Anti-Inflammatory and Antioxidant Properties of Bauhinia thailandica Leaf Extract in Microglial Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study explores the phytochemical makeup and neuroprotective effects of Bauhinia thailandica leaf extract in LPS-activated BV2 microglial cells. It shows a reduction in ROS generation, nitric oxide, and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), indicating antioxidant and anti-neuroinflammatory properties. The research emphasizes the potential of this lesser-known plant in controlling microglia-driven inflammation. Nonetheless, the extract's molecular characterization is limited, signaling pathways are not fully elucidated, pharmacological effectiveness is not measured, and the translational significance remains early-stage. Significant revisions are necessary before it can be published.

Major revisions are required.

 

Comment 1. I recommend rewriting the introduction to include a paragraph that explains the importance of flavonoids in biomedical applications as background information.

Comment 2. The introduction broadly describes oxidative stress and microglial activation but does not clearly position this study within recent advances in plant-derived modulators of microglial signaling pathways. Expand the discussion to include contemporary reports linking phytochemicals with NF-κB, Nrf2, and MAPK modulation in microglial systems. This will anchor the novelty of the work within current neuroinflammation research. (See and incorporate: Int. J. Mol. Sci. 2025, 26(15), 7381; 10.3390/ijms26157381)

Comment 3. You propose that polyphenols and flavonoids contribute to antioxidant activity; however, the introduction does not explain how specific structural classes (e.g., tannins vs flavonoids vs cardiac glycosides) may differentially influence inflammatory cascades. Provide a clearer mechanistic rationale for selecting Bauhinia thailandica based on bioactive class–function relationships.

Comment 4. The unusually high flavonoid value relative to total phenolics requires explanation. Re-express concentrations per gram of dry extract to avoid ambiguity.

Comment 5. The study relies entirely on crude ethanolic leaf extract, yet the phytochemical characterization remains limited to qualitative screening and total phenolic/flavonoid/tannin estimation (Table 1–2). This level of chemical definition is insufficient to support mechanistic biological claims. Perform LC–MS/MS profiling or HPLC fingerprinting to identify dominant constituents and standardize the extract. Without compositional resolution, reproducibility and mechanistic attribution remain uncertain.

Comment 6. The antioxidant claim is based on intracellular ROS reduction using LPS stimulation. Clarify whether the extract acts as a direct radical scavenger or modulates endogenous antioxidant systems. Measure SOD, catalase, GPx activity, or Nrf2/Keap1 expression levels. Without assessing endogenous redox machinery, the antioxidant mechanism remains incomplete.

Comment 7. NO suppression is central to the anti-inflammatory claim. Indicate whether the Griess reagent assay was calibrated with sodium nitrite standards and report exact nitrite concentrations (µM), not only relative changes. This will enhance the pharmacological interpretation.

 

Comment 8. Several sentences are too long, which lowers readability. Make the complex sentences more concise, remove informal transitions, and adjust verb tenses to improve scientific accuracy throughout the manuscript.

 

Looking forward to reviewing the revised version.

End of the report!

Author Response

Reviewer 1

Comments and Suggestions for Authors

This study explores the phytochemical makeup and neuroprotective effects of Bauhinia thailandica leaf extract in LPS-activated BV2 microglial cells. It shows a reduction in ROS generation, nitric oxide, and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), indicating antioxidant and anti-neuroinflammatory properties. The research emphasizes the potential of this lesser-known plant in controlling microglia-driven inflammation. Nonetheless, the extract's molecular characterization is limited, signaling pathways are not fully elucidated, pharmacological effectiveness is not measured, and the translational significance remains early-stage. Significant revisions are necessary before it can be published.

Response

Thank you for the reviewer’s thoughtful evaluation of our manuscript and for highlighting both its strengths and limitations. We appreciate the constructive feedback and the recognition of the potential significance of Bauhinia thailandica leaf extract in modulating microglia-mediated neuroinflammatory responses.

We acknowledge the points raised regarding the limited molecular characterization of the extract, the need for deeper investigation of the signaling pathways involved, and the early-stage translational relevance of the findings. These comments are valuable for improving the manuscript's clarity and scientific rigor.

We have revised the manuscript to address your concerns and have included detailed responses to each of your comments. The related changes and clarifications are now reflected throughout the updated manuscript.

Major revisions are required.

Comment 1. I recommend rewriting the introduction to include a paragraph that explains the importance of flavonoids in biomedical applications as background information.

Response 1: We sincerely thank the reviewer for this valuable suggestion. The revised text has been incorporated into the Introduction as

“These compounds are useful because they neutralize free radicals, modulate inflammatory pathways, reduce microglial activation, and improve cellular antioxidant defense systems [24]. Importantly, different groups of phytochemicals have unique structures that shape their biological effects. Flavonoids possess a polyphenolic backbone with specific hydroxyl groups that contribute to their strong antioxidant properties. These compounds are known to scavenge reactive oxygen species (ROS) and regulate key inflammatory signaling pathways, including nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response [25,26]. Tannins are another type of large polyphenols. They have strong antioxidant effects and can lower the production of inflammatory enzymes and cytokines. [27]. Other bioactive groups, such as alkaloids and terpenoids, affect signaling pathways involved in immune responses and oxidative stress [28]. Understanding these bioactive class/function correlations provides a molecular basis for selecting plant species with potential neuroprotective properties.”(Red font, Page 2, line 72-85).

We appreciate the reviewer’s constructive comment, which has helped improve the clarity and completeness of our manuscript.

Comment 2. The introduction broadly describes oxidative stress and microglial activation but does not clearly position this study within recent advances in plant-derived modulators of microglial signaling pathways. Expand the discussion to include contemporary reports linking phytochemicals with NF-κB, Nrf2, and MAPK modulation in microglial systems. This will anchor the novelty of the work within current neuroinflammation research. (See and incorporate: Int. J. Mol. Sci. 2025, 26(15), 7381; 10.3390/ijms26157381)

Response 2: We appreciate this valuable suggestion. In response to the reviewer’s recommendation, we have revised the Introduction to more clearly emphasize recent advances in plant-derived phytochemicals that modulate microglial signaling pathways, including NF-κB, MAPK, and Nrf2-related mechanisms in neuroinflammation. We have incorporated relevant contemporary studies to strengthen the background and clarify the novelty of the present work. The revised text is highlighted in red in the revised manuscript. (Page 2, line 77-80). As below.

“These compounds are known to scavenge reactive oxygen species (ROS) and regu- late key inflammatory signaling pathways, including nuclear factor kappa B (NF-κB), mi- togen-activated protein kinase (MAPK), and the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response [25,26].”

We carefully examined the suggested reference (Int. J. Mol. Sci. 2025, 26(15), 7381). However, the primary focus of this review is on anticancer mechanisms and nanoformulation-based delivery of isorhamnetin, which is not directly related to microglial activation or neuroinflammatory signaling pathways addressed in our study. Consequently, we did not incorporate this reference.

Comment 3. You propose that polyphenols and flavonoids contribute to antioxidant activity; however, the introduction does not explain how specific structural classes (e.g., tannins vs flavonoids vs cardiac glycosides) may differentially influence inflammatory cascades. Provide a clearer mechanistic rationale for selecting Bauhinia thailandica based on bioactive class–function relationships.

Response 3: We sincerely thank the reviewer for this insightful and constructive comment. We agree that a clearer mechanistic explanation linking specific phytochemical classes to their biological functions would strengthen the scientific rationale for selecting Bauhinia thailandica. In response, we have revised the introduction to better distinguish the functional roles of major phytochemical classes present in Bauhinia thailandica. (Red font, Page 2, line 74-85; page 3, line 86-109) As below.

“Importantly, different groups of phytochemicals have unique structures that shape their biological effects. Flavonoids possess a polyphenolic backbone with specific hydroxyl groups that contribute to their strong antioxidant prop- erties. These compounds are known to scavenge reactive oxygen species (ROS) and regu- late key inflammatory signaling pathways, including nuclear factor kappa B (NF-κB), mi- togen-activated protein kinase (MAPK), and the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response [25,26]. Tannins are another type of large polyphenols. They have strong antioxidant effects and can lower the production of inflammatory enzymes and cytokines. [27]. Other bioactive groups, such as alkaloids and terpenoids, affect sig- naling pathways involved in immune responses and oxidative stress [28]. Understanding these bioactive class/function correlations provides a molecular basis for selecting plant species with potential neuroprotective properties.

Bauhinia L. is a genus of the family Leguminosae (Fabaceae) and currently classified under the subfamily Cercidoideae according to modern phylogeny-based classification of legumes [29]. In the present study, we follow the currently accepted classification and consistently treat Bauhinia as a member of Cercidoideae. Species of Bauhinia are widely distributed in tropical and subtropical regions and are valued as ornamental, medicinal, and ethnobotanically important plants [29]. Previous phytochemical studies have re- ported that Bauhinia species contain diverse secondary metabolites, including phenolics, flavonoids, tannins, terpenoids, steroids, alkaloids, quinones, lactones, and aromatic acids. These phytochemicals are believed to contribute to their antioxidant, anti-inflammatory [30], and other pharmacological activities [31]. The documented abundance of flavonoids and phenolic compounds in this genus supports its potential relevance in modulating ox- idative stress and inflammatory signaling. Bauhinia thailandica (Siao-Phum) is a newly identified endemic species from Thai- land, classified within the subfamily Cercidoideae of the Leguminosae family [32]. It was first discovered in 2018. Phu Pha Yon National Park in Sakon Nakhon Province, Thailand, is currently the only known habitat for this species. It grows in dry deciduous forests and along roadsides in dry evergreen forests at elevations of 300 to 600 meters. Several species within the genus Bauhinia have been reported to contain bioactive compounds with anti- oxidant and anti-inflammatory properties. In contrast, Bauhinia thailandica is a newly de- scribed species, and information regarding its phytochemical composition and biological activities remains limited. Considering the phytochemical diversity observed in related Bauhinia species, especially the presence of flavonoids and phenolic compounds with known anti-inflammatory and antioxidant effects, Bauhinia thailandica is a promising can- didate for research into microglia-mediated neuroinflammation”

Comment 4. The unusually high flavonoid value relative to total phenolics requires explanation. Re-express concentrations per gram of dry extract to avoid ambiguity.

Response 4: Thank you for this helpful comment. In the revised manuscript, we reconstructed the standard curves for gallic acid and quercetin, revalidated the regression equations, and recalculated all values accordingly. In addition, TPC and TFC are now consistently expressed per gram of dry extract. The apparently higher TFC than TPC is attributable to differences in assay principles and calibration standards (GAE vs. QE), and therefore, these values are not directly comparable on a one-to-one basis. The manuscript has been revised to clarify both the unit expression and the interpretation of these data.

(Red font, Page 2, line 74-805; page 3, line 86-109) as below.

“The quantitative analysis of major phytochemical constituents in the ethanolic leaf extract of Bauhinia thailandica is presented in Table 2. The extract contained 70.55 ± 0.12 mg GAE/g dry extract of total phenolics, 249.47 ± 0.26 mg QE/g dry extract of total flavonoids, and 397.50 ± 0.00 mg TAE/g dry extract of total tannins. Among these, tannins were present at the highest level, followed by flavonoids and phenolics, indicating that the extract is particularly rich in polyphenolic constituents. “

Table 2. Total phenolic, flavonoid, and tannin contents of the ethanolic leaf extract of Bauhinia thailandica.

Parameter analyzed	Values obtained
Total phenolic content (mg GAE/g dry extract) Total flavonoid content (mg QE/g dry extract) Total tannin content (mg TAE/g dry extract)	70.55 ± 0.12 249.47 ± 0.26 397.50 ± 0.00

Notes: GAE = gallic acid equivalent; QE = quercetin equivalent; TAE = tannic acid equivalent. Values are presented as mean ± SD.

 

Comment 5. The study relies entirely on crude ethanolic leaf extract, yet the phytochemical characterization remains limited to qualitative screening and estimation of total phenolics, flavonoids, and tannins (Table 1–2). This level of chemical definition is insufficient to support mechanistic biological claims. Perform LC–MS/MS profiling or HPLC fingerprinting to identify dominant constituents and standardize the extract. Without compositional resolution, reproducibility and mechanistic attribution remain uncertain.

Response 5: We thank the reviewer for highlighting the importance of comprehensive phytochemical characterization. The principal novelty of our study is the investigation of a newly identified plant species that has not been previously examined in pharmacological or biological contexts. To our knowledge, this is the first report demonstrating its biological activity, specifically its capacity to reduce intracellular ROS levels and suppress pro-inflammatory cytokine production in activated microglia.

These findings provide the first experimental evidence that this plant extract has neuroprotective and anti-neuroinflammatory effects. Since there is no prior phytochemical or bioactivity data available for this species, our current objective was to establish basic biological activity and therapeutic potential. We believe this makes an important initial contribution to the field.

We agree that thorough phytochemical profiling would improve mechanistic interpretation. In our ongoing and future work, we plan to perform HPLC fingerprinting to standardize the extract, conduct LC–MS/MS analysis to identify major bioactive compounds, and explore the molecular mechanisms behind ROS reduction and cytokine suppression in microglial signaling pathways.

Therefore, the present study serves as an essential first step, providing biological validation and rationale for deeper mechanistic and chemical investigations.

We have written this point in the last paragraph of the discussion and in the conclusion of the revised manuscript. (Red font, Page 10, line 343-352; page 14, line 508-513)

 

“Taken together, the presence of phenolics, flavonoids, tannins, saponins, and cardiac glycosides in Bauhinia thailandica leaf extract may contribute to the antioxidant and anti-inflammatory activities observed in LPS-activated BV2 microglial cells. These phytochemical constituents have been reported to regulate oxidative stress and inflammatory responses through pathways associated with microglial activation, including NF-κB, MAPK, and the Nrf2-mediated antioxidant defense system [25–26]. Such mechanisms may partially explain the reductions in intracellular ROS, NO, and pro-inflammatory cytokines observed in this study. Therefore, the inhibitory effects of Bauhinia thailandica leaf extract on microglial activation may be attributed, at least in part, to the combined actions of these bioactive phytochemicals.

Further research is necessary to identify and characterize the active constituents of Bauhinia thailandica leaf extract using chromatographic profiling techniques such as high-performance liquid chromatography (HPLC). Additionally, studies should elucidate the molecular mechanisms responsible for its neuroprotective effects. Future investigations are encouraged to determine whether the extract modulates key signaling pathways associated with microglial activation and neuroinflammation, including the NF-κB, MAPK, and Nrf2/HO-1 pathways. These studies would enhance understanding of the mechanistic basis for the observed antioxidant and anti-inflammatory activities and could support the development of Bauhinia thailandica as a potential source of therapeutic agents for neuroinflammatory and neurodegenerative disorders.”

5. Conclusions

“The present study presents the first evidence that Bauhinia thailandica leaf extract exhibits substantial antioxidant and anti-inflammatory properties in LPS-stimulated BV2 microglial cells. The extract did not exhibit any cytotoxicity within the concentration range that was tested. It substantially diminished the production of intracellular ROS and NO and the release of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), which are critical mediators of neuroinflammation driven by microglia. The high levels of phytochemicals in the Bauhinia thailandica leaf extract, which include phenolics, flavonoids, tannins, cardiac glycosides, and saponins, are likely the cause of the observed biological activities. The present findings highlight the therapeutic potential of the leaf extract from Bauhinia thailandica as a natural source of bioactive compounds for the prevention of neuroinflammation. However, further research is required to isolate and characterize the individual active components and to clarify their specific molecular targets and signaling pathways. Phytochemical characterization through LC–MS/MS profiling or HPLC fingerprinting would specifically aid in identifying the predominant constituents and assist in the standardization of the extract. Furthermore, validation in in vivo models of neuroinflammatory and neurodegenerative disorders is necessary to establish its potential translational relevance”.

 

Comment 6. The antioxidant claim is based on intracellular ROS reduction using LPS stimulation. Clarify whether the extract acts as a direct radical scavenger or modulates endogenous antioxidant systems. Measure SOD, catalase, GPx activity, or Nrf2/Keap1 expression levels. Without assessing endogenous redox machinery, the antioxidant mechanism remains incomplete.

Response 6: We are grateful for your insightful recommendation. To prevent the antioxidant mechanism from being overinterpreted, we have carefully revised the manuscript in accordance with the reviewer's recommendations. The conclusions have been revised to more accurately reflect the scope of the current findings, and the statement that implied a definitive mechanism of antioxidant action has been removed. The altered text is highlighted in red in the revised manuscript (refer to page 14, lines 498–513).

 

Comment 7. NO suppression is central to the anti-inflammatory claim. Indicate whether the Griess reagent assay was calibrated with sodium nitrite standards and report exact nitrite concentrations (µM), not only relative changes. This will enhance the pharmacological interpretation.

Response 7: We appreciate your valuable recommendation. The Griess assay results have been recalculated in accordance with the reviewer's recommendation, and the nitrite concentrations are now reported as absolute values (µM). This was achieved by utilizing a sodium nitrite standard curve. The revised manuscript has been updated to reflect the corresponding values incorporated into the results section (page 6, lines 195–209) and the method section (page 13, lines 453–454). 

2.6. Impact of Bauhinia thailandica leaf extract on LPS-induced nitric oxide synthesis

To evaluate the anti-inflammatory efficacy of Bauhinia thailandica leaf extract, its impact on NO synthesis in LPS-activated BV2 microglial cells was examined. Figure 3 demonstrates that LPS stimulation markedly elevated NO levels in BV2 cells after 24 hours of treatment (44.6 ± 2.19 μM) compared with the untreated control group (4.6 ± 0.74 μM) (p < 0.01), indicating effective microglial activation. Co-treatment with Bauhinia thailandica leaf extract markedly suppressed LPS-induced NO generation in a concentration-dependent manner. After treatment with 12.5 μg/mL and 25 μg/mL of the extract, NO levels dropped to 31.1 ± 3.58 μM and 27.0 ± 3.93 μM, respectively. A statistically significant reduction (p < 0.05 and p < 0.01) was observed at the concentrations of 12.5 μg/mL and 25 μg/mL, indicating a considerable anti-inflammatory effect. Quercetin, employed as a positive control, markedly reduced NO generation to 25.6 ± 0.91 μM, compared with the LPS-treated group (p < 0.01). These results demonstrate that Bauhinia thailandica leaf extract efficiently inhibits NO-mediated inflammatory responses in activated microglial cells and may confer protective effects against neuroinflammation.

 

4.8. Nitric oxide (NO) assay

The levels of NO were determined by measuring nitrite accumulation in the cell culture supernatants using Griess reagent [1% sulfanilamide/0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride 2.5% phosphoric acid] (Invitrogen, Carlsbad, CA). In this assay, the nitrite-containing sample (150 µL/well) was placed in a 96-well plate, mixed with 20 µL of Griess reagent and 130 µL of deionized water, and incubated for 30 min at room temperature. Absorbance values were read at 540 nm using a microplate reader (Bio-Tek Instruments Inc., Winooski, VT) [62]. The concentrations of nitrite in the culture supernatants were determined from the standard curve and expressed as µM nitrite.

 

Comment 8. Several sentences are too long, which lowers readability. Make the complex sentences more concise, remove informal transitions, and adjust verb tenses to improve scientific accuracy throughout the manuscript.

Response 8: Thank you for this valuable suggestion. The manuscript has been thoroughly revised to improve readability and scientific precision. Long and complex sentences have been shortened, informal transitions have been removed, and verb tenses have been carefully standardized throughout the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In the current study, the authors have studied the qualitative and colorimetic quantitative phenolic content of the Bauhinia thailandica ethanolic extract. Further, they  examined the effect of Bauhinia thailandica ethanolic extract on BV2 cells by determining the concentrations that didn't affect the cell viability to examine their antioxidant and anti-inflammatory effect against LPS. The study have showed that Bauhinia thailandica at 25 ug/ml showed an antioxidant and anti-inflammatory activities on BV2 cells treated with LPS by reducing the deyecting ROS, NO, IL-6, IL 1B, TNF-alpah. 
The study is well designed; however, the data presenting needs to be supported by numerical result, particularly for results visualized using charts. Besides, the methodology have to be detailed for reproducibility, regarding the procedure and the concentrations of the LPS, quercetin, and the extract. More importantly, the phenolic content is lower than the flavonoids and the tannins, which is not logical. and need to clarified.

1- In the introduction:
highlight the research gap; illustrate why two different sub-families are mentioned (what is the taxonomic link between both); separate the first paragraph into shorter paragraphs as it is too long; illustrate why LPS is selected

2- In section 4.2, illustrate how the extraction was conducted and the temperature used to evaporate the ethanol.

3- Detail section 4.3.

4- Add references to the methodologies in section 4.4.

5- in line 393, did the authors used 95% O2?? Indicate whether cells were allowed to attach overnight before treatments began.

6- In line 395, superscript the 4; why serum-free DMEM is used, isn't it overstressed the cells?

7- Italize B in line 399; indicate the duration the cells are exposure to the extract

8- Spell out LPS in line 400; indicate its concentration.

9- Unify the BV2 or BV-2 spelling throughout the manuscript; mention what is the control used and showed in the results

10- In section 4.7, why did the authors measured at 540, add a reference, as the included reference have measured at 570. Besides, illustrate how the viability was determined

11- In section 4.8, illustrate how the NO was estimated/calculated and expressed.

12- The methodology in section 4.9 have to be detailed. Besides, the authors did not mentioned the mixtures used to treat the cells as mentioned in the results.

13- In section 4.10, illustrate how the supernatant was prepared; how the results are expressed.

14- It was better if the manuscript provide a phenolic identification using an HPLC, as a novel used plant extract to deepen the understanding of the extract effect.

15- In section 2.2, how the flavonoids is nearly 8 times the total flavonoids?????? It is not rational, as the flavonoid content is a part of the total phenolics. similarly for the tannins??
Revise the phenolic calculations.

16- Remove the No. from Table 2

17- in line 117, the inhibition % was at which concentration of the extract versus which concentration of vitamin c?

18-  In table 3, subscript the 50 in the third column; remove the units ug/ml after % inhibition

19- In section 2.4, add a numerical results, as the figure didn't show accurate numerical data. Kindly, apply throughout the results visualized using charts.

20- In figure 1 caption, add the BT after Bauhinia thailandica; remove the following from  the caption "We 135 treated BV2 cells with different concentrations of Bauhinia thailandica extract in serum-free medium 136 for 24 hours. The MTT assay was used to determine cell viability."

21- remove the following from the caption of figure 3 "The cells were treated with LPS (1 μg/mL) in the presence or absence of B. thailandica leaf 185 extract in serum-free medium for 24 h. The level of NO in the culture medium was determined using 186 the Griess reaction protocol."

22- Remove "Cells were 207 treated with LPS (1 μg/mL) in the presence or absence of B. thailandica leaf in serum-free medium 208 for 24 h. The level of IL-6 in the culture medium was determined using ELISA kits." from the caption of figure 4 

and "Cells were 229 treated with LPS (1 μg/mL) in the presence or absence of B. thailandica leaf in serum-free medium 230 for 24 h. The level of 1β in the culture medium was determined using ELISA kits." from figure 5 caption

and "Cells were 246 treated with LPS (1 μg/mL) in the presence or absence of B. thailandica leaf in serum-free medium 247 for 24 h. The level of TNF-α in the culture medium was determined using ELISA kits." from the caption of figure 6

23- The first paragraph of the discussion have to answer the research question.

24- Reference the sentences in lines 276-278; 306-308; 312-313

25- the abstract should be more informative, focusing on the significant results, showing a numerical significant data

27- Revise the English within the manuscript.

28- Check the references, some references did not show the article's name

 

 

Author Response

Reviewer 2

Comments and Suggestions for Authors

In the current study, the authors have studied the qualitative and colorimetic quantitative phenolic content of the Bauhinia thailandica ethanolic extract. Further, they  examined the effect of Bauhinia thailandica ethanolic extract on BV2 cells by determining the concentrations that didn't affect the cell viability to examine their antioxidant and anti-inflammatory effect against LPS. The study have showed that Bauhinia thailandica at 25 ug/ml showed an antioxidant and anti-inflammatory activities on BV2 cells treated with LPS by reducing the deyecting ROS, NO, IL-6, IL 1B, TNF-alpah. 
The study is well designed; however, the data presenting needs to be supported by numerical result, particularly for results visualized using charts. Besides, the methodology have to be detailed for reproducibility, regarding the procedure and the concentrations of the LPS, quercetin, and the extract. More importantly, the phenolic content is lower than the flavonoids and the tannins, which is not logical. and need to clarified.

Response:  We sincerely thank the reviewer for the positive assessment of our study and for the constructive comments and suggestions. These comments have been very helpful in improving the clarity and quality of our manuscript. We have carefully revised the manuscript and addressed all concerns raised by the reviewer. Our point-by-point responses to each comment are provided below.

Comment 1. In the introduction: highlight the research gap; illustrate why two different sub-families are mentioned (what is the taxonomic link between both); separate the first paragraph into shorter paragraphs as it is too long; illustrate why LPS is selected.

Response 1: We sincerely thank the reviewer for these helpful and constructive suggestions to improve the clarity and structure of the introduction.

First, following the reviewer’s suggestion, the introduction's first paragraph has been divided into shorter paragraphs to improve readability and logical flow.

Second, we have revised the introduction to highlight the research gap (in red font) more clearly. Specifically, we now emphasize that “Several species within the genus Bauhinia have been reported to contain bioactive compounds with antioxidant and anti-inflammatory properties. In contrast, Bauhinia thailandica is a newly described species, and information regarding its phytochemical composition and biological activities remains limited. Considering the phytochemical diversity observed in related Bauhinia species, especially the presence of flavonoids and phenolic compounds with known anti-inflammatory and antioxidant effects, Bauhinia thailandica is a promising candidate for research into microglia-mediated neuroinflammation.” (page 3, lines 102–109). 

Third, we have clarified the taxonomic relationship between the mentioned subfamilies. The manuscript now explains that the genus Bauhinia belongs to the family Leguminosae (Fabaceae) and is currently classified within the subfamily Cercidoideae according to modern phylogeny-based classification systems. The revised text clarifies this taxonomic placement to avoid confusion when different subfamily names appear in the literature. (page 3, lines 86–99). 

Finally, we have added a brief explanation describing why lipopolysaccharide (LPS) was selected as the inflammatory stimulus in this study. “LPS was selected as a stimulant because it is widely used to activate microglia in experimental models and mimics bacterial endotoxin exposure. It reliably induces inflammatory responses through activation of the toll-like receptor 4 (TLR4) signaling pathway [33]. This results in increased production of pro-inflammatory cytokines, nitric oxide, and ROS [33].”(page 3, lines 116–120). 

Comment 2. In section 4.2, illustrate how the extraction was conducted and the temperature used to evaporate the ethanol.

Response 2:  Thank you for this valuable comment. The extraction procedure and the temperature used for ethanol evaporation have now been added in Section 4.2. The revised text is highlighted in red in the manuscript (Page 11, Lines 360–363), as shown below.

“The leaves of Bauhinia thailandica were extracted with 95% ethanol at a sample-to-solvent ratio of 1:10 using ultrasound-assisted extraction (UAE) for three successive cycles. The combined filtrates were concentrated under reduced pressure using a rotary evaporator at 40 °C to obtain the crude ethanolic extract. The extraction yield was 15.67%.”

 

Comment 3. Detail section 4.3.

Response 3: Thank you for this valuable comment. Section 4.3 has been revised to provide a clearer and more detailed description of the qualitative phytochemical analysis. The corresponding changes have been highlighted in red font in the revised manuscript (Page 11, Lines 364–376).

“Qualitative phytochemical analysis of the Bauhinia thailandica leaf extract was conducted using standard screening methods to detect major classes of secondary metabolites [52]. Phenolic compounds were identified using the ferric chloride test, while flavonoids were examined by the ammonia test. Tannins were detected using the warm ferric chloride test. Cardiac glycosides were screened by the Keller–Killiani test, and saponins were evaluated using the frothing test. Terpenoids and steroids were assessed using the Salkowski test and Liebermann–Burchard reaction, respectively. Alkaloids were determined by Dragendorff’s test, whereas anthraquinones were analyzed using Borntrager’s test. The presence of each phytochemical class was interpreted based on the development of a characteristic color reaction, precipitate formation, or stable froth, depending on the respective assay. The results were expressed as positive (+) for presence and negative (−) for absence. All analyses were carried out in triplicate [59].”

 

Comment 4. Add references to the methodologies in section 4.4.

Response 4: Thank you for this helpful comment. Appropriate references have now been added to support the methodologies described in Section 4.4. The references [60] have been included in the revised manuscript (Page 11, Lines 393).

Iqbal, S.; Younas, U.; Ali, I. Phytochemical Profiling and Antioxidant Potential of Polyphenolic Compounds. Frontiers in Pharmacology 2022, 13, 865341. https://doi.org/10.3389/fphar.2022.865341.

Comment 5.  In line 393, did the authors used 95% O2?? Indicate whether cells were allowed to attach

overnight before treatments began.

Response 5: Thank you for this helpful comment. The culture conditions have been clarified in the revised manuscript. The incorrect statement regarding 95% O₂ has been corrected to indicate that the cells were cultured at 37 °C in a humidified incubator containing 5% CO₂.

The revised text is as follows.

“The cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. The culture medium was replaced twice weekly while the cells were growing. When the cells reached approximately 80% confluence, they were subcultured for further maintenance. For experimental assays, cells were plated at a density of 1x10⁴ cells/well in 96-well plates and allowed to attach overnight before treatments were initiated.”

 

These revisions have been included in the revised manuscript (Page 12, Lines 413–417).

 

 Comment 6. In line 395, superscript the 4; why serum-free DMEM is used, isn't it overstressed the cells?

Response6:  Thank you for your valuable comment.

The superscript formatting of “4” in line 416, page 12 has been corrected as suggested.

Cells in serum-free DMEM functioned as the untreated control. Serum-free conditions were maintained during the entire treatment period to avert any potential interference from serum-derived growth factors and cytokines that might affect LPS-induced inflammatory signaling and nitric oxide production. This method is often used in short-term inflammatory stimulation experiments to make sure that the effects that were reported are only because of LPS and the extract that was tested. The treatment lasted only 24 hours, and the MTT test was used to check the status of the cells. The results showed that neither LPS nor the extract had a large effect on the viability of BV2 cells when there was no serum present at the doses that were tested. As a result, the serum-free environment did not induce significant cellular stress, which might have complicated the interpretation of the results [61, 62].

We have clarified this point in the revised manuscript (as red fronts, page 12, lines 425-435).

 

Comment 7. Italize B in line 399; indicate the duration the cells are exposure to the extract

Response7: Thank you for this careful observation.

1. The letter B in line 399 has been italicized, as suggested in red, on page 12, line 420 in revised manuscript.
2. The duration of extract exposure has now been clearly specified in the revised manuscript (red font).

We appreciate the reviewer’s attention to detail, which has helped improve the accuracy and transparency of the manuscript.

 

Comment 8. Spell out LPS in line 400; indicate its concentration.

Response 8: Thank you for this helpful comment. In the revised manuscript, the abbreviation LPS has been spelled out as lipopolysaccharide (LPS) at its first occurrence in the introduction section (Page 3, Line 112)., and its concentration (1 µg/mL) has now been specified in the revised manuscript.  

 

Comment 9. Unify the BV2 or BV-2 spelling throughout the manuscript; mention what is the control used and showed in the results

Response 9:  Thank you for this important comment.

1. The spelling of the cell line has now been unified throughout the manuscript. We use BV2 throughout, including the Abstract, Methods, Results, Figures, and Figure legends.
2. Untreated BV2 cells served as the control, while cells stimulated with lipopolysaccharide (LPS, 1 µg/mL) without extract treatment served as the inflammatory control.

Comment 10. In section 4.7, why did the authors measure at 540, add a reference, as the included reference has measured at 570. Besides, illustrate how the viability was determined.

Response 10:  Thank you for your valuable comment. We apologize for the inconsistency. The absorbance was measured at 570 nm, in accordance with the cited reference and the standard MTT assay protocol. The previously stated wavelength (540 nm) was a typographical error and has been corrected in Section 4.7 of the revised manuscript.

Cell viability (%) was calculated as (Absorbance of treated cells / Absorbance of control cells) × 100, with the control group set as 100% viability.

This clarification has now been included in the Methods section to ensure methodological transparency and reproducibility.

 

We appreciate the reviewer’s careful evaluation, which has helped improve the clarity and accuracy of our manuscript.

 

Comment 11. In section 4.8, illustrate how the NO was estimated/calculated and expressed.

Response 11: Thank you for this comment. In the original manuscript, nitric oxide (NO) production was presented as a fold increase relative to the untreated control. In the revised manuscript, NO production has been recalculated using the nitrite standard curve obtained from sodium nitrite via the Griess assay. The concentrations of nitrite in the culture supernatants were determined from the standard curve and expressed as µM nitrite. The revised manuscript has been updated to reflect the corresponding values incorporated into the results section (page 6, lines 195–209) and the method section (page 13, lines 453–454). 

 

Comment 12. The methodology in section 4.9 have to be detailed. Besides, the authors did not mentioned the mixtures used to treat the cells as mentioned in the results.

Response 12: Thank you for this valuable comment. We have revised Section 4.9 to provide a more detailed description of the methodology used to measure intracellular ROS production.  (Page 13, section 4.9).

 

Comment 13. In section 4.10, illustrate how the supernatant was prepared; how the results are expressed.

Response 13: Thank you for your comment. We amended Section 4.10 to clarify how the culture supernatants were generated and how cytokine levels were calculated and expressed.

“After 24 hours of treatment, the culture medium from each well was collected and centrifuged to remove cellular debris. The resulting cell culture supernatants were used for cytokine analysis. Prior to ELISA analysis, the collected supernatants were diluted with sample diluent as follows: IL-1β samples were diluted 1:2, IL-6 samples were diluted 1:6, while TNF-α samples were analyzed without dilution. The levels of IL-6, IL-1β, and TNF-α in the culture supernatants were quantified using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, 50 µL of assay diluent was added to each well of the antibody-coated microplate, followed by 50 µL of either cytokine standards or prepared samples. The plates were covered and incubated at 37 °C for 2 hours, then washed four times with washing buffer. Subsequently, the biotinylated detection antibody was added, and the plates were incubated at room temperature for an additional 2 hours. After washing, substrate solution was added and incubated at room temperature for 30 min, and the reaction was stopped by adding the stop solution to each well. Absorbance was measured at 450 nm using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Cytokine concentrations were calculated based on the standard curves generated from the corresponding cytokine standards. The final concentrations were obtained by multiplying the calculated values by the respective dilution factors and were expressed as cytokine levels (pg/mL) in the culture supernatant. These methodological details have been included in the updated text (Page 13, section 4.10)

 

Comment 14.  It would be better if the manuscript provided phenolic identification using HPLC, as a novel plant extract, to deepen understanding of the extract's effects.

Response 14: Thank you for this valuable suggestion. We agree that the identification of phenolic compounds using HPLC analysis would provide deeper insight into the bioactive constituents responsible for the observed biological effects. In the present study, our objective was to provide evidence of the antioxidant and anti-inflammatory activities of Bauhinia thailandica leaf extract, along with basic phytochemical characterization through qualitative screening and analyses of total phenolic, flavonoid, and tannin contents.

We acknowledge that detailed chemical profiling would strengthen the mechanistic understanding of the extract’s activity. Therefore, HPLC-based phenolic identification and characterization of the major bioactive compounds are planned in our future studies. This point has now been clarified in the revised manuscript (Red font, Page 10, line 343-352; page 14, line 508-513)

We appreciate the reviewer’s constructive suggestion, which will help guide our future investigations.

Comment 15.  In section 2.2, how the flavonoids is nearly 8 times the total flavonoids?????? It is not rational, as the flavonoid content is a part of the total phenolics. similarly for the tannins??
Revise the phenolic calculations.

Response 15: Thank you for this important comment. We carefully re-examined the raw absorbance data, blank correction, calibration curves, and unit conversions used to determine the total phenolic content (TPC), total flavonoid content (TFC), and total tannin content (TTC). Following this review, the calculations were revised using the corresponding standard-curve equations for gallic acid, quercetin, and tannic acid, respectively, and the corrected values have now been updated in Section 2.2 and Table 2.

Based on the revised calculations, the Bauhinia thailandica leaf extract contained 70.55 ± 0.12 mg GAE/g dry extract for TPC, 249.47 ± 0.26 mg QE/g dry extract for TFC, and 397.50 ± 0.00 mg TAE/g dry extract for TTC. The revised manuscript (Page 4, Lines 133–144)

Comment 16.  Remove the No. from Table 2.

Response 16: The No. from Table 2 was removed.

 

 Comment 17. In line 117, the inhibition % was at which concentration of the extract versus which concentration of vitamin C?

Response 17: Thank you for this important comment.

The antioxidant activity of the Bauhinia thailandica leaf extract was evaluated using the DPPH radical scavenging assay. The extract demonstrated a concentration-dependent increase in radical scavenging activity, reaching 89.90 ± 1.36% inhibition at 1000 µg/mL. The IC₅₀ value of the extract was 513.60±7.20µg/mL. Vitamin C, used as the positive control, showed 80.91 ± 0.42% inhibition at 100 µg/mL and an IC₅₀ value of 68.25±2.31µg/mL. Based on the lower IC₅₀ value, vitamin C exhibited stronger DPPH radical scavenging activity than the extract. (Page 4, Lines 145–155)

 

Comment 18. In table 3, subscript the 50 in the third column; remove the units ug/ml after % inhibition.

Response 18: Thank you for this helpful comment. The value 50 in the third column of Table 3 has been formatted as a subscript, and the unit µg/mL after % inhibition has been removed as suggested. These corrections have been made in the revised manuscript. (Page 4)

 

Comment 19. In section 2.4, add a numerical results, as the figure didn't show accurate numerical data. Kindly, apply throughout the results visualized using charts.

Response 19: Thank you for this helpful suggestion. Numerical values corresponding to the graphical data have now been added in Section 2.4 and throughout the results section where data are presented in charts. These changes have been incorporated in the revised manuscript (red font).

 

Comment 20. In figure 1 caption, add the BT after Bauhinia thailandica; remove the following from  the caption "We 135 treated BV2 cells with different concentrations of Bauhinia thailandica extract in serum-free medium 136 for 24 hours. The MTT assay was used to determine cell viability."

Response 20: Thank you for your comment. The requested sentences have been removed from the caption of Figure 1 as suggested. And BT was added after Bauhinia thailandica.

 

Comment 21. Remove the following from the caption of figure 3 "The cells were treated with LPS (1 μg/mL) in the presence or absence of B. thailandica leaf 185 extract in serum-free medium for 24 h. The level of NO in the culture medium was determined using 186 the Griess reaction protocol."

Response 21: Thank you for your comment. The requested sentences have been removed from the caption of Figure 3 as suggested.

Comment 22. Remove "Cells were 207 treated with LPS (1 μg/mL) in the presence or absence of B. thailandica leaf in serum-free medium 208 for 24 h. The level of IL-6 in the culture medium was determined using ELISA kits." from the caption of figure 4 

and "Cells were 229 treated with LPS (1 μg/mL) in the presence or absence of B. thailandica leaf in serum-free medium 230 for 24 h. The level of 1β in the culture medium was determined using ELISA kits." from figure 5 caption

and "Cells were 246 treated with LPS (1 μg/mL) in the presence or absence of B. thailandica leaf in serum-free medium 247 for 24 h. The level of TNF-α in the culture medium was determined using ELISA kits." from the caption of figure 6

Response 22: Thank you for your comment. The requested sentences have been removed from the caption of Figure 3-Figure 6 as suggested.

Comment 23. The first paragraph of the discussion have to answer the research question.

Response 23: Thank you for this helpful suggestion. The first paragraph of the discussion has been revised to clearly address the research question and summarize the main findings of the study. (Page 8-9, Lines 272–282)

 

Comment 23. Reference the sentences in lines 276-278; 306-308; 312-313

Response 24:  Thank you for this comment. The sentences in lines 276–278, 306–308, and 312–313 have been revised accordingly.

Specifically, the sentence “Quercetin, employed as a positive control due to its recognized anti-inflammatory properties” has been modified to “Quercetin, employed as a positive control” in the revised manuscript (page 6, lines 205–206). The supporting reference has instead been incorporated into the discussion section in the sentence “This effect was comparable to that of quercetin, a well-known antioxidant flavonoid [46]” (page 9).

In addition, the previous sentences in lines 306–308 of the original discussion have been removed during revision. The sentence corresponding to lines 312–313 has been revised and now includes the appropriate reference in the revised manuscript (page 10, lines 323–326).

Comment 25.The abstract should be more informative, focusing on the significant results, showing a numerical significant data

Response 25: Thank you for this valuable suggestion. The abstract has been revised to provide more informative content by emphasizing the key significant findings of the study. In addition, relevant numerical values of the significant results have been included to clearly present the main outcomes of the study. These revisions have been incorporated into the revised manuscript.

 

Comment 26. Revise the English within the manuscript.

Response 26. I appreciate your insightful recommendation. The manuscript has been thoroughly revised to improve the English language and overall clarity. Additionally, professional language correction was performed using the MDPI editing service. The revised manuscript and the certified editing certificate are included as attachments.

 

Comment 27 Check the references; some references did not show the article's name.

Response 27: Thank you for your valuable comment. We have thoroughly reviewed the reference list and corrected entries with missing article titles. The references have now been carefully revised to ensure completeness and consistency. As red front in reference 63 in the revised manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed all comments. I recommend acceptance of the manuscript in its present form. 

Author Response

Thank you for your positive evaluation and recommendation to accept our manuscript. We also appreciate your helpful comments and suggestions, which have made our work clearer and stronger.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have carefully revised the manuscript; however, some issues need to be revised before publication.

1- In section 4.2, mention how the plant material was prepared (drying method/grinding/ particle size), the exact weight of the sample used, the duration and ultrasonic frequency or power of each extraction cycle, the temperature during ultrasound treatment, and the filtration method.

2- In section 4.3, the methodology lacks sufficient experimental details. The authors should specify the exact procedures followed for each phytochemical test, including reagent concentrations, volumes of extract used, incubation conditions, and observation criteria for positive reactions.

3- In section 4.8, illustrate what material/substrate is being used to prepare the standard curve. 

4- In section 2.2, the reported flavonoid and tannin contents appear considerably higher than the total phenolic content, which is unusual since flavonoids and tannins are subclasses of phenolic compounds. The authors should verify the calculations and clarify whether differences in calibration standards (GAE, QE, and TAE) may explain this discrepancy.

Author Response

Comments and Suggestions for Authors

 Reviewer 2:

The authors have carefully revised the manuscript; however, some issues need to be revised before publication.

 Response:

Thank you very much for your valuable comments and suggestions. We have carefully revised the manuscript in accordance with your recommendations, and the remaining issues have been addressed. All changes have been clearly indicated in the revised manuscript.

 

Comment 1. In section 4.2, mention how the plant material was prepared (drying method/grinding/ particle size), the exact weight of the sample used, the duration and ultrasonic frequency or power of each extraction cycle, the temperature during ultrasound treatment, and the filtration method.

Response 1:  Thank you for this helpful comment. Section 4.2 has been revised to provide a more detailed and reproducible description of the extraction procedure. The revised text now specifies that Bauhinia thailandica leaves were dried by microwave-assisted hot-air drying at 50 °C and 1000 W for 2 h, finely ground, and sieved through an 80-mesh sieve. It also includes the exact sample weight (100 g), extraction solvent (95% ethanol), solid-to-solvent ratio (1:10, w/v), ultrasonic frequency (20 kHz), extraction temperature (50 °C), extraction time (30 min per cycle for three cycles), and filtration through Whatman No. 1 filter paper. As red fonts, Page 10-11, lines 358-366.

 

Comment 2. In section 4.3, the methodology lacks sufficient experimental details. The authors should specify the exact procedures followed for each phytochemical test, including reagent concentrations, volumes of extract used, incubation conditions, and observation criteria for positive reactions.

Response 2:  We thank the reviewer for this helpful comment. Section 4.3 has been revised to provide detailed experimental procedures for each qualitative phytochemical assay, including reagent concentrations, extract volume, reagent volume, reaction conditions, and criteria for determining positive reactions. These additions improve the reproducibility and methodological transparency of the study. As red fonts, Page 11, lines 368-394.

 

Comment 3. In section 4.8, illustrate what material/substrate is being used to prepare the standard curve. 

Response 3: Thank you for your valuable comment. We have clarified this point in the revised manuscript. The standard curve for nitrite quantification was generated using a sodium nitrite (NaNO₂) standard provided in the Griess reagent kit. This information has now been added to the Methods section (page 13, lines 471-473). 

“The concentrations of nitrite in the culture supernatants were determined from a standard curve generated using sodium nitrite (NaNO₂) standards provided in the Griess reagent kit and expressed as µM nitrite.”

 

 Comment 4. In section 2.2, the reported flavonoid and tannin contents appear considerably higher than the total phenolic content, which is unusual since flavonoids and tannins are subclasses of phenolic compounds. The authors should verify the calculations and clarify whether differences in calibration standards (GAE, QE, and TAE) may explain this discrepancy.

Response 4:  

We thank the reviewer for this important and constructive comment. We carefully recalculated the total phenolic, flavonoid, and tannin contents using the replicate absorbance data, calibration curves, dilution factors, and unit conversions. After this re-evaluation, the values were confirmed as follows: total phenolic content = 70.55 ± 1.35 mg GAE/g dry extract, total flavonoid content = 249.47 ± 1.24 mg QE/g dry extract, and total tannin content = 397.50 ± 4.44 mg TAE/g dry extract.

We agree that flavonoids and tannins are chemically classified as subclasses of phenolic compounds, and therefore, their apparently higher values may seem unusual if interpreted as directly comparable absolute concentrations. However, these parameters were determined using different colorimetric assays and expressed as different calibration equivalents, namely gallic acid equivalents (GAE), quercetin equivalents (QE), and tannic acid equivalents (TAE). Because each assay is based on different reaction chemistries, calibration standards, and response factors, the resulting values are assay-dependent and should not be interpreted as directly comparable or additive measures of the same chemical pool. We have clarified this point in the revised manuscript and revised the reported mean ± SD values accordingly to avoid overinterpretation. As red fonts, Page 4, lines 133-142.

 

We sincerely appreciate the reviewer’s insightful comment, which helped us improve the manuscript's clarity and accuracy

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

the authors have carefully revised the manuscript