Isolation, Characterization, and HPTLC-Quantification of Compounds with Anticancer Potential from Loranthus Acaciae Zucc.

Round 1

Reviewer 1 Report

The manuscript describes the normal phase HPTLC separation of two compounds, betulinic acid and β-sitosterol. Such separations of plant extracts of more complex mixture are quite common in the literature but these particular two compounds have not been often separated by HPTLC. One relevant reference that does separate these compounds is cited at the end of this review. This article needs to be described and cited in the Introduction. In general, the Introduction needs to be rewritten providing context about other related HPTLC methods of plant extracts. In addition, the authors' method needs to be compared in the Discussion section to the method cited below. In general, HPTLC references 22-26 are not explained well in the text. Hopefully there are some advantages but it looks very similar. The analytical figures of merit describing this HPTLC method do seem to be very complete in this manuscript. The potential cancer hazard of chloroform needs to be made clear in the Experimental section of the manuscript. It would of been much better if the authors devised a method which does not use a chlorinated solvent; that would be a significant advantage over previous methods.

Cited paper: https://akjournals.com/view/journals/1006/26/3/article-p254.xml

Author Response

Reviewer 1:

The manuscript describes the normal phase HPTLC separation of two compounds, betulinic acid and β-sitosterol. Such separations of plant extracts of more complex mixture are quite common in the literature but these particular two compounds have not been often separated by HPTLC. One relevant reference that does separate these compounds is cited at the end of this review. This article needs to be described and cited in the Introduction. In general, the Introduction needs to be rewritten providing context about other related HPTLC methods of plant extracts. In addition, the authors' method needs to be compared in the Discussion section to the method cited below. In general, HPTLC references 22-26 are not explained well in the text. Hopefully there are some advantages but it looks very similar. The analytical figures of merit describing this HPTLC method do seem to be very complete in this manuscript. The potential cancer hazard of chloroform needs to be made clear in the Experimental section of the manuscript. It would of been much better if the authors devised a method which does not use a chlorinated solvent; that would be a significant advantage over previous methods.

Cited paper: https://akjournals.com/view/journals/1006/26/3/article-p254.xml

Reply:

We thank the respected reviewer for all valuable comments. The given reference “Maurya, Anupam & Srivastava, Santosh. (2013). A Simple and Reliable HPTLC Method for the Determination of Four Marker Components in the Quality Control of Alstonia scholaris. JPC - Journal of Planar Chromatography - Modern TLC. 26. 254-259. 10.1556/JPC.26.2013.3.9” has been inserted in the introduction part of the revised manuscript. Also, more information regarding the HPTLC analysis of betulinic acid as well as β-sitosterol has been added in the revised manuscript. The references quoted from 34-38 describes the advantages of HPTLC methods in the quality control of several herbal and cosmetic preparations. It also emphasizes on the operational cost benefits of HPTLC in comparison to other chromatographic analysis due to smaller amounts of mobile phase and standard compounds requirement as well as short analysis time. This explanation has been already added in the manuscript so if the respected reviewer allows us we would like to keep the given information the same in the manuscript.

We improved the introduction and rephrased and added  many sentences. Please have a look to the revision.

The potential cancer hazard of chloroform needs to be made clear in the Experimental section of the manuscript. It would of been much better if the authors devised a method which does not use a chlorinated solvent; that would be a significant advantage over previous methods.

Reply: We are totally in agreement with this comment. However, we think that the potential cancer hazard of chloroform and other chlorinated organic solvents is obvious and known for all researchers in our filed. Thus, we think there is no need to mention it in the part of extraction and isolation. It is not the proper place to mention that.

Author Response File: Author Response.pdf

Reviewer 2 Report

This paper reports the isolation and HPTLC-quantification of betulinic acid and β-sitosterol from Loranthus acaciae. The authors also examined the cytotoxicity and apoptosis inducing activity of betulinic acid. In this study, betulinic acid and b-sitosterol were isolated from Loranthus acacia for the first time. However, betulinic acid and b-sitosterol are well-known compounds in plant kingdom. The structural novelty of these compounds is of little significance. Also, the cytotoxicity and apoptosis inducing activity of betulinic acid are overlapped previous studies such as Foo et al. (J. Ethnopharmacol., 2015; 166: 270-278) and Majeed et al. (Mol. Carcinog., 2016; 55: 964-976). In my opinion, this work has not yet reached a stage at which publication in Separations and cannot be recommended for publication.

Comments:

1, Compounds 1 and 2 were known compounds. Usually, structural determination of known compounds is not described in the paper. Please delete.

2, The cytotoxic activity of the isolated compounds (betulinic acid and β-sitosterol) should be given as μM, not as μg/mL.

3, Please show the results of the positive control(s) on cytotoxic activity.

4, Please show the IC₅₀ values of the positive controls of the cytotoxic assay.

5, Figure 4 (D): There is a major peak of Rf 0.55.  What is this compound? This compound should be analysis and determine the chemical structure.

6, Please describe the reason why the authors use HPTLC to separate these two compounds. Why not use HPLC?

Author Response

Reviewer 2:

This paper reports the isolation and HPTLC-quantification of betulinic acid and β-sitosterol from Loranthus acaciae. The authors also examined the cytotoxicity and apoptosis inducing activity of betulinic acid. In this study, betulinic acid and β -sitosterol were isolated from Loranthus acacia for the first time. However, betulinic acid and b-sitosterol are well-known compounds in plant kingdom. The structural novelty of these compounds is of little significance. Also, the cytotoxicity and apoptosis inducing activity of betulinic acid are overlapped previous studies such as Foo et al. (J. Ethnopharmacol., 2015; 166: 270-278) and Majeed et al. (Mol. Carcinog., 2016; 55: 964-976). In my opinion, this work has not yet reached a stage at which publication in Separations and cannot be recommended for publication.

Response: We thanks the respected reviewer for the remarkable comments. We are partly in agreement with some of these comments.

Our hypothesis was very simple. It was to investigate if L. acaciae possess anticancer activity (was not reported previously) or not since several plant species of the Loranthaceae family such as L. micranthus, L. parasiticus and L. ferrugineus were found to exhibit anticancer activity. The respected reviewer knows as specialist in natural product chemistry that we sometimes work blindly. As phytochemists and pharmacognosists we try to follow certain pharmacological activities and to isolate the compounds responsible for that activity (activity-guided fractionation) and don’t know the chemical structure until we do NMR and Mass experiments. That was exactly what happened with us. We were happy that L. acacia extract showed anticancer activity and followed the activity in the hexane-fraction and isolated the major active compounds found responsible for the activity. What we did, is a bioactivity-guided fractionation and isolation for the anticancer active compounds from the hexane-extract which was the most active fraction. We found that the activity remained in one fraction which contained both compounds. Therefore, we isolated both compounds. Actually, the NMR- and Mass-experiments were the last experiments what we carried out. Unfortunately, both compounds were well-known and identified as reported. If we could know from the beginning that the isolated compounds are betulinic acid and β-sitosterol, we perhaps didn’t complete the work.  

We think that interesting results could be conveyed from this investigation namely:

• The plant ( acaciae) possesses anticancer activity (not previously reported).
• Betulinic acid is one of the major compounds responsible for the observed anticancer activity and found in the active hexane fraction of acacia (not previously isolated and reported).
• We established a chromatographic method (HPTLC) which can be applied for the concurrent analysis of betulinic acid and β-sitosterol in any plant species or marketed herbal formulations claiming the presence of the two biomarkers.

We believe that these remarkable findings make our manuscript somewhat interesting for the readers.

1. Compounds 1 and 2 were known compounds. Usually, structural determination of known compounds is not described in the paper. Please delete.

Response: it is true that the compounds are known however, both compounds are isolated for the first time as we previously mentioned. We didn't discuss the NMR data since the compounds are known but we think it is of great benefit for the readers if they find the NMR data of both compounds in this manuscript. Thus, we hope that the respected reviewer allows us to keep the table in manuscript.

2. The cytotoxic activity of the isolated compounds (betulinic acid and β-sitosterol) should be given as μM, not as μg/mL.

Response: We are totally in agreement with. The unit of cytotoxic activity of the isolated compounds was changed and calculated to μM accordingly.

3. Please show the results of the positive control(s) on cytotoxic activity.

Response: A new figure showing the cytotoxic activity of cisplatin (positive control) was   included in the revised manuscript.

4. Please show the IC50 values of the positive controls of the cytotoxic assay.

Response: The IC₅₀ of the positive control (Cisplatin) in μM was added in the IC₅₀ Table (2). Please have a look to the revision

5. Figure 4 (D): There is a major peak of Rf 0.55. What is this compound? This compound should be analysis and determine the chemical structure.

Response:  What we did, is a bioactivity-guided fractionation and isolation for the anticancer active compounds from the hexane-extract. We found that the activity almost remained in one fraction (fraction B) which contained both compounds. Therefore, we concentrated on that fraction and isolated both compounds. At this stage of our research We were not interested in other compounds including that compound at Rf 0.55, but We definitely have to continue on this plant and try to isolate and identify the structures of other compounds for future communication.

6. Please describe the reason why the authors use HPTLC to separate these two compounds. Why not use HPLC?

Response: HPTLC was not used for the separation of two compounds. The compounds were separated by using column chromatography. HPTLC was only used for the qualitative and quantitative analysis of the isolated compounds in the plant L. acacia because it is a very reliable chromatographic technique in the analysis of markers in the plant extracts as well as herbal preparations. We tried to develop the HPLC method for the analysis of above isolated compounds in L. acacia for future use.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors described an interesting study on two different molecules extracted by a plant and potentially useful as anticancer agents.

The authors stated that the two compounds have been extracted from fraction B. However, they separated seven different fractions. Please add more details on how they chose the fraction B. 

Data on HPTLC should be improved. In particular:

1. Authors referred to the ICH guidelines (2005), but the reference is missing
2. what the authors used as blank material for method validation?
3. Did the use any internal standards?
4. How many quality controls did they use for method validation? At which concentration?

Author Response

Reviewer 3:

The authors stated that the two compounds have been extracted from fraction B. However, they separated seven different fractions. Please add more details on how they chose the fraction B.

Response: The problem statement of our research was: does the plant L. acacia possess anticancer activity or not? And what are the compounds responsible for that activity? Consequently, what we did, is a bioactivity-guided fractionation and isolation for the anticancer active compounds from the hexane-extract. We found that the activity almost remained in one fraction (fraction B). Therefore, we concentrated on that fraction and isolated both compounds which were the main constituents. We added a sentence explaining that in the section "compound isolation and identification".

       Data on HPTLC should be improved. In particular:

1. Authors referred to the ICH guidelines (2005), but the reference is missing

Response: We are totally in agreement with. The reference has been added in the revised manuscript.

2. What the authors used as blank material for method validation?

Response: The present method was used for the quantitative analysis of betulinic acid and β-sitosterol in the plant extract only. We have not used this method for the quantification of the above-mentioned compounds in any biological samples obtained from any treated animals, as we have not performed any pharmacokinetic or pharmacodynamics experiments in animal species. Therefore, we did not use any blank material for the validation of the developed method.

3. Did the use any internal standards?

Response: We appreciate the comments raised by the reviewer, in the present investigation we have developed the HPTLC method for the analysis of betulinic acid and β-sitosterol in the hexane extract of aerial parts of the plant L. acacia by using the already established markers namely, Betulinic acid and β-sitosterol procured from standard source (Sigma Aldrich, St Louis, MO, USA). Therefore, we did not use any internal standard during the method development.

4. How many quality controls did they use for method validation? At which concentration?

Response: The authors appreciate the comment raised by the reviewer. In the present study, the method was validated by including the validation parameters such as recovery, precision and robustness. The recovery of the materials by using present method was performed at 40, 60, 80 and 100 µg/mL concentration levels. The precision was performed    by considering the three concentration levels (40, 60 and 80 µg/mL), while robustness of the method was checked at 40 µg/mL concentration. 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

This paper reports the isolation and HPTLC-quantification of betulinic acid and b-sitosterol from Loranthus acaciae. The responses to the reviewer comments were adequate. The manuscript has been much improved. This manuscript would be acceptable for for publication in Separations.