Optical Properties and Antioxidant Activity of Water-Ethanolic Extracts from Sempervivum tectorum L. from Bulgaria
, Nadezhda Petkova 5 and Iliana Milkova-Tomova 6Round 1
Reviewer 1 Report
- The introduction part should be reviewed. However, the introduction should better explain the originality of this paper, which results in a little lacking in this version.
- In the Material and Methods section, insert subsection 2.1. Plant materials and more details about the plant used (purchased from or year of sampling, etc.)
- Page 2, line 75. Ethanol instead of the chemical formula
- The fig. 1-3 are not necessary
- In table 1, the measurement units are missing
- Provide more detail about how chlorophyll and β -carotene were calculated, provide a reference
- There is not clear how correlations were made and also a lack of a description of statistical analysis (software used)
- This work focused on demonstrating experimental results; the discussions should be supplemented and compared with the other studies.
- In the Conclusions section, add more information on the practical application of the results obtained during the research.
- Overall it is not easy to follow the logical flow of the manuscript.
Author Response
Reviewer 1
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
( ) English language and style are fine/minor spell check required
(x) I don't feel qualified to judge about the English language and style
Yes Can be improved Must be improved Not applicable
Does the introduction provide sufficient background and include all relevant references?
( ) ( ) (x) ( )
Is the research design appropriate?
( ) ( ) (x) ( )
Are the methods adequately described?
( ) ( ) (x) ( )
Are the results clearly presented?
( ) ( ) (x) ( )
Are the conclusions supported by the results?
( ) ( ) (x) ( )
Comments and Suggestions for Authors
The introduction part should be reviewed. However, the introduction should better explain the originality of this paper, which results in a little lacking in this version.
In the Material and Methods section, insert subsection 2.1. Plant materials and more details about the plant used (purchased from or year of sampling, etc.)
Added in revised MS
Plant materials
The above ground parts of the Sempervivum tectorum L (leaves) were sampled randomly from Sofia parks in april 2021. Samples were botanically identified by Assoc. Prof. Iliya Slavov (Department of Botany, Faculty of Pharmacy, MU - Varna ). The plant Sempervivum Tectorum L. is cultivated ornamental plant, not presented until now in the Herbarium
Samples were washed with distilled water, dried at 40 °C to constant weight and further ground in a coffee grinder.
Page 2, line 75. Ethanol instead of the chemical formula
Changed in revised MS
The fig. 1-3 are not necessary
Figures 1, 2 and 3 have been removed in revised MS.
In table 1, the measurement units are missing
Added in the revised MS
Provide more detail about how chlorophyll and β -carotene were calculated, provide a reference
Chlorophyll and β-carotene are computed, based on the transmission spectrum of the sample and its color characteristics by specially developed soft wear application Lovibond™ PFX/PFXi Software Upgrade for Chlorophyll and Beta-Carotene. Цитат от апарата
There is not clear how correlations were made and also a lack of a description of statistical analysis (software used)
The linear dependences between the studied parameters were obtained by performing a one-way analysis of variance. To estimate the parameters of the regression model, the least squares method is applied. The coefficient of determination was determined and the adequacy of the model was checked by using IBM SPS software.
This work focused on demonstrating experimental results; the discussions should be supplemented and compared with the other studies.
We tried to add additional studies to confirm our experimental results.
In the Conclusions section, add more information on the practical application of the results obtained during the research.
The highest total phenolic content was found in 50% ethanol extract - 0.84 ± 0.08 mg GAE/ml. This extract also has the highest antioxidant potential as assessed by DPPH and FRAP assays. High correlation coefficients were found between total phenolics content and antioxidant activity of water-ethanolic extracts from Sempervivum tectorum L. The same is valid for the strong relationship between phenols content and concentrations of Na and K. Most probably bioavailable species of elements such as Fe, Zn, Ca, Mg are mostly aqueous soluble. On the opposite concentrations of compounds like β-carotene and chlorophyll increase with the increase of ethanol in the extract. The content of toxic elements As, Cd, Tl, Pb is far below the permissible limits for pharmaceutical products. Results from this study might be used for preliminary prognosis of pharmaceutical applications of extracts from Sempervivum tectorum L.
Overall it is not easy to follow the logical flow of the manuscript.
We tried to improve the quality of the revised MS
Author Response File: 
Author Response.pdf
Reviewer 2 Report
I will give all comments for manuscript "Optical properties and antioxidant activity of water-alcoholic extracts from Sempervivum Tectorum L. from Bulgaria" in order by line number:
Line 5 – "Milkova–Tomova" – please, use a hyphen instead of a dash --> "Milkova-Tomova"
Line 28 – "10%, 50%, and 95%" – please, put the space between the number and percentage (throughout the text) --> "10 %, 50 %, and 95 %"
Line 32 – please, put the space between the number and percentage
Line 33 – please, put the space between the number and percentage
Line 35 – please, put the space between the number and percentage
Line 35 – "As a result, toxic elements such as As, Tl, Cd, and Pb are missing." – please, rephrase
Lines 41 – 49 – A literature reference (or references) for this allegation is (are) missing. Please, correct the error.
Line 58 – please, use a dash instead of a hyphen
Line 73 – " was obtained from Sofia city" – please, clarify
Line 76 – please, put the space between the number and percentage
Line 134 – please, put the space between the number and percentage
Line 147 – "0.1mM" --> "0.1 mM"
Line 173 – please, put the dot at the end of the sentence
Line 181– please, put the space between the number and percentage
Line 182 – please, put the space between the number and percentage
Line 214 – please, put the space between the number and percentage
Table 4 – please, put the space between the number and percentage
Line 230 – please, put the space between the number and percentage
Line 241 – please, put the space between the number and percentage
Line 252 – please, put the space between the number and percentage
Line 267 – please, put the space between the number and percentage
Line 270 – please, put the space between the number and percentage
Line 274 – please, put the space between the number and percentage
Line 288 – please, put the space between the number and percentage
Line 295 – please, put the space between the number and percentage
Lines 295 – 300 – please, rephrase; please add in the conclusion to what the research referred (a full sentence).
Line 299 – please, put the space between the number and percentage
Line 360 – please, fix the formatting
Line 362 – redundant line
Author Response
Reviewer 2
- Open Review
- (x) I would not like to sign my review report
- ( ) I would like to sign my review report
- English language and style
- ( ) Extensive editing of English language and style required
- (x) Moderate English changes required
- ( ) English language and style are fine/minor spell check required
- ( ) I don't feel qualified to judge about the English language and style
- Yes Can be improved Must be improved Not applicable
- Does the introduction provide sufficient background and include all relevant references?
- ( ) (x) ( ) ( )
- Is the research design appropriate?
- (x) ( ) ( ) ( )
- Are the methods adequately described?
- ( ) (x) ( ) ( )
- Are the results clearly presented?
- (x) ( ) ( ) ( )
- Are the conclusions supported by the results?
- (x) ( ) ( ) ( )
- Comments and Suggestions for Authors
- I will give all comments for manuscript "Optical properties and antioxidant activity of water-alcoholic extracts from Sempervivum Tectorum L. from Bulgaria" in order by line number:
- Line 5 – "Milkova–Tomova" – please, use a hyphen instead of a dash --> "Milkova-Tomova"
Done in the revised MS
- Line 28 – "10%, 50%, and 95%" – please, put the space between the number and percentage (throughout the text) --> "10 %, 50 %, and 95 %"
Done in the revised MS
- Line 32 – please, put the space between the number and percentage
Done in the revised MS
- Line 33 – please, put the space between the number and percentage
Done in the revised MS
- Line 35 – please, put the space between the number and percentage
Done in the revised MS
- Line 35 – "As a result, toxic elements such as As, Tl, Cd, and Pb are missing." – please, rephrase
The content of As, Tl, Cd and Pb are below the detection limit of method used (DL 0.002 mg/kg). This result is later on discussed from the view point of permissible limits of toxic elements in plant extracts
- Lines 41 – 49 – A literature reference (or references) for this allegation is (are) missing. Please, correct the error.
Additional literature references mostly review papers have been included in revised MS
- Evidence – based validation of herbal medicine, Edited by Pulak K. Mukherjee, Amsterdam, Elsevier 2015, ISBN 978-012-800874-4.
- Waseem, H.; Hamsa, N.; Shakila, R.; Shehnaz, G.; Amjad Kamal, Mohammad, A.K.; Jean, P.K.; Bakht, Z.;, Joao, B.T.da Rocha. Oxidative Stress and Antioxidant Potential of One Hundred Medicinal Plants, Current Topics in Medicinal Chemistry, 2017, 17, 1336-1370(35).
- Sharma, S.K.; Singh, L.; Singh, S. A review on medicinal plants having antioxidant potential, Indian Journal of Research in Pharmacy and Biotechnology, 2013, 1 (3), 404- 409.
- Chanda, S.; Dave, R. In vitro models for antioxidant activity evaluation and some medicinal plants possessing antioxidant properties: An overview, African Journal of Microbiology Research, 2009, 3 (13), 981-996.
5.Christaki,E.; Bonos, E.; Giannenas,I.; Florou-Paneri, P. Aromatic Plants as a Source of Bioactive Compounds, Agriculture, 2012, 2, 228-243.
- Jayanta Kumar Patra, Gitishree Das, Siyoung Lee, Seok-Seong Kang, Han-Seung Shin. Selected commercial plants: A review of extraction and isolation of bioactive compounds and their pharmacological market value, Trends in Food Science & Technology, 2018, 82, 89-109
- Ammar Altemimi, Naoufal Lakhssassi, Azam Baharlouei, Dennis G. Watson, David A. Lightfoot. Phytochemicals: Extraction, Isolation, and Identification of Bioactive Compounds from Plant Extracts, Plants, 2017, 6, 42-65
Line 58 – please, use a dash instead of a hyphen
Done in the revised MS
Line 73 – " was obtained from Sofia city" – please, clarify
Clarified in the revised MS
The above ground parts of the Sempervivum tectorum L (leaves) were sampled randomly from Sofia parks in april 2021. Samples were botanically identified by Assoc. Prof. Iliya Slavov (Department of Botany, Faculty of Pharmacy, MU - Varna ).
.
Line 76 – please, put the space between the number and percentage
Done in the revised MS
Line 134 – please, put the space between the number and percentage
Done in the revised MS
Line 147 – "0.1mM" --> "0.1 mM"
Done in the revised MS
Line 173 – please, put the dot at the end of the sentence
Done in the revised MS
Line 181– please, put the space between the number and percentage
Done in the revised MS
Line 182 – please, put the space between the number and percentage
Done in the revised MS
Line 214 – please, put the space between the number and percentage
Done in the revised MS
Table 4 – please, put the space between the number and percentage
Done in the revised MS
Line 230 – please, put the space between the number and percentage
Done in the revised MS
Line 241 – please, put the space between the number and percentage
Done in the revised MS
Line 252 – please, put the space between the number and percentage
Done in the revised MS
Line 267 – please, put the space between the number and percentage
Done in the revised MS
Line 270 – please, put the space between the number and percentage
Done in the revised MS
Line 274 – please, put the space between the number and percentage
Done in the revised MS
Line 288 – please, put the space between the number and percentage
Done in the revised MS
Line 295 – please, put the space between the number and percentage
Done in the revised MS
Lines 295 – 300 – please, rephrase; please add in the conclusion to what the research referred (a full sentence).
The highest total phenolic content was found in 50% ethanol extract - 0.84 ± 0.08 mg GAE/ml. This extract also has the highest antioxidant potential as assessed by DPPH and FRAP assays. High correlation coefficients were found between total phenolics content and antioxidant activity of water-ethanolic extracts from Sempervivum tectorum L. The same is valid for the strong relationship between phenols content and concentrations of Na and K. Most probably bioavailable species of elements such as Fe, Zn, Ca, Mg are mostly aqueous soluble. On the opposite concentrations of compounds like β-carotene and chlorophyll increase with the increase of ethanol in the extract. The content of toxic elements As, Cd, Tl, Pb is far below the permissible limits for pharmaceutical products. Results from this study might be used for preliminary prognosis of pharmaceutical applications of extracts from Sempervivum tectorum L.
Line 299 – please, put the space between the number and percentage
Done in the revised MS
Line 360 – please, fix the formatting
Fixed in the revised MS
Line 362 – redundant line
Line 362 removed in revised MS
Author Response File: 
Author Response.docx
Reviewer 3 Report
The manuscript presents the results of the spectroscopic characterization and antioxidant activity of ethanolic extracts of Sempervivum tectorium, which contributes to the chemical validation of the therapeutic uses of this plant.
The fluorescence spectroscopy technique employed is not conclusive in associating the chemical composition with a particular group of metabolites. In these cases it is more advisable to record the UV-VIS absorption spectra, which can confirm the qualitative reaction of TPC.
The color registration technique is more recommended to analyze surfaces and determine the absorption of pigments, the justification for this type of analysis in a phytochemical is not sufficiently clear.
The analysis by means of the ICP-MS technique is not sufficiently described, it is not clear whether only a general scan of the ions is made or a standard of known concentration is used for each element reported. Additionally, in the methods the authors do not mention the treatment required to fix volatile elements such as As, and Hg.
Additionally I suggest the following changes
Page 1, line 3, change "Tectorium" to tectorium
Page 1, lines 23 to 25, I suggest that the text be changed or that substantial information directly related to the study species be indicated.
"In recent years, studies have been focused on the concentrations of potentially bioactive compounds in various plants and their antioxidant activity, detoxifying properties, and other essential properties for human health."
Page 2, Lines 69 to 70, In the objectives it is indicated that the optical characteristics and chemical composition will be determined, however, only a basic description of those parameters is referred, I suggest that the objectives refer only to the aspects that are included in this work.
Page 2, Line 73, I suggest that the taxonomic keys used for the identification of the plant be indicated and, if possible, its registration in a herbarium.
Page 8, Line 214, Elemental composition data are not described in the results.
Page 8, Line 213, It is suggested that the resolution and size of Figure 7c be improved.
Page 9, Line 232, pull apart "tectorumcould"
Page 9, line 239, correct "demonatrated" to demonstrated
Page 9, line 239 correct "oshowed" to showed
Page 9, line 240 correct "methal" to metal
Page 9, line 244 correct "methal" to metal
Page 9, lines 264 to 266, According to the fluorescence results, what are the main compounds associated with antioxidant activity?
Page 9, line 268, replace "Figure 2 and Figure 3" with Figure 6 and Figure 7
Author Response
Reviewer 3
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
Yes Can be improved Must be improved Not applicable
Does the introduction provide sufficient background and include all relevant references?
( ) (x) ( ) ( )
Is the research design appropriate?
( ) ( ) (x) ( )
Are the methods adequately described?
( ) (x) ( ) ( )
Are the results clearly presented?
( ) (x) ( ) ( )
Are the conclusions supported by the results?
( ) (x) ( ) ( )
Comments and Suggestions for Authors
The manuscript presents the results of the spectroscopic characterization and antioxidant activity of ethanolic extracts of Sempervivum tectorium, which contributes to the chemical validation of the therapeutic uses of this plant.
The fluorescence spectroscopy technique employed is not conclusive in associating the chemical composition with a particular group of metabolites. In these cases, it is more advisable to record the UV-VIS absorption spectra, which can confirm the qualitative reaction of TPC.
Most probably both UV-VIS and fluorescence spectroscopy are useful techniques to define chemical composition. In our study, we use fluorescence as we expect beta-carotene and chlorophyll as main constituents.
The color registration technique is more recommended to analyze surfaces and determine the absorption of pigments, the justification for this type of analysis in a phytochemical is not sufficiently clear.
In this study, we use color registration technique to define pigments content and most of all use this technique to calculate Chlorophyll and β-carotene content by using a specially developed soft wear.
The analysis by means of the ICP-MS technique is not sufficiently described, it is not clear whether only a general scan of the ions is made or a standard of known concentration is used for each element reported. Additionally, in the methods the authors do not mention the treatment required to fix volatile elements such as As, and Hg.
We apologize for non-consistent description of ICP-MS measurements in our study. We have used stock standard solutions: Multielement standard solution 5 for ICP (TraceCERT®, Merck), 1000 mg L−1 As (Fluka, Sigma-Aldrich) and 1000 mg L-1 Hg (Fluka, Sigma-Aldrich) to prepare working standard solutions in order to construct calibration graph for each element and to perform quantitative determination. Optimal instrumental parameters for ICP-MS measurements can be find in our previous study ref. â„–
The evaporation of ethanol extracts as well as their digestion was performed under IR lamp, at almost equilibrium conditions. In this way temperature used was always below 40 °C ensuring loss free determination of As and Hg.
Detailes of ICP-MS procedure are given in the revised MS and in refâ„–
Gentscheva, G.; Karadjova, I.; Radusheva, P.; Minkova, St.; Nikolova, Kr.; Sotirova, Y.; Yotkovska, I.; Andonova, V. Determination of the Elements Composition in Sempervivum tectorum L. from Bulgaria, Horticulture, 2021, 7, 306.
Additionally I suggest the following changes
Page 1, line 3, change "Tectorium" to tectorium
Done in revised MS
Page 1, lines 23 to 25, I suggest that the text be changed or that substantial information directly related to the study species be indicated.
Almost new abstract was included in the revised MS, following the referee suggestions:
Abstract: Sempervivum tectorum L. is an evergreen plant belonging to a large family of the Crassulaceae. The fresh juice of the plant is used as a folk remedy almost exclusively for external purposes. Combination of several instrumental methods – transmission and fluorescence spectroscopy, ICP-MS spectrometry and assays for the assessment of antioxidant activity were used for the characterization of water-ethanolic extracts from leaves of Sempervivum tectorum L. with different polarity (ethanol content in the extracts varied between 10 and 95%). The highest total phenolic content was found in 50 % ethanol extract- 0.84±0.08 mg GAE/mL. Furthermore, this extract also possessed the highest antioxidant potential evaluated by DPPH and FRAP assays-7.02±0.71 mM TE/ml and 6.15±0.25 mM TE/ml, respectively. High correlation coefficients were found between total phenolics content and antioxidant activity of water-ethanoic extracts from Sempervivum tectorum L. The same is valid for the strong relationship between phenols content and concentrations of Na and K. Most probably bioavailable species of elements such as Fe, Zn, Ca, Mg are mostly aqueous soluble. For all studied extracts toxic elements (As, Cd, Pb, Tl, Hg) content are much below the permissible limits for pharmaceutical products. On the opposite concentrations of compounds like β-carotene and chlorophyll increase with the increase of ethanol in the extract. Results from this study might be used for preliminary prognosis of pharmaceutical applications of extracts from Sempervivum tectorum L.
"In recent years, studies have been focused on the concentrations of potentially bioactive compounds in various plants and their antioxidant activity, detoxifying properties, and other essential properties for human health." This sentence was removed from abstract in revised MS
Page 2, Lines 69 to 70, In the objectives it is indicated that the optical characteristics and chemical composition will be determined, however, only a basic description of those parameters is referred, I suggest that the objectives refer only to the aspects that are included in this work.
We have shown various characteristics of studied water-ethanolic extracts by using transmission and fluorescence spectroscopy and ICP-MS spectrometry. In addition, assays for the assessment of oxidant activity and total phenolics content are included. The objectives of the study have been rewritten in the revised MS.
Page 2, Line 73, I suggest that the taxonomic keys used for the identification of the plant be indicated and, if possible, its registration in a herbarium.
The plant Sempervivum Tectorum L. is a cultivated, ornamental plant, not presented until now in the Herbarium
Page 8, Line 214, Elemental composition data are not described in the results.
Table 4 is presented in Results section with concentrations of essential and toxic elements content. Later on in discussion section, these results are discussed.
Page 8, Line 213, It is suggested that the resolution and size of Figure 7c be improved.
Done in the revised MS
Page 9, Line 232, pull apart "tectorumcould"
Done in the revised MS
Page 9, line 239, correct "demonatrated" to demonstrated
Done in the revised MS
Page 9, line 239 correct "oshowed" to showed
Done in the revised MS
Page 9, line 240 correct "methal" to metal
Done in the revised MS
Page 9, line 244 correct "methal" to metal
Done in the revised MS
Page 9, lines 264 to 266, According to the fluorescence results, what are the main compounds associated with antioxidant activity?
The main compounds associated with antioxidant activity were carotenoids and polyphenolic compounds (especially phenolic acids and flavonoids). In our case, the fluorescence spectra give a fluorescence maximum in the range (630-700) nm, which is associated with the presence of chlorophyll in the samples. For the sample with 50% ethanol content, there is a low-intensity fluorescent band in the range (500 - 580) nm, associated in the literature with the presence of β-carotene.
Page 9, line 268, replace "Figure 2 and Figure 3" with Figure 6 and Figure 7
Done in the revised MS. Taking into account removal of Figures 1-3 (proposed by the reviewer ), Figure 6 in the revised MS is new Figure 3 and Figure 7 is new Figure 4.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The paragraph from Page 11, lines 330-333, should be rephrased
Author Response
Reviewer 1
The paragraph from Page 11, lines 330-333, should be rephrased
Thank you for this suggestion. The paragraph was rephrased in the revised MS
Analysis of the transmission spectra, shows that intense absorption bands between 630 nm and 690 nm was observed only in 50% and 95% water-alcoholic extracts and is absent in 10% ethanolic extract of Sempervivum tectorum L (chlorophyll is not leached in this extract).
