Discovery of Anti-Inflammatory Triterpenoid Glucosides from the Heritiera littoralis Dryand
Round 1
Reviewer 1 Report
Journal Name: Molecules (ISSN 1420-3049)
Manuscript Number: molecules- 2085246
Type: Article
Title Optimization of the Isolation of Blackcurrant and Black Cumin 2 Seeds Oils using Supercritical Fluid Extraction - Box-Behnken Design of the Experiment
REVIEWER’S COMMENT
Major revision comments
1. Introduction
1. Line 46. Figure 1 regarding the structures of compounds 1-8 from H. littoralis is not given in the text of the manuscript and in the supplementary materials.
2. The aim of the study needs to be clearly stated.
2. Results and Discussion:
1. The structural formula of the identified triterpenoids should be given.
2. The results obtained for cell viability assays should be presented in more details.
3. Have the isolates been tested for cytotoxicity? If such testings were performed, the results of these studies should be presented and discussed. If no such studies have been performed, it should be explained why they have not been conducted.
4. The novelty of this study should be clearly explained. The strengths and the limitations of the study should be specified.
3. Experimental
Lines 151- 172: The sample preparation should be explained in more details: how were prepared the plant samples (weighting, mincing, preparing average sample, etc.); how was performed the extraction (liquid/liquid, device, duration, if mixing was applied intensity and time should be given); the column separation should be given in more details (column size, elution flow rate, device etc.); more details regarding HPLC semi preparative separation (flow rate, time, how was performed the monitoring of the selected fractions); for fractionation was it done automatically (device) or not, etc.
It is not clear whether extraction efficiency and extractions recovery were determined. This should be explained in Materials and Methods.
Lines 145-149: The conditions of spectrophotometric measurements (wavelength range or wavelength (if it was fixed), slit etc.), NMR and MS measurements.
Lines 184-188: Explanations and details regarding cytotoxicity assay, MTT assay, LPS stimulation, and calculation of cell viability should be given. It is not clear how the authors conclude that “all compound showed no cytotoxicity on RAW264.7 cells at the concentrations from 0 to 50 μm”. The Griess assay needs to be described in brief.
Overall Recommendation
The paper can in principle be accepted after major revision.
Author Response
Responses to reviewers’ comments and the list of changes
Dear Reviewer,
We appreciate you very much for the positive and constructive suggestions on our manuscript. All alterations are colored in the revised manuscript. Here, we reply to the questions point-by-point. The revision details are listed below.
Thank you very much for your work on the manuscript and your kind consideration!
Respectfully yours.
Yanjun Zhang
The answers to the reviewers, issues for Ms. molecules-2138096 are as follows:
- Introduction
Qestion (Q) 1: Line 46. Figure 1 regarding the structures of compounds 1-8 from H. littoralis is not given in the text of the manuscript and in the supplementary materials.
Answer (A) 1: Thank you for the comment. We have added the structures of compounds 1-8 in Figure 1 in the manuscript and the supplementary materials (Figure S17).
Q2: The aim of the study needs to be clearly stated.
A1: Thank you for the comment. Heritiera littoralis has been reported to have significant anti-inflammatory activity in the folk. However, there have been few reports on the biologically active constituents of H. littoralis. The present study was initiated to explore anti-inflammatory lead compounds of H. littoralis.
- Results and Discussion:
Q1: The structural formula of the identified triterpenoids should be given.
A1: Thank you for the comment. We have added the structural formula (Figure 1) of the identified triterpenoids in the manuscript.
Q2: The results obtained for cell viability assays should be presented in more details.
A2: Thank you for the comment. We have added the details of cell viability in the manuscript .
Q3: Have the isolates been tested for cytotoxicity? If such testings were performed, the results of these studies should be presented and discussed. If no such studies have been performed, it should be explained why they have not been conducted.
A3: Thank you for the comment. Cytotoxicity was tested by LPS-induced RAW 264.7 macrophages cells according to MTT assay. The macrophages were treated with different concentrations of the compounds, and the cell viability was observed. All isolates at concentrations below 50 μM showed the cell viability were greater than 90%.
Q4: The novelty of this study should be clearly explained. The strengths and the limitations of the study should be specified.
A4: Thank you for the comment. The novelty of this study as follow: two previously undescribed triterpenoid glucosides, and 6 known ones, were isolated from the leave of H. littoralis; one new compound showed anti-inflammatory activity inhibited the release of NO, with an IC50 value of 10.33 μM. Our study only carried out its anti-inflammatory in vitro by inhibiting the release of NO. However, its anti-inflammatory mechanism has not been studied yet. We will conduct mechanism research in the next step.
- Experimental
Q1: Lines 151-172: The sample preparation should be explained in more details: how were prepared the plant samples (weighting, mincing, preparing average sample, etc.); how was performed the extraction (liquid/liquid, device, duration, if mixing was applied intensity and time should be given); the column separation should be given in more details (column size, elution flow rate, device etc.); more details regarding HPLC semi preparative separation (flow rate, time, how was performed the monitoring of the selected fractions); for fractionation was it done automatically (device) or not, etc.
A1: Thank you for the comment. We have added the details of sample
preparation, performation of the extraction, column separation ect in the manuscript and the supplementary materials (Figure S20).
Q2: It is not clear whether extraction efficiency and extractions recovery were determined. This should be explained in Materials and Methods.
A2: In this experiment, Dried leaves (5.0 kg) of H. littoralis was extracted. The
extract was obtained with the weight of 0.52 kg, The extraction efficiency is about
10.4%. We have added the details in Materials and Methods. The yield was calculated as follows:
Yield extract % = weight of dry extract / weight of dried plant material × 100%
Q3: Lines 145-149: The conditions of spectrophotometric measurements (wavelength range or wavelength (if it was fixed), slit etc.), NMR and MS measurements.
A3: Thank you for the comment.We have added the details of the conditions of measurements in the manuscript (3.1. General experimental procedures).
Q4: Lines 184-188: Explanations and details regarding cytotoxicity assay, MTT assay, LPS stimulation, and calculation of cell viability should be given. It is not clear how the authors conclude that “all compound showed no cytotoxicity on RAW264.7 cells at the concentrations from 0 to 50 μm”. The Griess assay needs to be described in brief.
A4: Thank you for the comment. We have added the details of cell viability and the Griess assay in the manuscript (3.5. Cell viability and Anti-inflammatory activity test).
Reviewer 2 Report
As a descriptive article the manuscript is good; however, it is necessary to make some adjustments: The genus Heritiera currently belongs to the Malvaceae family, subfamily Scutellarioideae, this need to be updated. In the case of the results and discussion, it is important to make some comparative analyzes regarding the structures and their anti-inflammatory potential within genera and species, at least within Malvaceae, since the similarities and differences of structures and their function, considering taxa with relevant, available and comparable information, supports the development of hypotheses that can help to understand patterns and processes.
Author Response
Responses to reviewers’ comments and the list of changes
Dear Reviewer,
We appreciate you very much for the positive and constructive suggestions on our manuscript. All alterations are colored in the revised manuscript. Here, we reply to the questions point-by-point. The revision details are listed below.
Thank you very much for your work on the manuscript and your kind consideration!
Respectfully yours.
Yanjun Zhang
The answers to the reviewers, issues for Ms. molecules-2138096 are as follows:
Qestion: As a descriptive article the manuscript is good; however, it is necessary to make some adjustments: The genus Heritiera currently belongs to the Malvaceae family, subfamily Scutellarioideae, this need to be updated. In the case of the results and discussion, it is important to make some comparative analyzes regarding the structures and their anti-inflammatory potential within genera and species, at least within Malvaceae, since the similarities and differences of structures and their function, considering taxa with relevant, available and comparable information, supports the development of hypotheses that can help to understand patterns and processes.
Answer: Thank you for the comment. We have revised in the manuscript and changed the genus Heritiera to the Malvaceae family. The structures and their functions within Malvaceae are added in our manuscript (2.3. Similarities and differences of some triterpenoids and their anti-inflammatory activity from Malvaceae).
Reviewer 3 Report
The manuscript “molecules-2138096” entitled “Discovery of Anti-inflammatory Triterpenoid Glucosides from the Heritiera littoralis Dryand.” Describe the isolation of two new and six known triterpenoids from Heritiera littoralis Dryand. The authors used several spectroscopic techniques to identify the isolated compounds, such as 1D, 2D, and HR-MS spectroscopy. The anti-inflammatory activity of the isolated compounds was investigated in vitro in LPS-induced inflammation in RAW 264.7 cells. Although the authors use proper spectroscopic techniques to identify the isolates, many typos are found in the manuscript and are listed below.
Comments to authors:
Page 1, line 31; Following the scientific Latin name of the plant, the author’s name “Dryand” should be written with a period i.e., ‘’Dryand.” exactly as in the title of the manuscript.
Page 1, Line 41-42; .....In the statement “Furthermore, the isolates were evaluated their anti-inflammatory activities against ......etc. (Add for “evaluated for their...”)
Page 2; Figure 1 is missing! The caption “Figure 1. The structures of compounds 1-8 from H. littoralis” appeared but no figure is displayed.
Lines 48 and 173. ... heritiera A (1) and heritiera B (2) please use bold font for compound numbers.
Lines 49 & 176; Heritiera A (1) ...... please adjust the bold font of the round bracket to normal font.
Page 2, line 53, regarding the proton H-21 at δH 5.35, and the anomeric protons at δH 6.40 & 6.50, d (8.2), the authors described them as a doublet. If possible, please provide a good expansion for the 1H NMR spectrum (Figure S2 and S10) to properly display the splitting patterns of these protons. No need to display the full chemical shift scale from -3 to 16 ppm, but from 0-10 or 0-12 will be better.
Page 2; line 62, add a space before reference # [19]; line 65, add a space before “COSY”.
Page 2; Line 66, 67; please try to adjust the continuity of the provided the partial structures.
Page 2; line 83, in the columns’ title, δH( J = Hz )...adjust the spaces..change to δH (J = Hz).
Page 4; line 100, add a reference for the HPLC method for the detection of D-glucose.
Page 4; line 113-114, Thus, the structure of 2 was established-3β-hydroxy...correct to ”was established as....” and also correct the structure name 3β-hydroxy-23-(carboxy-O-β-D-glucopyranosyl ester)-erythro-diol. Line 120; correct to 3β,28-dihydroxy-oleana-11,13(18)-diene.
Line 126; Table 2....not bold font.
Page 5; line 131, use bold font in the numbers of compounds 1–8. Line 132, correct Cellsa to cells. Line 135; use black font for “as positive control”. Line 138; UV spectra were acquired with a TU-1901 spectrophotometer (used for what?). Line 254; and e n-BuOH fraction...remove the extra “e” letter. Line 157; was using RP-C18 silica gel .... was purified using RP-C18 silica gel. Line 158; afford five subfractions, correct to ... to afford five subfractions. (start a new sentence).
In section “3.3. Extraction and isolation”, the eluting mobile phase composition should be provided for each compound to allow reproducibility of the method or a flowchart for the isolation procedure can be provided in the supplementary file. For e.g., in line 158-159 &163 ... Sephadex LH-20 CC (eluted with MeOH), what was the mobile phase composition, was it chloroform-MeOH or water-MeOH, and in what ratio? Also, sometimes “CC” is used and other times “column” is used...please use unified style.
Line 161, subfractions(C-1~C-8), add a space.
Line 164, compounds 5 (5.6 mg), 7 (4.9 mg)....correct to compounds 5 (5.6 mg) and 7 (4.9 mg).
167; H2O-MeOH(40:60)....add a space after MeOH.
Lines 177 & 181; electronic supporting information materials ..correct to online supplementary material.
Line 179; add a space before Colorless.
Line 191; use production or release of NO instead of secretions of NO (as Nitrous oxide is not a secretion).
Line 196; Experimental details, including figures of this manuscript, are provided as supplementary material (correct to the full 1D, 2D NMR, HRESIMS).
In supplementary data, use the same abbreviation for HRESIMS as the manuscript (or vice vers).
Line 73; C-23, C-28, respectively.... add “and” between the carbon numbers.
Line 58, 13C-NMR was written differently (Please, use unified style i.e., 13C NMR).
In Figure 2, the configuration of C-23 (the α-ester bond) should be drawn as a dashed bond similar to compound (1). Also, the font of substituent groups in both structures should be the same. The configuration of protons at certain chiral centers is not illustrated, such as the protons H-13 in compound (1) and H-9 in (2) although they were determined by ROESY in Figure 3.
Line 80, please use italic font for the “O” in O-β-D-glucopyranosyl.
Page 7, the references section contains many errors in the style, alignment, indentation, spaces, and paragraphs’ justification.... etc., which need to be revised as per the journal format.
Examples of reference typos;
The spaces between the authors’ initials should be removed in all authors’ names.
The issue number is not required.
Line 21; 38 ,83-102 (The spaces should be after and not before the comma)
Line 242, please correct the title of the cited reference # [18] cinnamoylgly-coflavonid antimyco bacterial
Line 265; inappropriate spaces in Kim,J. W.
Author Response
Responses to reviewers’ comments and the list of changes
Dear Reviewer,
We appreciate you very much for the positive and constructive suggestions on our manuscript. All alterations are colored in the revised manuscript. Here, we reply to the questions point-by-point. The revision details are listed below.
Thank you very much for your work on the manuscript and your kind consideration!
Respectfully yours.
Yanjun Zhang
The answers to the reviewers, issues for Ms. molecules-2138096 are as follows:
Qestion (Q) 1: Page 1, line 31; Following the scientific Latin name of the plant, the author’s name “Dryand” should be written with a period i.e., ‘’Dryand.” exactly as in the title of the manuscript.
Answer (A) 1: Thank you for the comment. “Dryand” was revised as “Dryand.”
Q2: Page 1, Line 41-42; .....In the statement “Furthermore, the isolates were evaluated their anti-inflammatory activities against ......etc. (Add for “evaluated for their...”)
A2: Thank you for the comment. We accepted the referee’s advice and revised in the manuscript.
Q3: Page 2; Figure 1 is missing! The caption “Figure 1. The structures of compounds 1-8 from H. littoralis” appeared but no figure is displayed.
A3: Thank you for the comment. We have added the structures of compounds 1-8 from H. littoralis in Figure 1 in the manuscript and the supplementary materials (Figure S17).
Q4: Lines 48 and 173. ... heritiera A (1) and heritiera B (2) please use bold font for compound numbers.
A4: Thank you for the comment. We have revised the bold font for compound numbers in the manuscript.
Q5: Lines 49 & 176; Heritiera A (1) ..... please adjust the bold font of the round bracket to normal font.
A5: Thank you for the comment. We have adjusted the bold font of the round bracket to normal font in the manuscript.
Q6: Page 2, line 53, regarding the proton H-21 at δH 5.35, and the anomeric protons at δH 6.40 & 6.50, d (8.2), the authors described them as a doublet. If possible, please provide a good expansion for the 1H NMR spectrum (Figure S2 and S10) to properly display the splitting patterns of these protons. No need to display the full chemical shift scale from -3 to 16 ppm, but from 0-10 or 0-12 will be better.
A6: Thank you for the comment. We have made mistakes. We found that anomeric protons were double-double spits at δH 6.40 dd (8.0, 2.8); 6.50 dd (8.0, 2.8) in 1H NMR (600 MHz). We have revised in the manuscript. We have revised chemical shift scale from 0 to 12 ppm in the Figure S2 and S10.
Q7: Page 2; line 62, add a space before reference # [19]; line 65, add a space before “COSY”.
A7: Thank you for the comment. We have added a space before “COSY”.
Q8: Page 2; Line 66, 67; please try to adjust the continuity of the provided the partial structures.
A8: Thank you for the comment. We have adjusted the continuity of the provided the partial structures in the manuscript.
Q9: Page 2; line 83, in the columns’ title, δH( J = Hz )...adjust the spaces..change to δH (J = Hz).
A9: Thank you for the comment. We have adjusted the spaces in the manuscript.
Q10: Page 4; line 100, add a reference for the HPLC method for the detection of D-glucose.
A10: Thank you for the comment. We have added a reference for the HPLC method for the detection of D-glucose.
Q11: Page 4; line 113-114, Thus, the structure of 2 was established-3β-hydroxy
...correct to”was established as....” and also correct the structure name 3β-hydroxy -23-(carboxy-O-β-D-glucopyranosyl ester)-erythro-diol. Line 120; correct to 3β,28-
dihydroxy-oleana-11,13(18)-diene.
A11: Thank you for the comment. We accepted the referee’s advice and revised the structure names in the manuscript.
Q12: Line 126; Table 2....not bold font.
A12: Thank you for the comment. We have revised the compound numbers in bold font.
Q13: Page 5; line 131, use bold font in the numbers of compounds 1–8. Line 132, correct Cellsa to cells. Line 135; use black font for “as positive control”. Line 138; UV spectra were acquired with a TU-1901 spectrophotometer (used for what?). Line 254; and e n-BuOH fraction...remove the extra “e” letter. Line 157; was using RP-C18 silica gel .... was purified using RP-C18 silica gel. Line 158; afford five subfractions, correct to ... to afford five subfractions. (start a new sentence).
A13: Thank you for the comment. We have revised this part in the manuscript.
Q14: In section “3.3. Extraction and isolation”, the eluting mobile phase composition should be provided for each compound to allow reproducibility of the method or a flowchart for the isolation procedure can be provided in the supplementary file. For e.g., in line 158-159 &163 ... Sephadex LH-20 CC (eluted with MeOH), what was the mobile phase composition, was it chloroform-MeOH or water-MeOH, and in what ratio? Also, sometimes “CC” is used and other times “column” is used...please use unified style.
A14:Thank you for the comment. We have drew a flowchart for the isolation procedure (Figure S20) in the supplementary file. The sentence of “eluted with MeOH” means that the mobile phase is 100% MeOH.
Q15: Line 161, subfractions(C-1~C-8), add a space.
A15: Thank you for the comment. We have added a space between subfractions and (C-1~C-8).
Q16: Line 164, compounds 5 (5.6 mg), 7 (4.9 mg)....correct to compounds 5 (5.6 mg) and 7 (4.9 mg).
A16: Thank you for the comment. We have revised “compounds 5 (5.6 mg), 7 (4.9 mg)...” as “compounds 5 (5.6 mg) and 7 (4.9 mg)”.
Q17:167; H2O-MeOH(40:60)....add a space after MeOH.
A17: Thank you for the comment. We have added a space after MeOH.
Q18: Lines 177 & 181; electronic supporting information materials ..correct to online supplementary material.
A18: Thank you for the comment. We have revised “electronic supporting information materials ..” as “online supplementary material”.
Q19: Line 179; add a space before Colorless.
A19: Thank you for the comment. We have added a space before Colorless.
Q20: Line 191; use production or release of NO instead of secretions of NO (as Nitrous oxide is not a secretion).
A20: Thank you for the comment. The “secretions of NO” was revised as “production of NO”.
Q21: Line 196; Experimental details, including figures of this manuscript, are provided as supplementary material (correct to the full 1D, 2D NMR, HRESIMS).
A21: Thank you for the comment. Experimental details, figures of this manuscript, NMR and HRESIMS are all provided as supplementary material.
Q22: In supplementary data, use the same abbreviation for HRESIMS as the manuscript (or vice vers).
A22: Thank you for the comment. We have used the same abbreviation for HRESIMS as the manuscript.
Q23: Line 73; C-23, C-28, respectively.... add “and” between the carbon numbers.
A23: Thank you for the comment. We have added “and” between the carbon numbers.
Q24: Line 58, 13C-NMR was written differently (Please, use unified style i.e., 13C NMR).
A24: Thank you for the comment. The writing of 13C NMR was used unified style in the manuscript.
Q25: In Figure 2, the configuration of C-23 (the α-ester bond) should be drawn as a dashed bond similar to compound (1). Also, the font of substituent groups in both structures should be the same. The configuration of protons at certain chiral centers is not illustrated, such as the protons H-13 in compound (1) and H-9 in (2) although they were determined by ROESY in Figure 3.
A25: Thank you for the comment. The configuration of C-23 have been drawn as a dashed bond. The configuration of protons at certain chiral centers have been illustrated.
Q26: Line 80, please use italic font for the “O” in O-β-D-glucopyranosyl.
A26: Thank you for the comment. We have used italic font for the “O” in O-β-D-glucopyranosyl.
Q27: Page 7, the references section contains many errors in the style, alignment, indentation, spaces, and paragraphs’ justification.... etc., which need to be revised as per the journal format.
A27: Thank you for the comment. We have revised errors in the style, alignment, indentation, spaces, and paragraphs’ justification in reference and made them to the journal format.
Q28: Examples of reference typos;
The spaces between the authors’ initials should be removed in all authors’ names.
The issue number is not required.
Line 21; 38 ,83-102 (The spaces should be after and not before the comma)
Line 242, please correct the title of the cited reference # [18] cinnamoylgly-coflavonid antimyco bacterial
Line 265; inappropriate spaces in Kim,J. W.
A28: Thank you for the comment.The format of the references in the manuscript has been carefully modified in accordance with the requirements of the journal.
