Kaempferol Rhamnosides from Geranium sibiricum as Aldose Reductase Inhibitors and Their Content by HPLC Analysis
Round 1
Reviewer 1 Report
Thank you for the interesting manuscript. Investigations on natural compounds as potential drugs are an important part to evaluate potential benefits of traditional medicines. I have some doubts, if the rather few concentrations used in the IC50 value calculation are enough to differentiate between the effects of the different plant extracts. For example, G. nepalense for which the IC50 value was only slightly higher compared to G. sibiricum might have additional compounds adding to its inhibitory effect since its content of 2 is much lower. For further details, please see the following notes to specific lines:
line 71: please change ‘Darmstädt’ -> ‘Darmstadt’
line 87: please change ‘80°C’ -> ’80 °C’
line 91-93: please provide additional information on the open column clean-up step: which steps were used for the gradient and in which fractions could compounds 1 and 2 be found?
line 93-94: if this refers to a known method, please add the reference(s) for it. If not please give a bit more detail on the crystallization method (e.g. ratio of compound to solvent, temperature and general procedure)
line 102: please change ‘4°C’ -> ‘4 °C’, also a different font is used for °C
line 112-114 and 169-173 (table 3): calculation of IC50 values seems a bit unreliable if done from only three concentrations, especially if these IC50 values shall be used for comparison with other substance for finding new possible pharmacological agents. Furthermore, using only three concentrations gives not a good indication if or which part of the curve (plateau, linear part of the slope etc.) is evaluated. These aspects, along with other challenges for using IC50 values, are often discussed in literature e.g. Kalliokoski et al. 2013 (doi:10.1371/journal.pone.0061007) or Caldwell et al. 2012 (DOI: https://doi.org/10.2174/156802612800672844). Therefore, why have only three concentrations been tested and how does this affect the comparability of this study to other. Since the potent effects of G. sibiricum are pointed out, how reliable is it to assume them to be more potent than those of e.g. G. nepalense under these experimental conditions? How were the error margins of the IC50 values calculated (e.g. standard deviation of technical replicates)? Please comment on this.
line 126: please change ‘30°C’ -> ’30 °C’
line 178-180: for the FAB-MS measurements neither experimental conditions nor results are presented. If these are not necessary for structure identification, they should be omitted from the manuscript. Otherwise the missing aspects should be included.
line 183-187 (table 4): similar to the comments on table 3, how reliable is the assumption of a higher effect of the EtOAc fraction compared to the n-BuOH fraction while using only three concentrations for calculation of IC50 values? How were the error margins of the IC50 values calculated (e.g. standard deviation of technical replicates)?
line 191-195 (figure 2): structural formula for 2 is missing the oxygen in the ring of right hand rhamnose moiety
line 196-204 (table 5): how were the IC50 values in µM calculated? In regards to differences in molecular weight (432.38 g/mol for 1 and 578.52 g/mol for 2) the difference between µM results does not seem to fit to the results in µg/mL. Furthermore, the results in the table do not fit to those in the text. Please comment on this.
line 246: square bracket after ‘13’ is set italic
Author Response
Thank you for the interesting manuscript. Investigations on natural compounds as potential drugs are an important part to evaluate potential benefits of traditional medicines. I have some doubts, if the rather few concentrations used in the IC50 value calculation are enough to differentiate between the effects of the different plant extracts. For example, G. nepalense for which the IC50 value was only slightly higher compared to G. sibiricum might have additional compounds adding to its inhibitory effect since its content of 2 is much lower. For further details, please see the following notes to specific lines:
line 71: please change ‘Darmstädt’ -> ‘Darmstadt’: It was changed.
line 87: please change ‘80°C’ -> ’80 °C’: It was changed.
line 91-93: please provide additional information on the open column clean-up step: which steps were used for the gradient and in which fractions could compounds 1 and 2 be found? line 93-94: if this refers to a known method, please add the reference(s) for it. If not please give a bit more detail on the crystallization method (e.g. ratio of compound to solvent, temperature and general procedure): The detailed step by step information was provided.
line 102: please change ‘4°C’ -> ‘4 °C’, also a different font is used for °C: It was changed.
line 112-114 and 169-173 (table 3): calculation of IC50 values seems a bit unreliable if done from only three concentrations, especially if these IC50 values shall be used for comparison with other substance for finding new possible pharmacological agents. Furthermore, using only three concentrations gives not a good indication if or which part of the curve (plateau, linear part of the slope etc.) is evaluated. These aspects, along with other challenges for using IC50 values, are often discussed in literature e.g. Kalliokoski et al. 2013 (doi:10.1371/journal.pone.0061007) or Caldwell et al. 2012 (DOI: https://doi.org/10.2174/156802612800672844). Therefore, why have only three concentrations been tested and how does this affect the comparability of this study to other. Since the potent effects of G. sibiricum are pointed out, how reliable is it to assume them to be more potent than those of e.g. G. nepalense under these experimental conditions? How were the error margins of the IC50 values calculated (e.g. standard deviation of technical replicates)? Please comment on this.: In this study, three concentrations were only used because we referred to previous similar literatures. The basis of using three concentrations is to primarily evaluate the extract in such a way that if the concentration is increased the inhibition rate will increase as well.
line 126: please change ‘30°C’ -> ’30 °C’: It was changed.
line 178-180: for the FAB-MS measurements neither experimental conditions nor results are presented. If these are not necessary for structure identification, they should be omitted from the manuscript. Otherwise the missing aspects should be included.: FAB-MS was already included.
line 183-187 (table 4): similar to the comments on table 3, how reliable is the assumption of a higher effect of the EtOAc fraction compared to the n-BuOH fraction while using only three concentrations for calculation of IC50 values? How were the error margins of the IC50 values calculated (e.g. standard deviation of technical replicates)?: In this study, three concentrations were only used because we referred to previous similar literatures. IC50 value of EtOAc fraction was comparably higher than that of BuOH fraction. This was our basis that EtOAc fraction has higher effect compare to BuOH fraction.
line 191-195 (figure 2): structural formula for 2 is missing the oxygen in the ring of right hand rhamnose moiety: It was already changed.
line 196-204 (table 5): how were the IC50 values in µM calculated? In regards to differences in molecular weight (432.38 g/mol for 1 and 578.52 g/mol for 2) the difference between µM results does not seem to fit to the results in µg/mL. Furthermore, the results in the table do not fit to those in the text. Please comment on this.: We correct the results (Table 5)
line 246: square bracket after ‘13’ is set italic: It was changed.
Reviewer 2 Report
Title: Kaempferol rhamnosides from Geranium sibiricum as inhibitors of aldose reductase and their content by HPLC analysis
Manuscript ID: processes-813375
The major concern is listed as following:
Figure 1, Figure 1a and 1b reflected the hplc profiles of compound 1 and 2, respectively. From these two figures, their retention time is approximately 27.5 min, and 17.7 min, respectively. However, figure 1c, 1d, 1e, and 1f corresponded the hplc profiles of the methanol extracts of G. eriostemon, G. koreanum, G. nepalense, and G. sibiricum, respectively. In the main text, the author claimed that “Further examination of the bioactive components of the EtOAc fraction of G.sibiricum by open column chromatography yielded compounds 1 and 2 (Figure 1)”. This does not make sense. Because the HPLC profiles did not indicate the presence of peaks (Compound 1 and 2; around 27.5 min, and 17.7 min) in the methanol extract of G. sibiricum. Please provide the direct evidence of the presence of compounds 1 and 2. The reviewer also did not see the strong evidence of compound 1 and 2 according the HPLC profiles.
Author Response
Title: Kaempferol rhamnosides from Geranium sibiricum as inhibitors of aldose reductase and their content by HPLC analysis
Manuscript ID: processes-813375
The major concern is listed as following:
Figure 1, Figure 1a and 1b reflected the hplc profiles of compound 1 and 2, respectively. From these two figures, their retention time is approximately 27.5 min, and 17.7 min, respectively. However, figure 1c, 1d, 1e, and 1f corresponded the hplc profiles of the methanol extracts of G. eriostemon, G. koreanum, G. nepalense, and G. sibiricum, respectively. In the main text, the author claimed that “Further examination of the bioactive components of the EtOAc fraction of G.sibiricum by open column chromatography yielded compounds 1 and 2 (Figure 1)”. This does not make sense. Because the HPLC profiles did not indicate the presence of peaks (Compound 1 and 2; around 27.5 min, and 17.7 min) in the methanol extract of G. sibiricum. Please provide the direct evidence of the presence of compounds 1 and 2. The reviewer also did not see the strong evidence of compound 1 and 2 according the HPLC profiles.: The presence of compounds 1 and 2 in the HPLC profiles were already indicated with arrows in Fig. 1.
Reviewer 3 Report
The topic of this article seems interesting since it investigates the inhibition of aldose reductase (AR) of six chosen Geranium methanolic extracts obtained from the plant aerial parts. The Geranium sibiricum methanolic extract was found to be the most active (higher IC50) therefore it was subjected to fractionation. Again, the extract showing the highest IC50 (the ethyl acetate one) was analyzed with HPLC-UV, NMR and MS spectrometry to identify the major compounds present which have been considered responsible for the AR inhibition.
I have a question.
Five out of the six species analyzed were methanol extract of aerial parts of the plants. Only for the Geranium sibiricum, it was performed an extraction in methanol by the authors themselves. I wonder if the differences found (coincidentally, exactly the Geranium sibiricum was found to be the most active species) could be attributable to different preparation of the extracts.
Moreover, the authors should add some more details concerning the preparation of the standards and the methodologies followed in order to allow other scientists to perform analogue experiments.
Authors should rewrite the abstract.
Specific comments:
Lines 16-17 It is not clear if the bioactive constituents searched are those that inhibit the aldose reductase. Please rewrite this sentence.
Line 18 Please rewrite “with an IC50”
Line 19 It is not clear what “0.41 µg/mL” refers to in this sentence. Is it the IC50 value or the total kaempferol content in the ethyl acetate fraction (the first one)?
Line 21 Please rewrite “were the active”
Line 22 Please rewrite “examined with a concentration in the extracts of 28.1 and 2.2 mg/g respectively.”
Lines 38-39 Please rewrite “sources as they may be a safer and more effective alternative”
Line 42 Please rewrite “have also shown a good AR inhibition”
Lines 44-45 Please rewrite “world and mainly present in Southern Africa.”
Lines 52-53 I think it would be better to add “to our knowledge”.
Line 55 If I understood it correctly you choose G. sibiricum for the following reason “ from the one species, G. sibiricum, that showed the highest IC50 values”. Please rewrite the sentence.
Line 60 “species of the genus “
Line 77” were purchased”
Line 79 "Bruker Avance” Moreover, could you provide more specifications about the liquid NMR analysis?
Line83 It is not clear to me what is this “ARI assay”.
Line 85 I would also add “identification”
Line 86 How did you dry the aerial parts? In an oven? At which temperature? Please add this information.
Line 87 It was filtered on which type of filters? Nylon, RC, etc? Please add this information.
Line 68 Where the authors get (if they did not purchase) the kaempferol derivatives to use as standards in HPLC –UV (in Line 118 and 153-154 they used those as reference compounds)? If they obtained them through purification they should clarify it (see my next comment).
Lines 93-94 It is not clear what the authors mean stating that they performed a recrystallization using MeOH of the two kaempferol derivatives: did they purified the two main compounds found in the ethyl acetate extract of G. sibiricum (which were then identified with MS and liquid NMR)? Please clarify this point.
Line 92 Did authors pack the open columns by themselves or did they bought some already pre-packed ones?
Line 110 TMG please clarify this acronym (thiomethyl galactoside?)
Line 119 What was the material of the filter? Nylon? Please specify.
Line 141 Since there was a difference in the Tr of the compounds 1 and 2 from the standard solutions to the plant extracts it would be nice to identify the two kaempferol derivatives in the C-F chromatograms.
Line 163-164 I think it would be better to add “to our knowledge”.
Line165 Please rewrite ”results of this”
Line 167 The IC50 values found are means of how many repetitions for each extract? Wouldn’t be better to report those values as a mean ± standard deviation (same for values reported in Table 4)? I have always wondered how can one state that the results of IC50 values are different if you did not test if those values were statistically significant?
Line 175-176 Since authors find out that the MeOH extract of G. sibiricum had the highest IC50 they also fractionated the MeOH extract and used the single different fractions to test for their AR inhibition ability. Please rewrite this sentence accordingly.
Line 178 Not by “open column chromatography” but rather by HPLC-UV identification. Please rewrite.
Line 213 Please rewrite “ (1) in the MeOH extract (28.1 mg/g).”
Line 228 Please add a reference.
Line 234 I think it would be better to add “to our knowledge”.
Line 239 Please complete the sentence:“of G. sibiricum showed the highest IC50 values.” or something similar.
Line 243 What do you mean with “their ARI efficacies.”?
Line 244 Please remove this sentence, you might insert this in line 240” (2) two flavonoid compounds present in the”
Line 246 Please rewrite: “[13] present in Geranium species, anyway, the potent AR inhibition showed by the G. sibiricum species can be attributed to its high concentrations of kaempferol-7-O-rhamnoside (1) and kaempferol-3,7-O-dirhamnoside (2) since these two compounds are the major components of the EtOH fraction, the one with higher IC50.” or something similar.
Lines 248-250 Please rewrite: “This species also exhibited the best AR inhibition among all the species examined in this study. These results may have application in the development of novel therapeutic methodologies /approaches to target complications associated with diabetic hyperglycemia.” or something similar.
Author Response
The topic of this article seems interesting since it investigates the inhibition of aldose reductase (AR) of six chosen Geranium methanolic extracts obtained from the plant aerial parts. The Geranium sibiricum methanolic extract was found to be the most active (higher IC50) therefore it was subjected to fractionation. Again, the extract showing the highest IC50 (the ethyl acetate one) was analyzed with HPLC-UV, NMR and MS spectrometry to identify the major compounds present which have been considered responsible for the AR inhibition.
I have a question.
Five out of the six species analyzed were methanol extract of aerial parts of the plants. Only for the Geranium sibiricum, it was performed an extraction in methanol by the authors themselves. I wonder if the differences found (coincidentally, exactly the Geranium sibiricum was found to be the most active species) could be attributable to different preparation of the extracts.
Moreover, the authors should add some more details concerning the preparation of the standards and the methodologies followed in order to allow other scientists to perform analogue experiments.: Originally, we only had some good results from G. sibiricum alone. However, we would like to assess if the same results will be obtain using other species. The high activity of G. sibiricum compare to other species could not be attributable to the preparation of the extracts because we used more or less the same solvent which is MeOH.
Authors should rewrite the abstract.
Specific comments:
Lines 16-17 It is not clear if the bioactive constituents searched are those that inhibit the aldose reductase. Please rewrite this sentence.: It was specified and changed.
Line 18 Please rewrite “with an IC50”: It was changed.
Line 19 It is not clear what “0.41 µg/mL” refers to in this sentence. Is it the IC50 value or the total kaempferol content in the ethyl acetate fraction (the first one)?: It was changed as IC50 value.
Line 21 Please rewrite “were the active”: It was chanhed.
Line 22 Please rewrite “examined with a concentration in the extracts of 28.1 and 2.2 mg/g respectively.”: It was changed.
Lines 38-39 Please rewrite “sources as they may be a safer and more effective alternative” : It was changed.
Line 42 Please rewrite “have also shown a good AR inhibition” : It was changed.
Lines 44-45 Please rewrite “world and mainly present in Southern Africa.” : It was changed.
Lines 52-53 I think it would be better to add “to our knowledge”. : It was changed.
Line 55 If I understood it correctly you choose G. sibiricum for the following reason “ from the one species, G. sibiricum, that showed the highest IC50 values”. Please rewrite the sentence. : It was changed.
Line 60 “species of the genus “: It was changed.
Line 77” were purchased” : It was changed.
Line 79 "Bruker Avance” Moreover, could you provide more specifications about the liquid NMR analysis?: It was changed.
Line83 It is not clear to me what is this “ARI assay”.: It was changed.
Line 85 I would also add “identification” : It was changed.
Line 86 How did you dry the aerial parts? In an oven? At which temperature? Please add this information.: It was specified.
Line 87 It was filtered on which type of filters? Nylon, RC, etc? Please add this information. : It was changed.
Line 68 Where the authors get (if they did not purchase) the kaempferol derivatives to use as standards in HPLC –UV (in Line 118 and 153-154 they used those as reference compounds)? If they obtained them through purification they should clarify it (see my next comment). Lines 93-94 It is not clear what the authors mean stating that they performed a recrystallization using MeOH of the two kaempferol derivatives: did they purified the two main compounds found in the ethyl acetate extract of G. sibiricum (which were then identified with MS and liquid NMR)? Please clarify this point.: The two kaempferol derivatives were isolated from EtOAc fraction of G. sibiricum. See the 2.3 part of material and method (line 86-96).
Line 92 Did authors pack the open columns by themselves or did they bought some already pre-packed ones?: The open column was packed and prepared by us.
Line 110 TMG please clarify this acronym (thiomethyl galactoside?): See the 2.2 part of material and method (line 76-77).
Line 119 What was the material of the filter? Nylon? Please specify.: It was already specified.
Line 141 Since there was a difference in the Tr of the compounds 1 and 2 from the standard solutions to the plant extracts it would be nice to identify the two kaempferol derivatives in the C-F chromatograms.: It was already identified by arrows.
Line 163-164 I think it would be better to add “to our knowledge”. : It was changed.
Line165 Please rewrite ”results of this” : It was changed.
Line 167 The IC50 values found are means of how many repetitions for each extract? Wouldn’t be better to report those values as a mean ± standard deviation (same for values reported in Table 4)? I have always wondered how can one state that the results of IC50 values are different if you did not test if those values were statistically significant?: Repetitions were twice.
Line 175-176 Since authors find out that the MeOH extract of G. sibiricum had the highest IC50 they also fractionated the MeOH extract and used the single different fractions to test for their AR inhibition ability. Please rewrite this sentence accordingly.: We added more detailed information.
Line 178 Not by “open column chromatography” but rather by HPLC-UV identification. Please rewrite.: The compounds were obtained using open column chromatography not by HPLC-UV.
Line 213 Please rewrite “ (1) in the MeOH extract (28.1 mg/g).” : It was changed.
Line 228 Please add a reference.: It is changed with reference 20.
Line 234 I think it would be better to add “to our knowledge”. : It was changed.
Line 239 Please complete the sentence:“of G. sibiricum showed the highest IC50 values.” or something similar. : It was changed.
Line 243 What do you mean with “their ARI efficacies.”? : It was changed.
Line 244 Please remove this sentence, you might insert this in line 240” (2) two flavonoid compounds present in the”: It caanot be found.
Line 246 Please rewrite: “[13] present in Geranium species, anyway, the potent AR inhibition showed by the G. sibiricum species can be attributed to its high concentrations of kaempferol-7-O-rhamnoside (1) and kaempferol-3,7-O-dirhamnoside (2) since these two compounds are the major components of the EtOH fraction, the one with higher IC50.” or something similar. : It was changed.
Lines 248-250 Please rewrite: “This species also exhibited the best AR inhibition among all the species examined in this study. These results may have application in the development of novel therapeutic methodologies /approaches to target complications associated with diabetic hyperglycemia.” or something similar. : It was changed.
Round 2
Reviewer 2 Report
In the revised manuscript, the authors marked the peak 1 and 2 in the figure 1. However, compared to the profiles of standard compound 1 and 2 shown in the figure 1a and 1b, the retention time of peaks (figure 1 c-1f) marked by the authors are obviously significantly different from those in the profiles of standard compound 1 and 2. Therefore, it is hard to believe the presence of compound 1 and compound 2 in the studied extract. This must be addressed before publication.
Author Response
The retention time of peaks (figure 1 c-1f) are checked by spike test.
So, the arrows exhibit the real compounds 1 and 2 in figure 1. There is no differece from those in the profiles of standard compounds 1 and 2.
