STAT3 Inhibitory Activities of Lignans Isolated from the Stems of Lindera obtusiloba Blume

Round 1

Reviewer 1 Report

The purpose of this study is to evaluate Lindera obtusiloba Blume's therapeutic potential for addressing STAT3-related diseases caused by IL-6. Seven compounds of lignan have been identified and characterized. Compounds 1 and 5 showed significant inhibition of IL-6-stimulated STAT3 activation in Hep3B cells, resulting in a reduction in the expression of inflammation-related genes in mRNA. In IL-6-induced U266 cells, p-STAT3 and p-JAK2 were found to be regulated in Western blot assays. Lindera obtusiloba has emerged as a promising bioactive candidate for treating diseases caused by IL-6 and STAT3, which may lead to potential therapeutic interventions. The manuscript requires some considerations:

1. In "2.2. Extraction and Isolation," please provide information on the initial weight of the dried stems of Lindera obtusiloba used for the extraction with 50% ethanol.

2. In "3. Results and Discussion," please explicitly reference Figure 1, Figure 2, and Table 1 in the text to facilitate readers in locating the relevant data during their reading.

3. In "3. Results and Discussion," please offer an in-depth discussion of the results illustrated in Figure 2.

4. Please format Table 1 to fit on a single page.

5. Please include instructions on calculating the IC50 in the table.

6. In Figure 3, indicate the specific compound used for cell treatment.

7. Kindly discuss the potential compounds present in both the extracts and the fractions.

8. Based on the results from Figure 5, please provide the quantification of protein expression.

Author Response

Dear. Reviewer #1.

We appreciate your review of our manuscript entitled “STAT3 inhibitory activities of lignans isolated from the stems of Lindera obtusiloba Blume (scipharm-2704450)”.

We have revised the manuscript based on the reviewer comments and all corrections are marked in red.

Reviewer #1.

Comments and Suggestions for Authors

The purpose of this study is to evaluate Lindera obtusiloba Blume's therapeutic potential for addressing STAT3-related diseases caused by IL-6. Seven compounds of lignan have been identified and characterized. Compounds 1 and 5 showed significant inhibition of IL-6-stimulated STAT3 activation in Hep3B cells, resulting in a reduction in the expression of inflammation-related genes in mRNA. In IL-6-induced U266 cells, p-STAT3 and p-JAK2 were found to be regulated in Western blot assays. Lindera obtusiloba has emerged as a promising bioactive candidate for treating diseases caused by IL-6 and STAT3, which may lead to potential therapeutic interventions. The manuscript requires some considerations:

1. In "2.2. Extraction and Isolation," please provide information on the initial weight of the dried stems of Lindera obtusiloba used for the extraction with 50% ethanol.

-->Thank you for comment. We added the initial weight of the dried stems of Lindera obtusiloba used for the extraction with 50% ethanol in text.

2. In "3. Results and Discussion," please explicitly reference Figure 1, Figure 2, and Table 1 in the text to facilitate readers in locating the relevant data during their reading.

--> Thank you for comment. We added reference Figure 1, Figure 2, and Table 1 in the text.

3. In "3. Results and Discussion," please offer an in-depth discussion of the results illustrated in Figure 2.

--> Thank you for comment. Additionally, it was explained that the results in figure 2 are used when separating compounds.

4. Please format Table 1 to fit on a single page.

--> Thank you for comment. We modified the table in the text.

5. Please include instructions on calculating the IC50 in the table.

--> Thank you for comment. At line 291, added instructions on the IC50.

6. In Figure 3, indicate the specific compound used for cell treatment.

--> Thank you for comment. Cirsiliol used a compound published in the 2019 paper and was used as a positive control. The reference is attached below.

Reference (Lim, Hyung Jin, et al. "In vitro inhibitory effects of cirsiliol on IL-6-induced STAT3 activation through anti-inflammatory activity." Bioorganic & Medicinal Chemistry Letters 29.13 (2019): 1586-1592.)

7. Kindly discuss the potential compounds present in both the extracts and the fractions.

-->Thank you for comment. It was mentioned in the paper that isolated compounds were separated from the fraction that showed significant inhibitory effects.

8. Based on the results from Figure 5, please provide the quantification of protein expression.

-->Thank you for comment. We added the quantification data of protein expression in the text. In our recent paper, protein quantification values were displayed as shown in Figure 5. Above all, please understand that the results cannot be displayed in this way due to the change of experimenter.

Reference (Bak et al. “ Ishige okamurae Extract: Diphlorethohydroxycarmalol with Efect of Atopic Dermatitis‑Like Skin Infammation” Revista Brasileira de Farmacognosia 2023: s43450-023-00488-2)

Author Response File: Author Response.pdf

Reviewer 2 Report

Lee and coworkers described the isolation of compounds 1-7 from L. obtusiloba and evaluated their IL-6-induced STAT3 inhibitory. In view of the reviewer, the manuscript in its present state leaves certain questions, in particular pertaining to the novelty of this work, unanswered and would benefit significantly from an additional revision.

The following points of concern could be addressed:

1.     Most compounds 1-7 were reported previously, and the stems of Lindera obtusiloba Blume are known to have inhibitory effects on various inflammatory responses such as neuroprotective activity and anti-allergic activity. Can authors highlight the novelty of this work in the manuscript?

2.     Lines 244-247, the chiral centers 7,7’ of compound 1 are not consistent with reference 11. Didn’t find compounds 3 and 4, 6in reference 21 and 23, respectively.

3.     Line 250-264 Section 3.2, the extract and fractions are a mixture and may contain unknown components in the mixture. It doesn’t make any sense to evaluate the inhibition of IL-6-induced pSTAT3 luciferase activity of the mixture.

4.     Line 283, Authors demonstrated that compounds 1 and 5 could be promising IL-6/STAT3 inhibitors. What’s the target of these compounds? IL-6, IL-6Rs, JAK or STAT3?

5.     Figure 3, Please label A, B, and C for each figure. For each subfigure, which column means compound 1, which means compound 5?

6.     SI, for all the 1HNMR, please show the spectrum from 0-10 ppm; for 13CNMR please show the spectrum from 0-160 ppm. As compounds 1-7 have been reported, please add a table to compare the NMR with previous data.

There are some small grammatical errors in the manuscript, please double-check.

Author Response

We appreciate your review of our manuscript entitled “STAT3 inhibitory activities of lignans isolated from the stems of Lindera obtusiloba Blume (scipharm-2704450)”.

We have revised the manuscript based on the reviewer comments and all corrections are marked in red.

Reviewer #2.

Comments and Suggestions for Authors

Lee and coworkers described the isolation of compounds 1-7 from L. obtusiloba and evaluated their IL-6-induced STAT3 inhibitory. In view of the reviewer, the manuscript in its present state leaves certain questions, in particular pertaining to the novelty of this work, unanswered and would benefit significantly from an additional revision.

The following points of concern could be addressed:

1. Most compounds 1-7 were reported previously, and the stems of Lindera obtusiloba Blumeare known to have inhibitory effects on various inflammatory responses such as neuroprotective activity and anti-allergic activity. Can authors highlight the novelty of this work in the manuscript?

--> Thank you for comment. Thank you for your feedback. However, there are no reports on whether the stem extract of Lindera obtusoloba Blume and compounds derived from it act on IL-6-induced STAT3 activation, one of the important cytokines in inflammatory response. We think there is something novel about this.

2. Lines 244-247, the chiral centers 7,7’ of compound 1are not consistent with reference 11. Didn’t find compounds 3 and 4, 6 in reference 21 and 23, respectively.

--> Thank you for comment. The compound structure at chiral center 7, 7’ was modified, and asarinin episesamin are the same compound. Compound was reported in the indicated reference 4 and compounds 4 and 6 were reported in reference 24. Rewritten to match the reference.

3. Line 250-264 Section 3.2, the extract and fractions are a mixture and may contain unknown components in the mixture. It doesn’t make any sense to evaluate the inhibition of IL-6-induced pSTAT3 luciferase activity of the mixture.

--> Thank you for comment. In separating compounds, isolation was conducted focusing on fractions that showed activity. As a result, active compounds were isolated in fractions 5 and 7.

4. Line 283, Authors demonstrated that compounds 1 and 5 could be promising IL-6/STAT3 inhibitors. What’s the target of these compounds? IL-6, IL-6Rs, JAK or STAT3?

--> Thank you for comment. Through this study, the effect of inhibiting JAK and STAT3 phosphorylation induced by IL-6 was confirmed. However, we plan to conduct additional research in the future to determine whether it directly inhibits IL-6 or IL-6r or inhibits receptor-ligand interaction.

5. Figure 3, Please label A, B, and C for each figure. For each subfigure, which column means compound 1, which means compound 5?

--> Thank you for comment. Figure 3 was modified. Labels are indicated, and compounds 1 and 5 are expressed separately.

6. SI, for all the 1HNMR, please show the spectrum from 0-10 ppm; for 13CNMR please show the spectrum from 0-160 ppm. As compounds 1-7 have been reported, please add a table to compare the NMR with previous data.

--> Thank you for comment. The spectrum presented by modifying it to the requested ppm value and added a table to compare the NMR with previous data in Supplementary data.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript has been edited accordingly. No more comments. 

Author Response

Thank you for your review, and we will be able to present better research results.

Reviewer 2 Report

Recommendation: The current version is suitable for publication.

All the comments are well addressed. The manuscript has improved significantly and is suitable for publication.

Author Response

Thank you for your review, and we will be able to present better research results.