThenticate report.

Author Response

Dear reviewer,

We are very pleased to receive your letter with the reviewer’s comments for our manuscript “foods-3847498”. We thank the reviewers for thoroughly reviewing our manuscript and making thoughtful comments. The manuscript has been revised carefully, and the detailed corrections requested by the reviewers have been done and listed below point by point.

 

1. Previous investigations have reported that Premna Microphylla Turcz leaf components, such as polysaccharides and phenolics, relieve inflammation-related symptoms. In particular, the protective effect of pectin extracted from Premna microphylla Turcz leaves has been investigated against gut microbiota dysbiosis. In this regard, the present manuscript does not offer new essential comprehension into this research subject.

Response: We are grateful to the reviewer for these insightful comments and for providing the highly relevant references. We acknowledge the significant contributions of previous studies demonstrating the anti-inflammatory and microbiota-modulatory effects of purified polysaccharides and pectin from PMTL. Our study was designed to extend this existing knowledge by addressing several critical and novel aspects:

Translation from Extract to Whole-Leaf Dietary Application: While the valuable cited studies focused on isolated compounds (e.g., purified polysaccharides or pectin), our research investigates the physiological effects of incorporating whole PMTL leaf powder into the diet itself. This approach models a direct and practical use of PMTL as a functional food ingredient, allowing us to evaluate the synergistic effects of its complete phytochemical matrix (e.g., polysaccharides, polyphenols, fiber, minerals) rather than a single component. This provides essential translational data for its potential use in functional foods.

Novelty in Mechanism within a Constipation-Specific Model: The previously published work primarily explored general anti-inflammatory mechanisms or metabolic disorders. Our study uniquely elucidates the laxative effects and mechanisms in a diphenoxylate-induced constipation model, a pathophysiological context not previously investigated for PMTL. We provide original evidence on how dietary PMTL restores gut function not only by modulating gut microbiota but also by specifically regulating aquaporin water channels (AQP3, AQP4) and key gut neurotransmitters, offering a distinct and more comprehensive mechanistic pathway.

Discovery of a Non-linear Dose-Response Relationship: A pivotal and novel finding of our work is the identification of a non-linear dose-response relationship, where the 10% PMTL dose showed superior efficacy compared to both lower (5%) and higher (20%) doses. This moves beyond the conventional dose-dependent paradigm and highlights the critical importance of identifying an optimal, rather than maximal, dosage for dietary interventions, a significant new concept for PMTL application.

 

2. In addition, the manuscript presents low-quality figures and a high percent match (40%) in iThenticate report.

Response: We sincerely apologize for the oversight regarding the elevated similarity score. This issue was primarily attributable to insufficient paraphrasing in the methodological sections and the use of conventional phrasing in the introduction. We have comprehensively revised the manuscript to improve textual originality and ensure adequate paraphrasing throughout. The similarity index has now been successfully reduced and falls well within the acceptable range stipulated by the journal.

In addition, all figures have been replaced with higher-resolution versions in the updated manuscript to enhance clarity and visual accuracy.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Introduction. Could the authors mention if there are antinutrients in the leaves of Premna microphylla turcz (PMTL)?

Metodology. It is recommended to check the international system of units (min).

In the analysis of polyphenols and flavonoids, the methodology must be detailed. It is only mentioned that, using a colorimetric method, the result obtained is not adequate to simply state mg/g.

In Table 2, it is recommended to check the units, since the total fat and protein values ​​should be percentages (%). Furthermore, the results assume that soluble polysaccharides are pectin or dietary fiber, which is not true; dietary fiber can be soluble or insoluble. The amounts of these components in the leaves must be determined individually.

Results. Improve the clarity of figures (1, 2B, 3, 4, 5, and 6)

References. Review the format and number of references.

Author Response

Dear reviewer,

We are very pleased to receive your letter with the reviewer’s comments for our manuscript “foods-3847498”. We thank the reviewers for thoroughly reviewing our manuscript and making thoughtful comments. The manuscript has been revised carefully, and the detailed corrections requested by the reviewers have been done and listed below point by point.

 

1. Introduction. Could the authors mention if there are antinutrients in the leaves of Premna microphylla turcz (PMTL)?

Responses: We sincerely appreciate your valuable suggestion. In accordance with your comment, the relevant references have been revised as follows:

lines 48-50: “PMTL contains a large number of pectins, crude fibers, carbohydrates, plant proteins, amino acids, minerals, and polyphenols [18-20], which is cultivated on a large scale as a momentous commercial crop due to its multifarious applications and benefits [21-22].” was revised as: “PMTL is cultivated on a large scale as a commercially significant crop due to its multifarious applications and nutritional benefits [21–22]. It contains abundant pectins, crude fibers, carbohydrates, plant proteins, amino acids, minerals, and polyphenols [18–20]. Although the potential presence of antinutritional factors—such as phytates, tannins, or oxalates, which may interfere with nutrient absorption—was considered, available phytochemical analyses indicate that the major bioactive constituents in PMTL are predominantly associated with beneficial health effects [23].”

 

Relevant references were added as follows:

23. Gao, J.; Zhang, M.; Zhang, L.; Wang, N.; Zhao, Y.; Ren, D.; Yang, X. Dietary Pectin from Premna microphylla Turcz Leaves Prevents Obesity by Regulating Gut Microbiota and Lipid Metabolism in Mice Fed High-Fat Diet. Foods2024, 13, 2248.

 

2. Metodology. It is recommended to check the international system of units (min).

Responses: We sincerely appreciate your valuable suggestion. In accordance with your comment, the relevant references have been revised as follows:

lines 108-109: “After 9 weeks, all the rats were sacrificed, and their bloods were collected and centrifuged at 4 °C, 10000 g for 15 minutes to obtain the serum samples and then placed in a -80 °C refrigerator.” was revised as: “After 9 weeks, all the rats were sacrificed, and their bloods were collected and centrifuged at 4 °C, 10000 g for 15 min to obtain the serum samples and then placed in a -80 °C refrigerator.”

 

lines 122-123: “After they were fasted for 12 h, all the rats were oral administrated with 1 mL carmine, and then 30 minutes later, ...” was revised as: “After they were fasted for 12 h, all the rats were oral administrated with 1 mL carmine, and then 30 min later, …”

 

lines 153-154: “After that, the loading buffer was added into each sample and boiled at 100 °C for 10 minutes to denature the proteins, …was revised as: “After that, the loading buffer was added into each sample and boiled at 100 °C for 10 min to denature the proteins …”

 

3. In the analysis of polyphenols and flavonoids, the methodology must be detailed. It is only mentioned that, using a colorimetric method, the result obtained is not adequate to simply state mg/g.

Responses: Thanks for the reviewer’s patience and detailed comments which have enabled us to improve our manuscript. According to the reviewer’s suggestion, this sentence has been corrected as follow:

lines 77-82: “In particular, contents of total flavonoids and total phenolics in PMTL were assayed by colorimetric method. The total soluble polysaccharides content in PMTL were measured by phenol sulfuric acid method. Gallic acid, rutin and glucose were used as standards, and the results were expressed as their equivalents (mg/g). The protein content was evaluated by Kjeldahl nitrogen method. The crude fat in PMTL was extracted with petroleum ether and determined using a Soxhlet apparatus.” was revised as: “Specifically, the total phenolic content was assayed according to the Folin–Ciocalteu method, with gallic acid as the standard. The results were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g). Similarly, the total flavonoid content was quantified using an aluminum chloride colorimetric assay, with rutin as the standard, and expressed as milligrams of rutin equivalents per gram of dry weight (mg RE/g). The total soluble polysaccharides were measured by the phenol–sulfuric acid method using glucose as the standard, and the results are given as milligrams of glucose equivalents per gram (mg GE/g). Protein content was determined using the Kjeldahl method and converted to crude protein content using a factor of 6.25. Crude fat was extracted with petroleum ether (boiling point 60–90 °C) in a Soxhlet extractor for 6 h and reported as percent fat relative to the dry weight of the sample.”

 

4. In Table 2, it is recommended to check the units, since the total fat and protein values should be percentages (%). Furthermore, the results assume that soluble polysaccharides are pectin or dietary fiber, which is not true; dietary fiber can be soluble or insoluble. The amounts of these components in the leaves must be determined individually.

Responses: We sincerely thank the reviewer for these critical comments. In response, we revised the relevant descriptions in the manuscript to more accurately reflect that soluble polysaccharides represent a component of dietary fiber rather than being synonymous with a broader category.

Furthermore, we have carefully checked and corrected the units for total fats and total soluble proteins in Table 2, converting the values to percentages (% w/w) as appropriate for these components. All necessary corrections have been implemented throughout the manuscript to ensure both conceptual and numerical precision.

lines 200-204: “By measuring the chemical composition of Premna microphylla turcz leaf (PMTL) as dried powder, it was found that the total polyphenols, total flavonoids, total soluble polysaccharides, total soluble proteins and total fats in PMTL were 116.8 ± 1.3 mg/g, 150.6 ± 4.1 mg/g, 459.9 ± 3.2 mg/g, 41.3 ± 3.7 and 41.6 ± 1.9 mg/g, respectively (Table 2), indicating that PMTL mainly rich in the polysaccharides or pectin.” was revised as: “By measuring the chemical composition of Premna microphylla Turcz leaf (PMTL) in dried powder form, it was determined that the contents of total polyphenols, total flavonoids, total soluble polysaccharides, total soluble proteins, and total fats in PMTL were 116.8 ± 1.3 mg GAE/g dw, 150.6 ± 4.1 mg RE/g dw, 459.9 ± 3.2 mg GE/g dw, 41.3 ± 0.37% (w/w), and 41.6 ± 0.19% (w/w), respectively (Table 2), indicating that PMTL is particularly rich in soluble polysaccharides, which are a major component of soluble dietary fiber and may include compounds such as pectin.”

Table 2. Composition analysis of total polyphenols, total flavonoids, total soluble polysaccharides, total soluble proteins and total fats in Premna microphylla turcz leaves (PMTL).

Assigned identity	Regression curve	Content (mean ± SD)
Total polyphenols	y = 38.1x + 0.0502	116.8 ± 1.3 mg GAE/g dw
Total flavonoids	y = 6.718x - 0.2861	150.6 ± 4.1 mg RE/g dw
Total soluble polysaccharides	y = 2.3629x + 0.0405	459.9 ± 3.2 mg GE/g dw
Total soluble proteins		41.3 ± 0.37 % (w/w)
Total fats		41.6 ± 0.19 % (w/w)

 

5. Results. Improve the clarity of figures (1, 2B, 3, 4, 5, and 6)

Responses: We sincerely thank the reviewer for the helpful suggestion. In response, we have replaced all mentioned figures (Figures 1, 2B, 3, 4, 5, and 6) with higher-resolution versions to improve clarity and readability. The captions and in-text citations remain unchanged.

 

6. References. Review the format and number of references.

Responses: We sincerely thank the reviewer for this valuable comment. We have carefully reviewed and unified the reference style throughout the manuscript according to the journal’s guidelines. All citations and corresponding entries in the reference list have been double-checked for correctness and consistency. The manuscript has been thoroughly updated to reflect these changes, and we have performed a final check to ensure the accuracy and completeness of all references in the newly uploaded version.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been substantially improved. It must be accepted in its present form.

Author Response

Dear Reviewer,

We are very pleased to receive your letter with the reviewer’s comments for our manuscript “foods-3847498”. We sincerely appreciate the time and effort expended by the reviewers in evaluating our work.

 

The manuscript has been substantially improved. It must be accepted in its present form.

Responses: We would like to express our sincerest gratitude for your time and for the positive assessment of our revised manuscript. We are truly delighted to hear that you find the manuscript "substantially improved" and recommend its acceptance in its present form. Your insightful comments throughout the review process were invaluable in helping us enhance the quality of our work.

Thank you once again for your constructive guidance.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

-The manuscript still presents low-quality figures.

-A representative picture of Premna microphylla turcz leaf would be attractive.

-Lines 88-90: ‘The total soluble polysaccharides were measured by the phenol–sulfuric acid method using glucose as the standard, and the results are given as milligrams of glucose equivalents per gram (mg GE/g)’

The phenol–sulfuric acid method measures the total carbohydrate content in a sample, but it does not allow for distinguishing between mono-, oligo-, and polysaccharides. In this regard, the results must be reported as total carbohydrate content instead of total soluble polysaccharides.

-Lines 96-99: The specific characteristics of the regular animal feed used must be described.

- Are 5, 10, and 20 % PMTL in g PMTL/100 g regular animal feed?

-Line 118: 1 mL of diphenoxylate solution (5 mg/kg·bw). Describe bw.

-It is necessary to revise Table 2 as the calculus seems incorrect:

Total soluble polysaccharides 459.9 ± 3.2 mg GE/g dw, which means 0.4599 g GE/g dw and corresponds to 45.99 g GE/ 100 g dw.

Total polyphenols 116.8 ± 1.3 mg GAE/g dw, corresponding to 11.68 g GAE/100 g dw

Total flavonoids 150.6 ± 4.1 mg RE/g dw corresponding to 15.06 g RE/100 g dw

Total soluble proteins 41.3 ± 0.37 % (w/w) corresponding to 41.3 g/100 g dw

Total fats 41.6 ± 0.19 % (w/w) corresponding to 41.6 g/100 g dw

In Table 2, the total composition of PMTL would be over 100%.

-In Table 2: 'Assigned identity' could be replaced by 'Total Component', and the word ‘Total’ could be eliminated for each component.  For each regression curve, R² (the coefficient of determination) must be included.

-It is necessary to improve the Conclusions section. It must detail the most critical findings.

Author Response

Dear reviewer,

We are very pleased to receive your letter with the reviewer’s comments for our manuscript “foods-3847498”. We extend our sincere appreciation to the reviewers for their thoughtful and thorough review. All suggested revisions have been incorporated into the manuscript, and we address each comment in detail below.

 

1. The manuscript still presents low-quality figures.

Responses: We thank the reviewer for pointing out the issue with the figure quality. We sincerely apologize for this oversight. In response, we have thoroughly reviewed and replaced all figures in the manuscript. The new figures have been created with higher resolution, improved clarity, and a more professional layout to ensure they meet the journal's standards. We believe these revisions have significantly enhanced the quality of the manuscript.

 

2. A representative picture of Premna microphylla turcz leaf would be attractive.

Responses: We are grateful to the reviewer for the valuable suggestion to include a representative plant image. In response, we have added a new high-quality photograph showing the morphological characteristics of a fresh Premna microphylla Turcz leaf (Figure 1A). We believe this addition will provide readers with a clear visual reference and strengthen the manuscript.

 

3. Lines 88-90: ‘The total soluble polysaccharides were measured by the phenol–sulfuric acid method using glucose as the standard, and the results are given as milligrams of glucose equivalents per gram (mg GE/g)’

The phenol–sulfuric acid method measures the total carbohydrate content in a sample, but it does not allow for distinguishing between mono-, oligo-, and polysaccharides. In this regard, the results must be reported as total carbohydrate content instead of total soluble polysaccharides.

Responses: We thank the reviewer for their comments, the sentence has been revised as suggested:

Lines 88-90: “The total soluble polysaccharides were measured by the phenol–sulfuric acid method using glucose as the standard, and the results are given as milligrams of glucose equivalents per gram (mg GE/g)” was revised as:“Total carbohydrates were measured by the phenol–sulfuric acid method using glucose as the standard, and the results are given as milligrams of glucose equivalents per gram (mg GE/g).”

 

4. Lines 96-99: The specific characteristics of the regular animal feed used must be described.

- Are 5, 10, and 20 % PMTL in g PMTL/100 g regular animal feed?

Responses: We thank the reviewer for the insightful comments. According to the reviewer’s guide, this sentence has been revised as follow:

Lines 96-99: “Specifically, different grams of PMTL were added in the regular animal feed (corn, wheat, fish meal, chicken meal, soybean meal, soybean oil, amino acids, vitamins, and minerals complex, Xietong Feed Factory) to make experimental rat chow containing 5%, 10% and 20% PMTL.” was revised as: “The experimental diets were prepared by thoroughly mixing powdered PMTL with the standard rodent chow at concentrations of 5%, 10%, and 20% (w/w, g PMTL/100 g feed). The standard chow (provided by Xietong Feed Factory) contained a balanced mix of corn, wheat, fish meal, chicken meal, soybean meal, soybean oil, amino acids, vitamins, and minerals, with approximate nutritional values of 20% crude protein, 5% crude fat, and 55% carbohydrates.

 

5. Line 118: 1 mL of diphenoxylate solution (5 mg/kg·bw). Describe bw.

Responses: We thank the reviewer for their comments, the sentence has been revised as suggested:

Line 118: “Briefly, rats in the NC group received 1 mL of normal saline, whereas those in the CM, 5% PMTL, 10% PMTL, and 20% PMTL groups were administered 1 mL of diphenoxylate solution (5 mg/kg·bw). was revised as:“Briefly, rats in the NC group received 1 mL of normal saline, whereas those in the CM, 5% PMTL, 10% PMTL, and 20% PMTL groups were administered 1 mL of diphenoxylate solution (5 mg/kg body weight, bw).”

 

6. It is necessary to revise Table 2 as the calculus seems incorrect:

Total soluble polysaccharides 459.9 ± 3.2 mg GE/g dw, which means 0.4599 g GE/g dw and corresponds to 45.99 g GE/ 100 g dw. 

Total polyphenols 116.8 ± 1.3 mg GAE/g dw, corresponding to 11.68 g GAE/100 g dw

Total flavonoids 150.6 ± 4.1 mg RE/g dw corresponding to 15.06 g RE/100 g dw

Total soluble proteins 41.3 ± 0.37 % (w/w) corresponding to 41.3 g/100 g dw

Total fats 41.6 ± 0.19 % (w/w) corresponding to 41.6 g/100 g dw

In Table 2, the total composition of PMTL would be over 100%.

In Table 2: 'Assigned identity' could be replaced by 'Total Component', and the word ‘Total’ could be eliminated for each component.  For each regression curve, R² (the coefficient of determination) must be included.

Responses: We thank the reviewer for this important comment. We agree that the components should not be summed as they are not mutually exclusive. To prevent any misunderstanding, we have clarified in the revised manuscript that bioactive compounds (e.g., polyphenols and flavonoids) are secondary metabolites contained within the plant's nutritional matrix (primarily the carbohydrate fraction). This explanation has been added as a note to Table 2. We believe this effectively resolves the ambiguity.

Table 2. Composition analysis of polyphenols, flavonoids, carbohydrates, soluble proteins and fats in Premna microphylla turcz leaves (PMTL).

Total Component	Regression curve	R2	Content (mean ± SD)
polyphenols	y = 38.1x ＋ 0.0502	0.9992	116.8 ± 1.3 mg GAE/g dw
flavonoids	y = 6.718x － 0.2861	0.9985	150.6 ± 4.1 mg RE/g dw
carbohydrates	y = 2.3629x ＋ 0.0405	0.9996	459.9 ± 3.2 mg GE/g dw
Soluble proteins			41.3 ± 0.37 % (w/w)
fats			41.6 ± 0.19 % (w/w)

Note: The components listed are not mutually exclusive mass fractions. Bioactive compounds (polyphenols and flavonoids) are secondary metabolites biosynthesized within the plant matrix and are contained within the mass of the nutritional components (e.g., carbohydrates).

 

7. It is necessary to improve the Conclusions section. It must detail the most critical findings.

Responses: We are grateful for the reviewers' insightful comments. In accordance with your suggestions, the conclusion section has been revised as follows:

In conclusion, this study demonstrates that PMTL effectively alleviates diphenoxylate-induced constipation in rats through multi-faceted mechanisms. The most critical findings include the significant enhancement of intestinal motility, restoration of gut barrier integrity, and a notable reshaping of the gut microbiota composition, which collectively led to favorable changes in associated metabolites and neurotransmitters.

To enhance the translational value of this study, future work should: (1) investigate synergistic effects between PMTL and probiotics/prebiotics; (2) optimize the structure of PMTL-derived polysaccharides to improve their bioavailability; and (3) apply metagenomic sequencing with PICRUSt2/KEGG analysis to functionally link gut microbiota remodeling to PMTL's physiological effects.

 

Author Response File: Author Response.docx