In Vitro Cytotoxic Effects of Secondary Metabolites Present in Sarcopoterium Spinosum L.

Round 1

Reviewer 1 Report

Dear Author, I carefully read the manuscript. I think that in this form is not suitable for publication. I attack PDF version of the manuscript with my suggestion. The title is not clear, please be more specific. In the abstact the aim of the work is really confused. You don’t perform a purification of compound and you consider only three compound present in the extract. The material and methods is incomplete.  It is strange that you perform a column chromatography and you collect only 2 fractions. From the paper I understand that you load 60 G of extract in a column 2X50 cm. I think it is not realistic. How you perform the identification of the three compound? How you quantified them? Why you do spectra of pure compound, even you can found them in literature? There is no the section in which you discuss how you calculate cell survival. What is the rational to mix this compound? NMR part should be discuss properly. NMR table are imprecise without multiplicity and J coupling. There are missing data in the tables of in vitro activity. Discussion are partial because it gives information on the activity of the compounds in general ( like in a introduction) but there is not discussion of obtained data of this work. The conclusion is weak because the aim is not clear.

Comments for author File: Comments.pdf

Author Response

Thank you very much for your comments, we accepted them all and edited the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled "Individual and combined in vitro cytotoxic effects of secondary metabolites from Sarcopoterium spinosum L. in cancer cells" by Hudec et al. describes the cytotoxic activity of an ethyl acetate fraction of S. spinosum and the three major compounds identified in it. The work is well written, and the results are cklear, but thereare some minor comments to do.

1- The work reported cytotoxicity agains Hela, MCF-7, and A-549 cell lines, but toxicity has been observed also against normal cells. This could be a great problem, the authors should better clarify this aspect in discussion section, because this is not clear.

2- Conclusions are not enough for the work. The authors should improve this section adding specific comments.

 I think this work is suitable for publication after these specific additions.

Author Response

Thank you very much for your comments, we accepted them all and edited the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

This original research article submitted by Hudec et al., presents their current discovery about the discovery of three molecules identified for the first time from the extracts of Sarcopoterium spinosum and their cytotoxic effects as single molecule or in combination against human cancer cell lines. The manuscript is clear and very well written; however, I consider that at the present time the manuscript requires some corrections, additional information and modifications, including an improvement of the picture quality before acceptance.

Editing:

Line 30 – 33: please check your manuscript.

Line 76: Please correct “play”.

Line 81 – 82: please check your manuscript.

Line 106, 253, 266: the quality and resolution of the figures must be improved.

Line 120: what are the yields (in gr) of fractions 18a and 18b?

Line 233 and 234, please delete “233” and “234”.

Line 234, what mean M8R and M8N? please mention the abbreviation in the legend of your table.

Line 306 and line 310: please be coherent with “anti-proliferative” and “antiproliferative”. Please check your manuscript.

Comments:

Line 150: How did you prepare the extracts for bioassays? Did you resolubilize them in water, DMSO or any other solvent? What was the percentage (extract/ final volume, v/v) for the bioassays? Please also include a control solvent only for the cell proliferation assay, as well a positive control.

Line 271: Why did you perform synthesis of stachydrine and not use the one you isolated from the plant? Please detail the reason in your manuscript.

Line 294: You determined the IC50 for HeLa cells. However, the two values 81.2 and 28.1 % are respectively for 10 and 100 µM, a 10-fold factor! Please re-determine the IC50 using at least 2 intermediate concentrations (e.g. 25 and 75 µM).

Why didn’t you use A549 and Caco-2 cells in this experiment, while you use them in the first biological screening. Please explain.

Why didn’t you include rutin in the mixture of stachydrine and chloride benzalkonium?

What is the rationale behind your choice of a ratio of stachydrine – benzalkonium chloride (1:1) for this experiment while you mention the ratio stachydrine – 89%, benzalkonium chloride – 7%, and rutin 4% (line 277). Please explain the reason.

Did you try to test different ratio of mixture in order to see if some ratio are more cytotoxic than other one and see if one molecule can be for example a sensitizing agent (rutin e.g.)?

Author Response

Thank you very much for your comments, we accepted them all and edited the manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear authors,

I read the resubmitted version of your manuscript and I observed that many of my comments and suggestions were completely ignored.

The manuscript in this last version is not suitable for publication because the chemical part was unacceptable. Please provide new version with the requested changes.

Line 31-33: “The first time, in these extracts were identified by the NMR analyses in the most cytotoxic sub-fraction of S. spinosum three compounds (stachydrine, benzalkonium chloride, and rutin).” This sentence is not clear. Please add a section in material and method section how do you perform the identification of compound. This sentence is not in agreement compared to line 250-253: “H NMR spectra was obtained and it was possible to obtain a general idea of M8R composition, but an identification of the components present in sample from the actual 1H spectra was not possible.”.  

Line 34-35: please be specific. you test the pure compound but you don’t perform the purification. Please correct the sentence.

line 72: “β-Dglucopyranoside” please correct

Line 86-89: “The purpose of the present study was to investigate aerial parts of S. spinosum, to isolate presumably cytotoxic compounds, and perform the identification and subsequent synthesis of the molecule, as a possible way to obtain the basic material for the synthesis of new anti-cancer compounds.” Please mention the in vitro assay that you perform. Please change the sentence because in this work you don’t describe the isolation of compound. Please explain the method that you used for the identification of the compound.

Line 115: “The resulting suspension was successively partitioned 3 more times with ethyl acetate and 1-butanol and the respective extracts combined.” Please be more specific in the description of this part. I suppose that ethanol extract was suspended in water. Please add the ratio between solvents for the partition.

Line 120-121: “Since the highest cytotoxic activity was found in the AF, this fraction was subjected to column chromatography (CC) using silica gel (75-150 μm, 2x50 cm, flow rate 0.7 mL/min). You write that fraction AF is 60g. I understand that you load 60 g of extract in a column 2X50 cm. I think it is not realistic.

The section 2.6 NMR analysis contains errors. In particular, here I report line 179: “Varian VNMRS 600 instrument at the Faculty of Chemical and Food Technology STU in Bratislava” and line 191: “NMR spectra of the major compound of sub-fraction M8R, proline betaine, synthesized as iodide at the Faculty of Science, Palacký University in Olomouc were obtained using a JEOL 400 MHz instrument (JEOL, Japan). Please write clearly the instrument that you use for analysis.

Line 197-206: this paragraph is not unacceptable in this form. Please write clearly the instrument that you use, the sample that you analyzed, how you set the instrument for analysis, the chromatographic column, the eluent gradient, the use of standard compound.

Line 199: electrospray interference (ESI) please change with ion sources

Line 237, Table 1: please write what correspond to M8 and M10. I don’t found any information in the text.

Line 252-253. Please write how you identify compound.

Figure 2: please provide information on maximized part of the spectra. Explain figure in the caption.

Legend of figure2 : alkane chain can be correlated with the aliphatic chain of benzalconium chain. Why you assigned selected signals to phtalate?

Figure 2: Please improve the quality of the NMR figure.

Figure 2: please use numbers to identify NMR peaks that correspond to each compound.

Table 2: table contains data already present in literature.  please move them in supplementary materials.

Line 263, legend of figure 2: please acquire NMR spectra of pure compound using MeOD_4, the same solvent that you use for NMR of fraction M8R. in this way you can do comparison of spectra.

Line 279: “M8R sub-fraction were as following: stachydrine – 89%, benzalkonium chloride – 7%, and rutin – 4%”. Add in ‘material and methods’ section the techniques that you use to measure compounds in fraction M8R.

Author Response

Comments to Reviewer 1

Thank you for an insightful review and all suggestions. We agree the suggested changes will contribute to the improvement of our paper. We hope you will find our improvements appropriate and comprehensive.

The abstract, methods and other sections have been reformatted to better present information and was added or specified also some details according to the comment of the reviewer.

Line 29: Yes, we meant ethanol. Everything is OK because according to the diagram in Fig. 1 petroleum ether and ethanol were first used and these fractions were tested (MTT assay), followed by ethylacetate, 1- butanol and water and those fractions were tested, and the subfractions obtained after CC fractionation were again tested.

Lines 30-35 have been modified: ..MTT assay. The ethanol extract was subsequently re-extracted with ethylacetate and in its sub-fraction obtained by column chromatography three compounds (stachydrine, benzalkonium chloride and rutine) were the first time identified by NMR analyses. The most active sub-fraction showed cytotoxic activity against HeLa, MCF-7, and Caco-2 cell lines. The three compounds mentioned, as standards of HPLC quality, were studied individually and in combination. Cytotoxic.... (Corrected)

To the original lines 34-35 : The amounts of raw material and the most active sub-fraction isolated from it was sufficient only to identify the active compounds and to determine cytotoxic properties of sub-fraction. Stachydrine subsequently has become the basic molecule to obtain new, more potent cytostatics. Therefore it was necessary to prepare stachydrine in bigger quantities by synthesis. Also was used for cell proliferation assay.

The sentence from lines 39-40 was removed: … extract of S. spinosum remains under-investigated, so this research describes that the three major compounds identified in the ethyl acetate extract can exert a significant dose dependent in vitro cytotoxicity.

Line  72 : corrected - .... of beta-D-glucopyranoside    (dash between D and g)

Line 77 : corrected - ...medicinal practice   

Line 81 : corrected: Durodola showed significant tumor inhibitory activity of crude extract of S. spinosum in experimental animals [20].

Line 86-89 were corrected : „The purpose of the present study was to investigate aerial parts of S. spinosum, perform an in vitro cell proliferation assay testing all extracted fractions and subfractions, perform the identification and subsequent synthesis of the molecule, as a possible way to obtain the basic material for the synthesis of new anti-cancer compounds, and study three identified compounds in the most cytotoxic subfraction individually and in combination.“              

Line 115 : L 112-115 modified - ... successsively partitioned in 3 sequentially partitions with ethyl acetate .....    

Line 117-121 : supplemented -  ...(CC) , repeated many times due to the larger amount of extract, using silica gel (75-150 μm, 2x50 cm, flow rate 0.7 mL/min), the dose of solid AF was 0.5-0.7 g/column, firstly eluted ...   

Line 120-121: Since the highest cytotoxic activity was found in the AF, this fraction was subjected to column chromatography (CC) using silica gel (75-150 μm, 2x50 cm, flow rate 0.7 mL/min). You write that fraction AF is 60 g. I understand that you load 60 g of extract in a column 2X50 cm. I think it is not realistic.  

The loading rate per column was about 500 to 700 mg, but the operation was repeated approximately hundred times, so that the total amount corresponds. However, the final active extract was so few that it was only sufficient to identify the substances present and the biological tests described.

Line 127 : TLC was not used.

Line 151: MTT is a standard colorimetric laboratory assay for quantifying cellular growth, which can be employed for determining the cytotoxicity of potential medicinal agents and other toxic materials. Is based on the reduction of yellow MTT bromide, to purple formazan in the mitochondria of viable cell and conversion can be directly associated with the number of viable cells. The efficacy of the agent that causes cell death can be inferred by the comparison of the amount of purple formazan produced by treated cells, as well as the amount of formazan produced by untreated control cells. 

Line 157: The calculation formula is: cell proliferation rate = (experimental group A540 nm- zero adjustment group A540 nm)/(Control group A540 nm-zero adjustment group A540 nm)×100%.

2.6 section : Everything is listed correctly. The sub-fraction was analyzed in Bratislava. Stachydrine was synthesized at another workplace and therefore to identification of product they used another, their own instrument in Olomouc. 

Varian VNMRS 600 instrument at the Faculty of Chemical and Food Technology STU in Bratislava was used for the identification of the molecule presents in the extract. Based on the results was proline betaine (stachydrin) synthesised at Palacky Universtity. For the confirmation of this structure was used JEOL 400 MHz instrument. This synthesised molecule was analysed by UPLC-MS analysis and MS was measured. (describe in the section 2.7) Method served for the structure confirmations.

Line 175: this is not an assignment of pure compounds, but assignment of them in the M8R fraction.

Line 180: added explanation to the 2D TOCSY method

In the manuscript is written that additional 2D-experiments  were performed  - gCOSY, gDQF- COSY, TOCSY, HSQC ,  (1H-, 13C-) HMBC and (1H-, 15N-) HMBC. TOCSY was used for clarification or aliphatic part from 2 to 4 ppm. 2D TOCSY gives correlations between all protons within a given spin system (for example suggar part or alkyl chain).  

The structure of molecules in the mixture was designed on the results of menitioned  2D-NMR experiments.

Line 186: M8R fraction was analyzed also on Varian VNMRS system at Slovak University of Technology.

Line 233, 234: Sorry, our bad control – number of line is incorrectly in text (233 and 234) – corrected

Dash in the table 1 – was not tested.

Asterisk after the number – statistical significance (p<0.01)

Line 237 : M was written in title (L.106) ...of S. spinosum L. (M) (1.6 kg),

Fr. 18 was designed as M8 (L.127) – was written and Fr. 20 was designed as M10 –it was not written – now under Tab. 1

Line 240 : supplemented in text  - ...than the others, Fr. 18 in all tested cells.

Line 249:  It should be: From series of NMR spectra (standard ¹H and ¹³C, 2-dimensional COSY, HSQC and HMBC) it was possible to obtain a general idea of M8R composition.

L.251 : Therefore were used ¹³C NMR method and adding of standards method. corrected

Fig. 2. and legend of Fig. 2 : Alkane chain is correlated with aliphatic chain of benzalkonium chain. Phtalate signals – indicate the presence of phtalates in the plastic material used to transfer of the sample for analysis and mainly the use of silicone lubricants on glass cuts. corrected

Figure 2.

- Expanded part of spectrum provides better resolution of the 2 methyl signals of stachydrin

- Alkan and benzalkonium alkyl chains are in the spectrum not resolved as is indicated in the spectrum

- This is an overview spectrum and peak picking would it made very messy. Peak picking is shown in supplementary information.

Line 259: Signal assignment is shown in detail in supplementary information.

Line 262: In Table 2, there are shown chemical shifts of stachydrin and rutin compounds present in M8R fraction in methanol.

It should be:

^a Chemical shift in δ scale (ppm). ^b Location (structure in Figure 3). NMR spectra of M8R sub-fraction were obtained at 600 MHz in deuterated methanol (Met-d4).

 Line 263 : Comparisons are also possible with date from catalogs using the same conditions as specified therein.

Line 279 : supplemented in Material and Methods: Quantification of the compounds in Fr. 18 was performed using the synthesized stachydrine and purchased standards of rutin and benzalkonium chloride, which simultaneously repeatedly confirmed their presence in fraction.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Dear author, thank you for provide the implemented version.

I think that the manuscript is improved but it is not acceptable for publication. The chemical part in the material and method section is not acceptable.  In section 2.7 the sample preparation is completely missing. How you prepare sample for analysis?

Line 199: “Analysis of synthesized stachydrine hydroiodide was also performed using a Waters 199 Acquity UPLC-PDA-QDA system (Waters, USA) equipped with an ion trap mass analyser.” QDA detector is different from ion trap analyser.  You write that you use photodiode array detector, but in the text you never mention it. Please be specific.

Line 200: “The system used electrospray interference (ESI) in positive and negative mode.” Electrospray ION SOURCE.

Line 202:” Analyses were performed for the following conditions: pre-separation on X Select HSS T3 column (2.5 μm, 3x50 mm, 30 °C). “ what you mean with pre-separation?

“Mobile phase: linear gradient from 20 to 80% of acetonitrile in 0.01 M ammonium acetate” please add time of gradient.

“MS parameters: TESI 600 °C, Tcap 450 °C” these temperatures are not realistic with my experience in the field.

Line 205 “Major compounds: UPLC-(+ESI)-MS: 0.607 min and UPLC-(-ESI)-MS: 0.449 min.” what are the major compound? Then you report two retention time (I suppose). I don’t understand what you mean. Please add the ion that you use for identify each substance. Please add chromatogram.

The preparation of standard solution and the calibration curves for quantification are missing.

Table 2: multiplicity of NMR signals and J coupling is missing. There is mistake in the format of the table.

I appreciated the content of supplementary material

In table 3 you report the survival of cell %. In material and methods part there isn’t present any paragraph that how you perform this assay.

As I report above, there is many weaknesses of the chemical part, in particular in the UPLC-MS part.  Overall the manuscript content was confused and difficult to understand. There is information missing in material and methods. The reader is not able to understand how you perform experiments.

Author Response

Dear Reviewer 1

Thank you for your insightful feedback. The section 2.7 has been modified and all required information was added in the text. We hope you will find our improvements appropriate and comprehensive.

Line 199: “Analysis of synthesized stachydrine hydroiodide was also performed using rt modified:a Waters 199 Acquity UPLC-PDA-QDA system (Waters, USA) equipped with an ion trap mass analyser.” QDA detector is different from ion trap analyser.  You write that you use photodiode array detector, but in the text you never mention it. Please be specific.

This part modified:

Analysis of stachydrine hydroiodide was also performed using a Waters Acquity  UPLC-PDA-QDA system (Waters, USA). The    system used electrospray ion source  (ESI) in positive and negative mode. Analyses were performed for the following conditions:  security cartrig C18-4x3 mm (Phenomenex) and  X Select HSS T3 column (2.5 196 μm, 3x50 mm, 30 °C). Mobile phase: linear gradient from 20 to 80% of acetonitrile in 0.01 197 M ammonium acetate, and a flow rate 600 μL/min was used. MS parameters: TESI 600 °C, 198 Tcap 450 °C, (±) ESI ±3.0 kV. For the analysis was used solution of  stachydrine hydroiodide 0,5 mg in 1 mL of the solvent (20% acetonitrile and 80% water). Time of the analysis 5.5 min.

Major component:

UPLC-(+ESI)-MS: m/z 144 (M+H⁺); 166 (M+Na⁺); 287 (2M+H⁺); 309 (2M+Na⁺)

UPLC-(-ESI)-MS: m/z 126.8;  140.8; 276.7  

The positive mode was used for the confirmation of stachydrine, and the negative mode was used for the confirmation of the presence of hydrogen iodide.

Line 200: “The system used electrospray interference (ESI) in positive and negative mode.” Electrospray ION SOURCE. supplemented, see above

Line 202:” Analyses were performed for the following conditions: pre-separation on X Select HSS T3 column (2.5 μm, 3x50 mm, 30 °C). “ what you mean with pre-separation?

“Mobile phase: linear gradient from 20 to 80% of acetonitrile in 0.01 M ammonium acetate” please add time of gradient.

“MS parameters: TESI 600 °C, Tcap 450 °C” these temperatures are not realistic with my experience in the field.

Waters Acquility  is used for the rutine analysis at the Department of chemistry. There is  a several methods for the analysis. One of them is a described method. Method was used for the MS identification of the compound together with NMR.

Line 205 “Major compounds: UPLC-(+ESI)-MS: 0.607 min and UPLC-(-ESI)-MS: 0.449 min.” what are the major compound? Then you report two retention time (I suppose). I don’t understand what you mean. Please add the ion that you use for identify each substance. Please add chromatogram.

The preparation of standard solution and the calibration curves for quantification are missing.

Major component:

UPLC-(+ESI)-MS: m/z 144 (M+H⁺); 166 (M+Na+); 287 (2M+H⁺); 309 (2M+Na⁺)

UPLC-(-ESI)-MS: m/z  126.8;  140.8; 276.7  

The positive mode was used for the confirmation of stachydrine, and the negative mode was used for the confirmation of the presence of hydrogen iodide.

• chromatogram was added in supplementary material.

Table 2: multiplicity of NMR signals and J coupling is missing. There is mistake in the format of the table.

Indicating the multiplicity of signals and J coupling (especially if correlation 2D NMR methodologies were used) is not necessary at all.

I appreciated the content of supplementary material -  this material was supplemented

In table 3 you report the survival of cell %. In material and methods part there isn’t present any paragraph that how you perform this assay. We think that's irrelevant.

As I report above, there is many weaknesses of the chemical part, in particular in the UPLC-MS part.  Overall the manuscript content was confused and difficult to understand. There is information missing in material and methods. The reader is not able to understand how you perform experiments.

Thank you for an insightful review and all suggestions. We agree the suggested changes will contribute to the improvement of our paper. We hope you will find our improvements appropriate and comprehensive.

Author Response File: Author Response.pdf