An In Vitro Anticancer, Antioxidant, and Phytochemical Study on Water Extract of Kalanchoe daigremontiana Raym.-Hamet and H. Perrier

Round 1

Reviewer 1 Report

The research article by Justyna et al., entitled “In vitro anticancer, antioxidant and phytochemical study on water extract of Kalanchoe daigremontiana Raym.-Hamet & H. Perrier” has been reviewed.

The major issue of this manuscript is the novelty and significance parts. The novelty of the manuscript is not very high. The authors did mention and discuss the previous study (Ref. 31 in the manuscript) which may make this manuscript a followed-up study or extended study, but the content of the manuscript showed just simply a small extension of the previous one. In authors’ previous study (Ref. 31), they investigated the effects of bersaldegenin-1,3,5-orthoacetate on DNA damage, cell death and cell cycle arrest in HeLa cells. Though using bersaldegenin-1,3,5-orthoacetate instead of the K. daigremontiana water extract, several parts are similar to the previous one. In current manuscript, the authors investigated the water extract of K. daigremontiana on DNA damage, cell death and cell cycle arrest in SKOV3 cells. However, according to the information of Table 1, bersaldegenin-1,3,5-orthoacetate is one of the dominant compounds in K. daigremontiana water extract, ~32.47% content (757.4 ± 18.7/2332.1 ± 30).

In addition, the structure and coherence of the manuscript are weak. The authors did perform a lot of experiments, however, the organization of the results and the hypothetic model are not properly represented in this manuscript. Also, the abstract lacked a conclusive pat and should be more organized with a focus on the cytotoxic mechanism of K. daigremontiana water extract against SKOV3 cells.

 

In addition, the following are details that are missing or required revision.

1. Although the method used (MTT) is ubiquitous to estimate proliferation and viability, it is actually an indirect measure of these parameters. There are many factors that would influence the MTT activity such as metabolic status, mitochondrial activity, and so on. Moreover, MTT couldn’t detect dead cells. Therefore, whether it is appropriate to speak of the results of MTT equal to the antiproliferative activity needs to be considered. (Page 3, line 96.)
2. Why the authors choose SKOV3 cells, an ovarian cancer cell line, in this manuscript?
3. Figure 1. The labels and legend of figure 1 are so confusing. I would suggest the authors revise this figure thoroughly. In addition, the statistics is required to see the differences between treatments.
4. Page 1, line 115. Although the authors said that they did not observe significant changes in the drug's effect in vitro in the presence of the extract, I noticed that there seems difference between kd extract alone and kd+cisplatin in “Figure 1C, group 9”.
5. Page 4, line 114-117. “We also estimated the effect of daigremontiana water extract on the cytostatic action of paclitaxel and cisplatin in SKOV-3 cells.” please add the references for cytostatic action of paclitaxel and cisplatin. And, the authors should describe the mechanism of these two well-known cytostatic dugs, and compare and discuss the results.
6. Page 4, line 121. Please add the references for action of annexin and 7-aminoactinomycin (7-AAD).
7. Page 4, line 121. “40.0-300.0 μg/mL” please correct to “40-300 μg/mL”
8. Figure 3.: 1.) Please add the information of treatment for 3A, 3B and 3C, respectively.; 2.) Although the authors said that arrows indicate the late apoptotic/necrotic cells (high annexin and high PI staining), I couldn’t see the differences between cell with arrow in 3B or 3C.; 3.) To my knowledge, the methods used annexin V with 7-ADD (Figure 2) or PI (Figure 3) are basically the same, therefore, it’s redundant to show both figure 2 and 3.
9. Figure 5. 1.) The authors should explain how to translate the caspase-3/7 activity to live/early/late/dead cells in figure 5. ; 2.) 2.) The representative histogram should be shown side by the bar chart. (ex. figure 2).; 3.) The authors should compare and discuss the meaning of results showed in Figures 2 & 5.
10. Figure 6. 1.) label error: two 100 ug/mL; 2.) the author should describe how they plot the boundary to define the four quadrants.
11. Figure 8. 1.) Why do the authors choose HaCaT cells to investigate the ROS effect of the extract and the evaluation and correlation with the effects in SKOV3 cells.; 2.) Please add the abbreviations for ME, k.d.; 3.) The total percentages (ROS+ plus ROS-) are not 100% and are even not equal between treatments. The shortened percentage may be due to the dead cells. This would lead to mis-calculate the statistics.
12. Page 9, line 222. 1.) Please add a reference for ascorbic acid as a positive control. ; 2.) there is no result of ascorbic acid in figure 9.
13. Figure 9. The authors should explain the statistical significance showed in lower panel.
14. Page 10, line 234. Please add a reference for “Phosphorylation of histone H2A.X at serine 139 is an important indicator of DNA damage.”
15. Page 10, line 238. Please add a reference for etoposide (a well-known DNA damage drug)
16. Figure 10. The authors should discuss why percentage of non-expressing H2A.X is gradually increased upon increasing dose of the water extract.
17. Figure 11. The overall percentage is not 100%. It may be due to the sub-G1 phase? The authors may show the representative cell cycle profile histograms or add the percentages of sub-G1 in the bar chart.
18. Page 12, line 263. Please add a reference for qPCR human apoptosis array.
19. Figure 12. Whether the authors did perform 3 independent experiments for figure 12? Would the authors provide the raw data of Figure 12.
20. Page 13, line 303. “arrest the cell cycle of cancer cells in the G2/M phase”, suggested to revise to specific HeLa S3 cells.
21. Page 13, line 328-3. The authors should rephrase the sentence “Our gene expression analysis revealed that SKOV-3 cell death is induced by death receptor (DR) Fas (CD95) and TNFRSF6/10…”.

The authors did observe the upregulation of TNF receptor family (death receptor (DR), Fas (CD95), and TNFRSF6/10) upon treatment with the water extract. However, the authors did not demonstrate the correlation between the water extract-induced death of SKOV3 and the TNF receptor family.

22. The authors should further investigate whether the daigremontiana water extract-induced cell death of SKOV3 is mediated by TNF receptor family.
23. In this manuscript, the authors showed that the water extract may induce increases is ROS and also possess antiradical activity. The authors should discuss the possible mechanisms for daigremontiana to exert both ROS-induced and antiradical activities.

Author Response

REVIEWER 1

We would like to thank for critical reading this manuscript and valuable suggestions. We have carefully considered all of the suggestions and made the appropriate corrections.

 

The research article by Justyna et al., entitled “In vitro anticancer, antioxidant and phytochemical study on water extract of Kalanchoe daigremontiana Raym.-Hamet & H. Perrier” has been reviewed.

The major issue of this manuscript is the novelty and significance parts. The novelty of the manuscript is not very high. The authors did mention and discuss the previous study (Ref. 31 in the manuscript) which may make this manuscript a followed-up study or extended study, but the content of the manuscript showed just simply a small extension of the previous one. In authors’ previous study (Ref. 31), they investigated the effects of bersaldegenin-1,3,5-orthoacetate on DNA damage, cell death and cell cycle arrest in HeLa cells. Though using bersaldegenin-1,3,5-orthoacetate instead of the K. daigremontiana water extract, several parts are similar to the previous one. In current manuscript, the authors investigated the water extract of K. daigremontiana on DNA damage, cell death and cell cycle arrest in SKOV3 cells. However, according to the information of Table 1, bersaldegenin-1,3,5-orthoacetate is one of the dominant compounds in K. daigremontiana water extract, ~32.47% content (757.4 ± 18.7/2332.1 ± 30).

In addition, the structure and coherence of the manuscript are weak. The authors did perform a lot of experiments, however, the organization of the results and the hypothetic model are not properly represented in this manuscript. Also, the abstract lacked a conclusive pat and should be more organized with a focus on the cytotoxic mechanism of K. daigremontiana water extract against SKOV3 cells.

Response: In our manuscript we present the cytotoxic activity of K. daigremontiana water extract which is commonly used in the treatment of many disorders and is more and more popular in many countries. The species has biological activities and among them is cytotoxic action, reported in the literature but not studied in more detail. Thus, we decided to show selected cellular factors that play a role in this cytotoxic activity despite our previous research on bersaldegein-1,3,5-orthoacetate - the main steroidal compound in the complex of the plant bufadienolides. Generally, plant extracts may act in a different way than their main metabolites. Others compounds (in K. daigremontiana there are mainly flavonoids and phenolic acids) may be responsible for the biological effect of the whole extract. To check if this way is different or similar to the main metabolites, we performed research in which we have experiences for many years. Indeed, we agree that the further study should be continued and the role of cellular factors in cell death triggered by the extract should be further developed, however this issue can be the topic of a separate project. Furthermore, we present phytochemical analysis of bufadienolides in the water extract and estimate antioxidant activity – in the case of this plant water extract all these new findings have not been described.

 

In addition, the following are details that are missing or required revision.

1. Although the method used (MTT) is ubiquitous to estimate proliferation and viability, it is actually an indirect measure of these parameters. There are many factors that would influence the MTT activity such as metabolic status, mitochondrial activity, and so on. Moreover, MTT couldn’t detect dead cells. Therefore, whether it is appropriate to speak of the results of MTT equal to the antiproliferative activity needs to be considered. (Page 3, line 96.)

 

Response: The discussion on MTT assay has been around for years. We know that this method in not ideal and has many disadvantages (please see also our work:  Stefanowicz-Hajduk et al. Toxicology Reports 2020, 7:335-344), however it is still used in many studies as the basic method to estimate proliferation and viability of cells treated with compounds/extracts. In Figure 1 we have changed the percentage of cell proliferation to “cell viability” as it is shown in most papers concerning cytotoxic activity of compounds/extracts and also we described the results in the text as percentage of cells viability.

 

 

 

1. Why the authors choose SKOV3 cells, an ovarian cancer cell line, in this manuscript?

 

Response: We have chosen SKOV-3 cell line due to the results obtained in our previous study (Justyna Stefanowicz-Hajduk , Anna Hering , Magdalena Gucwa , Rafał Hałasa, Agata Soluch , Mariusz Kowalczyk , Anna Stochmal, Renata Ochocka. Biological activities of leaf extracts from selected Kalanchoe species and their relationship with bufadienolides content, Pharmaceutical Biology, 58:1, 732-740, DOI: 10.1080/13880209.2020.1795208). In that study, we performed screening of cytotoxic activity of different Kalanchoe extract on HeLa, SKOV-3, MCF-7 and A375 cells. In the case of K. daigremontiana extracts, the water extract appeared to be the most active on SKOV-3 cell lines, thus we decided to continue the study with this extract to show its effect on selected cellular factors having an important role in ovarian cell death. This manuscript is the continuation of the topic that we have dealed with for several years.     

 

 

1. Figure 1. The labels and legend of figure 1 are so confusing. I would suggest the authors revise this figure thoroughly. In addition, the statistics is required to see the differences between treatments.

 

Response: The Figure 1 has been corrected. The statistical analysis (Anova with post hoc Tuckey’s test, p<0.05) did not show differences among the results.

 

1. Page 1, line 115. Although the authors said that they did not observe significant changes in the drug's effect in vitro in the presence of the extract, I noticed that there seems difference between kd extract alone and kd+cisplatin in “Figure 1C, group 9”.

 

Response: We have performed statistical analyses (ANOVA with post hoc Tuckey’s test, p<0.05) and the results did not show differences among the results. This sentence has been added in the text.

 

1. Page 4, line 114-117. “We also estimated the effect of daigremontiana water extract on the cytostatic action of paclitaxel and cisplatin in SKOV-3 cells.” please add the references for cytostatic action of paclitaxel and cisplatin. And, the authors should describe the mechanism of these two well-known cytostatic dugs, and compare and discuss the results.

 

Response: The references have been added in the text. We also shortly discussed the mechanism of action of the cytostatic drugs and the possible explanation of lack of synergistic action of the extract with drugs. However, it was not our goal in this study to compare the mechanism of action of the extract and cytostatics, we show only the effect of this combination on the cells viability.   

 

1. Page 4, line 121. Please add the references for action of annexin and 7-aminoactinomycin (7-AAD).

 

Response: The references have been added in the text.

 

1. Page 4, line 121. “40.0-300.0 μg/mL” please correct to “40-300 μg/mL”

 

Response: We have corrected this.

 

1. Figure 3.: 1.) Please add the information of treatment for 3A, 3B and 3C, respectively.; 2.) Although the authors said that arrows indicate the late apoptotic/necrotic cells (high annexin and high PI staining), I couldn’t see the differences between cell with arrow in 3B or 3C.; 3.) To my knowledge, the methods used annexin V with 7-ADD (Figure 2) or PI (Figure 3) are basically the same, therefore, it’s redundant to show both figure 2 and 3.

 

Response: We have corrected and added necessary information in the figure legend. The Fig. 3  is presented in this manuscript to confirm the effect of the extract on SKOV-3 cells presented in the Fig. 2 and to highlight the tendency in the extract action. We have also improved quality of this picture to show increasing amount of late/necrotic cells in the tested cell populations (treated with the extract at concentrations from 0 to 70 µg/mL).    

 

 

1. Figure 5. 1.) The authors should explain how to translate the caspase-3/7 activity to live/early/late/dead cells in figure 5. ; 2.) 2.) The representative histogram should be shown side by the bar chart. (ex. figure 2).; 3.) The authors should compare and discuss the meaning of results showed in Figures 2 & 5.

 

Response: We have added this information and placed histograms in the figure. We also compared the results showed in Fig. 2 and 5.

 

1. Figure 6. 1.) label error: two 100 ug/mL; 2.) the author should describe how they plot the boundary to define the four quadrants.

 

Response: The Figure 6 has been corrected. To improve quality we show only the plots from control and samples treated for 24 h (according to the comment of other Reviewer). We also shortly described the rule of boundary settings. 

 

1. Figure 8. 1.) Why do the authors choose HaCaT cells to investigate the ROS effect of the extract and the evaluation and correlation with the effects in SKOV3 cells.; 2.) Please add the abbreviations for ME, k.d.; 3.) The total percentages (ROS+ plus ROS-) are not 100% and are even not equal between treatments. The shortened percentage may be due to the dead cells. This would lead to mis-calculate the statistics.

 

Response: In our study we determined ROS induction in cancer SKOV-3 cell line. Also, we wanted to perform the experiments with non-cancer cells, thus we chose human keratinocytes HaCaT. The abbreviation for ME has been added. The total percentage are not 100%, since in the treatment of the cells with the higher doses of the extract the amount of cellular debrises significantly increases. We performed this flow cytometry analyses according with the manufacturer’s instruction: https://www.luminexcorp.com/muse-oxidative-stress-kit/?wpdmdl=36084).

 

 

1. Page 9, line 222. 1.) Please add a reference for ascorbic acid as a positive control. ; 2.) there is no result of ascorbic acid in figure 9.

 

Response: We have added the references in the text and results of ascorbic acid in Fig. 9.

 

1. Figure 9. The authors should explain the statistical significance showed in lower panel.

 

Response: We have explained the statistical significance in this Figure.

 

 

1. Page 10, line 234. Please add a reference for “Phosphorylation of histone H2A.X at serine 139 is an important indicator of DNA damage.”

 

Response: The reference has been added.

 

 

1. Page 10, line 238. Please add a reference for etoposide (a well-known DNA damage drug)

 

Response: We have added the reference.

 

1. Figure 10. The authors should discuss why percentage of non-expressing H2A.X is gradually increased upon increasing dose of the water extract.

 

Response: We have described this in the Results section.

 

1. Figure 11. The overall percentage is not 100%. It may be due to the sub-G1 phase? The authors may show the representative cell cycle profile histograms or add the percentages of sub-G1 in the bar chart.

 

Response: We have added histograms in the Fig. 11.

 

1. Page 12, line 263. Please add a reference for qPCR human apoptosis array.

 

Response: The references have been added in the text.

 

 

1. Figure 12. Whether the authors did perform 3 independent experiments for figure 12? Would the authors provide the raw data of Figure 12.

 

Response: We performed two independently experiments. Below we provide the raw data from RT-PCR analysis.

 

 

Control			Sample K. daigremontiana extract
the results of CT (average values from two experiments)			the results of CT (average values from two experiments)
Target Name	Cт		Target Name	CT
18S	15,47148991		18S	14,96766
GAPDH	23,97251987		GAPDH	23,98244
HPRT1	27,96638012		HPRT1	26,95391
GUSB	28,93729496		GUSB	28,95425
BIRC2	28,48172092		BIRC2	27,96991
APAF1	31,48576641		APAF1	31,98786
BAD	33,9848938		BAD	34,98297
BAK1	30,46984291		BAK1	29,98886
BAX	29,9771595		BAX	30,47644
BBC3	34,47091103		BBC3	33,98269
BCAP31	27,97041225		BCAP31	26,96907
BCL10	31,44435787		BCL10	31,45245
BCL2	33,96666336		BCL2	33,96705
BCL2A1	36,93249321		BCL2A1	35,93728
BCL2L1	30,49832058		BCL2L1	30,49552
BCL2L11	33,94406319		BCL2L11	33,44438
BCL2L13	30,45150948		BCL2L13	30,94988
BCL2L2	32,47553539		BCL2L2	31,97751
BCL3	29,96706486		BCL3	30,98421
BID	33,48080635		BID	32,98712
BIK	35,46869278		BIK	35,95736
NAIP	36,46675682		NAIP	36,9812
BIRC3	31,95112514		BIRC3	30,94749
XIAP	31,4533577		XIAP	30,95033
BIRC5	28,48004055		BIRC5	28,97497
BIRC6	29,95993233		BIRC6	28,94761
BNIP3	27,94617748		BNIP3	27,9297
BNIP3L	26,95663071		BNIP3L	26,96047
BOK	33,48851395		BOK	33,96825
NOD1	35,94499779		NOD1	34,94915
CARD6	33,97440529		CARD6	33,972
CASP1	32,94318962		CASP1	31,93044
CASP10	32,94798088		CASP10	33,44522
CASP2	31,46324062		CASP2	31,96295
CASP3	28,94543266		CASP3	28,94352
CASP4	29,45704937		CASP4	28,46916
CASP6	29,94808102		CASP6	30,94056
CASP7	30,96648788		CASP7	30,96974
CASP8	30,45553303		CASP8	29,94928
CASP8AP2	31,93758011		CASP8AP2	31,94632
CASP9	33,51523399		CASP9	36,00907
CFLAR	29,958745		CFLAR	29,95956
CHUK	35,95380974		CHUK	35,44851
CRADD	32,98007393		CRADD	31,98581
DAPK1	32,96313667		DAPK1	32,96502
DEDD	29,96847248		DEDD	29,96998
DEDD2	33,4739151		DEDD2	33,97695
DIABLO	29,95250988		DIABLO	29,96319
IFT57	29,4632082		IFT57	28,95826
FADD	32,48779869		FADD	33,47309
FAS	32,9632988		FAS	31,96275
HIP1	34,4681015		HIP1	34,97891
HTRA2	31,96972752		HTRA2	31,97581
IKBKB	31,4405098		IKBKB	31,97578
IKBKE	33,4475174		IKBKE	33,93613
IKBKG	30,96337509		IKBKG	30,96002
LRDD	34,97124481		LRDD	35,97417
LTA	36,47193718		LTA	36,43986
LTB	36,97594833		LTB	35,97554
MCL1	29,47623634		MCL1	28,48695
NLRT1	34,48204041		NLRT1	33,98666
NFKB1	29,96063805		NFKB1	29,47987
NFKB2	30,9777422		NFKB2	29,9961
NFKBIA	29,47056103		NFKBIA	28,96069
NFKBIB	33,46644402		NFKBIB	32,98025
NFBIE	34,96891403		NFBIE	34,46626
NFKBIZ	28,9449234		NFKBIZ	29,94668
PA15	27,97576618		PA15	26,96478
PMAIP1	28,98020077		PMAIP1	27,47952
REL	30,91921806		REL	30,94453
RELA	29,45736122		RELA	28,96925
RELB	33,98875999		RELB	32,98657
RIPK1	30,97151279		RIPK1	31,47114
RIPK2	29,44279003		RIPK2	28,93424
TBK1	29,94403172		TBK1	29,96714
TNF	32,98012924		TNF	32,48788
TNFRSF10	32,96786308		TNFRSF10	31,98492
TNFRSF10B	30,46632099		TNFRSF10B	29,47158
TNFRSF1A	28,47664165		TNFRSF1A	28,47233
TNFRSF1B	36,9646492		TNFRSF1B	37,00249
TNFRSF21	29,9684391		TNFRSF21	29,96752
TNFRSF25	32,98087502		TNFRSF25	33,48155
TNFSF10	30,93126965		TNFSF10	32,93324
TRADD	33,47594643		TRADD	33,95331

 

 

 

 

1. Page 13, line 303. “arrest the cell cycle of cancer cells in the G2/M phase”, suggested to revise to specific HeLa S3 cells.

 

Response: This information has been added in the text.

 

 

1. Page 13, line 328-3. The authors should rephrase the sentence “Our gene expression analysis revealed that SKOV-3 cell death is induced by death receptor (DR) Fas (CD95) and TNFRSF6/10…”.

The authors did observe the upregulation of TNF receptor family (death receptor (DR), Fas (CD95), and TNFRSF6/10) upon treatment with the water extract. However, the authors did not demonstrate the correlation between the water extract-induced death of SKOV3 and the TNF receptor family.

22. The authors should further investigate whether the daigremontianawater extract-induced cell death of SKOV3 is mediated by TNF receptor family.

 

Response to 21 and 22: In our study we tested a panel of genes and observed that the extract increased the expression level of the genes that belong to the TNF receptor family. Based on these RT-PCR results, we show in this manuscript only the possible beginning of induction of cells death after the extract treatment. In Discussion section we present that molecular mechanism connected with TNF receptor and non-apoptotic death is unclear. We agree that this result should be evaluated, however this important and extensive topic can be estimated in the next research project.

 

 

23. In this manuscript, the authors showed that the water extract may induce increases is ROS and also possess antiradical activity. The authors should discuss the possible mechanisms for daigremontianato exert both ROS-induced and antiradical activities.

 

Response: We have discussed this in the Discussion section.

 

We thank the Reviewer for all suggestions and hope that the revised manuscript is now appropriate for publication.

Sincerely,

Justyna Stefanowicz-Hajduk, Ph.D.

Assistant Professor,

Department of Biology and Pharmaceutical Botany,

Medical University of Gdansk, Gdansk, Poland

 

Reviewer 2 Report

In this manuscript titled “In vitro anticancer, antioxidant and phytochemical study on 2 water extract of Kalanchoe daigremontiana Raym.-Hamet & H. 3 Perrier” is reported the phytochemical composition of K. daigremontiana water extract by the UHPLC-QTOF-MS and estimated cytotoxic activity of the extract  on human ovarian cancer SKOV-3 cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo lium bromide) assay, flow cytometry, luminometric, and fluorescent microscopy techniques. The antioxidant activity was assessed with flow cytometry on human keratinocytes HaCaT cell line. The DPPH  (2,2-diphenyl-1-picrylhydrazyl) radical and FRAP (Ferric Reducing Antioxidant Power) assays  were also applied as conferm of antioxidant activity.

 

The subject is suitable for publication in the journal. However, some changes should be introduced.

 

 

-Lines 81: rewrite the sentence according to the right order of instrument. The analysis with HPLC coupled to mass spectrometry.

-In Table 1, some compounds are reported as <LOQ. In the manuscript is not reported the LOQ an LOD of the method of UHPLC-QTOF-MS analysis.  Where is reported the validation of the methods?

-Figures  2, 6, and 7 need to be redone. The quality of the images even by computer is not

optimal.

Author Response

REVIEWER 2

We would like to thank for critical reading this manuscript and valuable suggestions. We have carefully considered all of the suggestions and made the appropriate corrections.

 

In this manuscript titled “In vitro anticancer, antioxidant and phytochemical study on 2 water extract of Kalanchoe daigremontiana Raym.-Hamet & H. 3 Perrier” is reported the phytochemical composition of K. daigremontiana water extract by the UHPLC-QTOF-MS and estimated cytotoxic activity of the extract  on human ovarian cancer SKOV-3 cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo lium bromide) assay, flow cytometry, luminometric, and fluorescent microscopy techniques. The antioxidant activity was assessed with flow cytometry on human keratinocytes HaCaT cell line. The DPPH  (2,2-diphenyl-1-picrylhydrazyl) radical and FRAP (Ferric Reducing Antioxidant Power) assays  were also applied as conferm of antioxidant activity.

 

The subject is suitable for publication in the journal. However, some changes should be introduced.

 

 

-Lines 81: rewrite the sentence according to the right order of instrument. The analysis with HPLC coupled to mass spectrometry.

Response: We have corrected the sentence.

-In Table 1, some compounds are reported as <LOQ. In the manuscript is not reported the LOQ an LOD of the method of UHPLC-QTOF-MS analysis.  Where is reported the validation of the methods?

Response: The method was not yet fully validated. Thus, there is no report on validation results included with the manuscript. We calculated the limits of quantitation according to the ICH guidelines based on the standard deviation of the response (LOQ=3.3*a/s, where a is the slope of calibration curve and s is the response SD), obtained from the residual standard deviation of the regression line.

-Figures  2, 6, and 7 need to be redone. The quality of the images even by computer is not

optimal.

Response: The figures have been redone. In Fig. 2 we have enlarged the plots, in Fig.7 we have reduced the number of plots and enlarged others to improve the quality, in Fig. 7 we also enlarged the plots. However, the plots from flow cytometry analysis presented in the manuscript are copied from Muse Software and in this case we can not improve the smallest letters and numbers in the graphs.

 

We thank the Reviewer for all suggestions and hope that the revised manuscript is now appropriate for publication.

Sincerely,

Justyna Stefanowicz-Hajduk, Ph.D.

Department of Biology and Pharmaceutical Botany,

Medical University of Gdansk, Gdansk, Poland

Reviewer 3 Report

After careful revision of the manuscript by Stefanowicz-Hajduk entitled ‘In vitro anticancer, antioxidant and phytochemical study on water extract of Kalanchoe daigremontiana Raym.-Hamet & H. Perrier’, I suggest to accept the manuscript for publication after revision of few  minor points, as reported below. Indeed, although some studies on this topic have been already done, the findings reported in the present work could be of interest for researchers in the field.

 

-Abstract: use only abbreviation or full name for MTT, FRAP, DPPH and so on. The information of both full name and abbreviation is better in the main text;

-I suggest to add a sentence about future perspectives/possible applications, in the bottom of the introduction section. Moreover, it would be better to revise the last sentence (page 2, lines 76-77;

-Page 2, lines 80-82: revise part of the sentence;

-Page 3, line 96: use italics for scientific name of species. Check this in overall text;

-Figure 1: I suggest to revise Fig. 1, in order to report on x axis, the concentration: (i. e. ctrl, 5, 10, 15, 20, 30, 50, 100 and 200 μg/mL) instead of numbers from 1 to 9, as already done for Fig. 2. Revise also Fig. legend accordingly. Moreover, do the values represent the means ± SEM of at least three independent experiments? This information is required also for other Figures. Add in the Figure legend;

-Page 4, lines 123-128: add ‘%’ or ‘μg/mL’ after each number; check this and modify in overall text;

-Page 4, lines 114-117: the authors estimated the effect of K. daigremontiana water extract on the cytostatic action of both paclitaxel and cisplatin in SKOV-3 cells, concluding that no significant changes were observed alone or in combination. How the authors justify these findings? Moreover, why the authors decide to assay different concentration paclitaxel and cisplatin? Wouldn't it have been better to test the same concentrations? Please, explain/ clarify;

-Page 4, lines 127-128: change ‘highest extract concentrations - 200 and 300 μg/mL, respectively 127 (Figure 2)’ by ‘higher extract concentrations (200 μg/mL and 300 μg/mL, respectively), Figure 2’;

-Figure 3. Please provide images with higher resolution. Revise also the arrows (better if smaller), moreover, add the scale bar reporting also the µm of scale bar and the microscope in the Figure legend;

-When statistical analysis was performed, indicate the method used in the Figure legend; and revise the meaning of asterisks: e. g. *p < 0,05; **p < 0,01; *** p < 0,001; **** p < 0,0001 vs. control, depending on analysis and number of asterisks.

-I suggest to add a sentence about future perspectives/possible applications, in the bottom of the discussion section.

Author Response

REVIEWER 3

We would like to thank for critical reading this manuscript and valuable suggestions. We have carefully considered all of the suggestions and made the appropriate corrections.

 

After careful revision of the manuscript by Stefanowicz-Hajduk entitled ‘In vitro anticancer, antioxidant and phytochemical study on water extract of Kalanchoe daigremontiana Raym.-Hamet & H. Perrier’, I suggest to accept the manuscript for publication after revision of few  minor points, as reported below. Indeed, although some studies on this topic have been already done, the findings reported in the present work could be of interest for researchers in the field.

 

-Abstract: use only abbreviation or full name for MTT, FRAP, DPPH and so on. The information of both full name and abbreviation is better in the main text;

Response: According to Molecules guidelines for Authors, any “abbreviations in the abstract should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table”. 

-I suggest to add a sentence about future perspectives/possible applications, in the bottom of the introduction section. Moreover, it would be better to revise the last sentence (page 2, lines 76-77;

Response: We have corrected this and added a sentence.

-Page 2, lines 80-82: revise part of the sentence;

Response: We have revised this sentence.

-Page 3, line 96: use italics for scientific name of species. Check this in overall text;

Response: We have corrected this.

-Figure 1: I suggest to revise Fig. 1, in order to report on x axis, the concentration: (i. e. ctrl, 5, 10, 15, 20, 30, 50, 100 and 200 μg/mL) instead of numbers from 1 to 9, as already done for Fig. 2. Revise also Fig. legend accordingly. Moreover, do the values represent the means ± SEM of at least three independent experiments? This information is required also for other Figures. Add in the Figure legend;

Response: We have redone the Fig. 1 and added necessary information in the other figure legends.

-Page 4, lines 123-128: add ‘%’ or ‘μg/mL’ after each number; check this and modify in overall text;

Response: We have corrected this.

-Page 4, lines 114-117: the authors estimated the effect of K. daigremontiana water extract on the cytostatic action of both paclitaxel and cisplatin in SKOV-3 cells, concluding that no significant changes were observed alone or in combination. How the authors justify these findings? Moreover, why the authors decide to assay different concentration paclitaxel and cisplatin? Wouldn't it have been better to test the same concentrations? Please, explain/ clarify;

Response: We performed statistical analysis of the results (Anova with post hoc Tuckey’s test, p<0.05) and did not observe significant differences among the results. We decided to tested different concentrations of cytostatics due to different IC₅₀ values of these drugs on SKOV-3 cells found in the literature (according to Smith J.A., Ngo H., Martin M.C., Wolf J.K. An evaluation of cytotoxicity of the taxane and platinum agents combination treatment in a panel of human ovarian carcinoma cell lines. Gynecologic Oncology 2005, 98, 141-145).

-Page 4, lines 127-128: change ‘highest extract concentrations - 200 and 300 μg/mL, respectively 127 (Figure 2)’ by ‘higher extract concentrations (200 μg/mL and 300 μg/mL, respectively), Figure 2’;

Response: We have corrected this.

-Figure 3. Please provide images with higher resolution. Revise also the arrows (better if smaller), moreover, add the scale bar reporting also the µm of scale bar and the microscope in the Figure legend;

Response: We have improved the quality of these images and added the necessary information.

-When statistical analysis was performed, indicate the method used in the Figure legend; and revise the meaning of asterisks: e. g. *p < 0,05; **p < 0,01; *** p < 0,001; **** p < 0,0001 vs. control, depending on analysis and number of asterisks.

Response: We have corrected this.

-I suggest to add a sentence about future perspectives/possible applications, in the bottom of the discussion section.

Response: We have added this sentence.

 

We thank the Reviewer for all suggestions and hope that the revised manuscript is now appropriate for publication.

Sincerely,

Justyna Stefanowicz-Hajduk, Ph.D.

Department of Biology and Pharmaceutical Botany,

Medical University of Gdansk, Gdansk, Poland