Potential of Cissampelos pareira L. Pectin as a Bioactive Compound in Moisturizing and Anti-Aging Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comments: The antiaging potential of a Cissampelos pareira pectin fraction has been evaluated in the present study. After extracting and characterizing the fraction, its antiaging potential was evaluated both at cellular level and in an 8-week clinical trial. The methods used are clearly explained, at the cutting edge of currently used methods and allow firm conclusions to be drawn. I am personally convinced by the results presented by the Authors, their scientific values ​​as well as application potential. My only negative criticism would be on the presentation of the results is very descriptive, it would be interesting to discuss the data with other bioactives (possibly and in particular pectin fraction of other plants).

Specific comments: Whereas both ethanol and isopropanol could be used as extraction solvent as pointed by the authors in paragraph 2.1, isopropanol toxicity is well documented, so this is an additional point to choose ethanol as extraction solvent.

Figure 1: add ethanol used as extraction solvent

On Figure 2 presenting FTIR results, a higher intensity signal associated with methylesterified pectin seems to be observed after 30 minutes extraction which is in contrast to the results from Table 1, if I’m not mistaken. Could it be an indication of the presence of coextracted proteins or other compound(s) with this sample?

Letters corresponding to the statistical analysis are not presented in Table 4.

Author Response

Comment 1: General comments: The antiaging potential of a Cissampelos pareira pectin fraction has been evaluated in the present study. After extracting and characterizing the fraction, its antiaging potential was evaluated both at cellular level and in an 8-week clinical trial. The methods used are clearly explained, at the cutting edge of currently used methods and allow firm conclusions to be drawn. I am personally convinced by the results presented by the Authors, their scientific values as well as application potential. My only negative criticism would be on the presentation of the results is very descriptive, it would be interesting to discuss the data with other bioactives (possibly and in particular pectin fraction of other plants).

Response 1: Thank you for your feedback and thoughtful suggestion. We agree that a more comparative discussion with other bioactives, particularly pectin fractions from other plants, would provide valuable context. In the revised manuscript, we have included a discussion that compares our findings with existing literature on pectin from other plants and other bioactive compounds with similar moisturizing and anti-aging potential. The change can be found in the following sections.

• 1 Preparation of pectin C. pareira extract (PCP), page 3, lines 96-100.
• 2.2 Methoxy content (%), degree of esterification (%), and galacturonic acid (%), page 4, lines 145-146.
• 3.2 Total polysaccharides content, page 5, lines 178-185.
• 4 Antioxidant activity, page 6, lines 220-227.
• 7.2 Evaluation of moisturizing and anti-skin aging efficacy, page 9, lines 344-353.

 

Comment 2: Whereas both ethanol and isopropanol could be used as extraction solvent as pointed by the authors in paragraph 2.1, isopropanol toxicity is well documented, so this is an additional point to choose ethanol as extraction solvent.

Response 2: Agree. We have revised the manuscript to address this consideration. The change can be found on page 3, lines 89-91, and now as follows:

"Whereas isopropanol toxicity is well documented, ethanol was chosen as a safer extraction solvent for pectin in this study."

 

Comment 3: Figure 1: add ethanol used as extraction solvent

Response 3: Thank you for the suggestion. The name of Figure 1 has been revised and its updated placement as Figure 3 in the revised manuscript. This change is detailed on page 3, line 106, and now as follows:

"Figure 3. The percentage yield of the PCP extracted by using 95% ethanol as the extraction solvent, under each condition at 15, 30, and 45 minutes."

 

Comment 4: On Figure 2 presenting FTIR results, a higher intensity signal associated with methylesterified pectin seems to be observed after 30 minutes extraction which is in contrast to the results from Table 1, if I’m not mistaken. Could it be an indication of the presence of coextracted proteins or other compound(s) with this sample?

Response 4: We apologize for the oversight in the previous version of the manuscript, where the FTIR fingerprints were presented on different intensity scales, leading to a misunderstanding of their intensities. We have corrected this issue, and the revised figure (currently, Figure 4) now uses a consistent intensity scale. The updated figure aligns with the data presented in Table 1. Additionally, we would like to clarify that the FTIR fingerprints of the PCP at different extraction times are quite similar and are not intended to be directly compared for intensity variations.

 

Comment 5: Letters corresponding to the statistical analysis are not presented in Table 4
Response 5: Thank you for pointing this out. There were no statistically significant differences in pH and viscosity between T0, T3, and T6, which is why letters were not presented in Table 4. To clarify this, we have added the following note below the table:

“Not sharing the letters in the different columns indicates no significant differences (p > 0.05).”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript describes the extraction of Cissampelos pareira L. Pectin for serum formulation and in vivo human testing. The manuscript is properly written and the methodology is robust. Few comments to be addressed:

Suggest to provide a picture of the plant and the pectin extracted for visualisation.

2.2.1.: The direct comparisons of intensity may not be accurate. Intensity ratio should be used to compare a peak change relative to a constant peak. The regions of 1800-1600 & 1300-1000 cm-1.

Table 2: indicate the concentration of PCP at the relevant property. For N/A result, they may be removed.

2.3.3.: What are the solubility in glycerin & propylene glycol?

Table 3: indicate the concentration of PCP

2.6.2: Did the authors monitor the colour & smell change? May add these results in Table 4.

2.7.1.: The serums tested included all FB, F1-5? Please state clearly.

Fig 5/2.7.2: Any comparisons across different timepoints for the same serum, especially for hydration?

2.7.2.: the authors claimed the pollution issue for the insignificant result. However, pollution could also affect the hydration and TEWL. It is wise to blame pollution on the insignificant result OR it has no contribution on the parameters measured, especially anti-aging related.

3.9.2. /3.9.3/ 3.9.4.: Were the same 35 volunteers involved in both safety and efficacy tests? If so, how both tests were being conducted, concurrently or any gap?

Provide the demographic data of the volunteers e.g. gender, ethnic etc. Specify the inclusion and exclusion criteria.

Author Response

Comment 1: Suggest to provide a picture of the plant and the pectin extracted for visualization.

Response 1: We have included a picture of the plant [Figure 1 on page 2] and the pectin extracted [Figure 2 on page 3].

 

Comment 2: 2.2.1.: The direct comparisons of intensity may not be accurate. Intensity ratio should be used to compare a peak change relative to a constant peak. The regions of 1800-1600 & 1300-1000 cm-1.

Response 2: We agree that direct comparisons of intensity may not be accurate. We apologize for the oversight in the previous version of the manuscript, where the FTIR fingerprints were presented on different intensity scales, leading to a misunderstanding of their intensities. We have corrected this issue, and the revised figure (currently, Figure 4 on page 4) now uses a consistent intensity scale. Additionally, we would like to clarify that the FTIR fingerprints of the PCP at different extraction times are quite similar and are not intended to be directly compared for intensity variations.

 

Comment 3: Table 2: indicate the concentration of PCP at the relevant property. For N/A result, they may be removed.

Response 3: The concentration of PCP has been identified for pH and viscosity in Table 2 on page 5. The N/A result has been removed.

 

Comment 4: 2.3.3.: What are the solubility in glycerin & propylene glycol?

Response 4: The PCP was not soluble in glycerin and propylene glycol; thus, they were not used for further study. We have removed them from Table 2 and the method 3.5.3. The sentence in 2.3.3 has revised to be “The dried crude extract exhibited a high solubility in water, with a value of 99.93 ± 0.01% (Table 2).” (Page 6, lines 193-194)

 

Comment 5: Table 3: indicate the concentration of PCP

Response 5: The concentration of PCP has indicated in Table 3 and now is “Table 3. Stability of 1.0% w/v PCP solution after heating-cooling cycles.” (Page 8, line 275)

 

Comment 6: 2.6.2: Did the authors monitor the colour & smell change? May add these results in Table 4.

Response 6: Changes in color and smell were monitored by observing visually and smelling without any measurements. There was no change in color and smell after the heating-cooling test. Thus, we just mentioned it as “After stability testing, there was no change in color and smell.”  on page 8, line 290.

 

Comment 7: 2.7.1.: The serums tested included all FB, F1-5? Please state clearly.

Response 7: The serums tested included FB, and F1 to F5. We have stated in sections 2.7.1 and 3.9.3. The changes are made as follows.

“Therefore, all serums (FB and F1 to F5) can be further used to study their efficacy in human volunteers.” [2.7.1 on page 9, lines 306-307.]

“The serums FB and F1 to F5, as test samples, were filled in finn chambers prior to the application on the anterior upper arm of volunteers.” [3.9.3. on page 16, lines 589-590]

 

Comment 8: Fig 5/2.7.2: Any comparisons across different timepoints for the same serum, especially for hydration?

Response 8: Figure 5 (now updated as Figure 7 in the revised manuscript) has been revised to display the statistical differences between FB and each formulation within the same week. Comparisons of skin hydration and TEWL across different time points for the same serum have been made but the statistical differences were not presented in the figure.  Additionally, the explanation in Section 2.7.2 has been revised to reflect these updates and provide a clearer interpretation of the results. The changes are made as follows.

 “Comparing each serum across different time points, all formulations improved skin hydration over time, with varying patterns of significance across different week pairs. Only the serum containing 0.50 %w/w sodium hyaluronate (F5) demonstrated significant improvement in hydration earlier, starting in week 2 and continuing through week 8. In contrast, the serums FB, F1, F2, F3, and F4 significantly improved skin hydration (p < 0.05) from week 1, with effects sustained through weeks 3 to 8. However, Figure 7a illustrates that serums containing PCP (F2 and F3) exhibited significant differences (p < 0.05) compared to FB, similar to F4 and F5 in week 3. Therefore, it can be concluded that the efficiency of 0.5 %w/w PCP and 1.0 %w/w PCP were similar to positive controls.” [Page 9, lines 322-330]

“The decrease in TEWL of all products was observed with no significant difference (p > 0.05) compared to FB in the same week. After applying each serum at different time points, serums FB, F1, and F3 showed no significant differences in TEWL across all weeks. In contrast, F2 showed a significant decrease (p < 0.05) in TEWL since week 3 and maintained its prevention of TEWL until week 8, similar to pectin from apples (F4). Whereas sodium hyaluronate (F5) showed a significant decrease (p < 0.05) in TEWL in week 8.” [Page 9, lines 336-342]

 

Comment 9: 2.7.2.: the authors claimed the pollution issue for the insignificant result. However, pollution could also affect the hydration and TEWL. It is wise to blame pollution on the insignificant result OR it has no contribution on the parameters measured, especially anti-aging related.

Response 9: Thank you for your insightful feedback. We agree with your observation and have removed this point from the revised manuscript to ensure clarity and avoid unsupported claims regarding the impact of pollution on the measured parameters.

 

Comment 10: 3.9.2. /3.9.3/ 3.9.4.: Were the same 35 volunteers involved in both safety and efficacy tests? If so, how both tests were being conducted, concurrently or any gap?

Response 10: Yes, the same 35 volunteers participated in both the safety and efficacy tests. The safety tests were conducted first, followed by the efficacy tests with an appropriate gap between the two. This gap allowed us to ensure that any safety-related reactions did not affect the results of the efficacy tests. The specific timing and details of the testing procedure have been clarified in the revised manuscript.

“Participants underwent a two-week rest period after the epicutaneous patch test and were assessed for any adverse events at the test site before. Only those without adverse events were included in the subsequent study.” [Page17, lines 602-605]

 

Comment 11: Provide the demographic data of the volunteers e.g. gender, ethnic etc. Specify the inclusion and exclusion criteria.

Response 11: We have provided the demographic data of the volunteers, including the exclusion criteria and exclusion criteria in section 3.9.2.

“Thirty-five healthy Thai male and female volunteers aged between 25 - 50 years old were screened and enrolled according to inclusion and exclusion criteria. The inclusion criteria required participants to be healthy, with no lesions on the tested area, not currently involved in other clinical trials, and without a history of allergies to any ingredients in the formulation. The exclusion criteria included individuals with any skin diseases or abnormalities and those currently taking other medications.” [Page15, lines 579-584]

Author Response File: Author Response.pdf