Studies Regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb

Round 1

Reviewer 1 Report

The authors introduce an interesting topic regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb especially in the application of Oral environment.

However, to further proceed into accepted version, still more clarification as well as further experiment need to be obtained to support the conclusion. 

1. Materials: If the Epilobium parviflorum Schreb has already been dried for one month after the extracting method applied using the ethanol with relatively high concentration solvent, how can you differentiate the antibacterial effect of the ethanol and the Schreb, as both of them may reduce the adhesive of the bacteria, and the Schreb supposed to be dead also. 

2. Methods: Please provide the reference for the bacterial test, as normally we may use CDC imaging to obtain the CFU/ml for the bacterial characterization. As for the human test, how did you control many other factors? the sample seems not large enough as well.

3. Format & template: Please pay attention to the alignment of the format, e.g., Section 2.2 and 2.3 are not aligned properly.

4. Results: Regarding the ANOVA statistical analysis, the standard deviation (SD) result seems too big comparing with the average, which means such results may not be valid to support the conclusion that drawn from the experiment.

5. Discussion: So many experimental factors which may affect the final result, although it is able to prove that such scherb has an antibacterial effect, but the supportive evidence is too weak. Even there is no microscopy observation.

6. If you can narrow down which component within the extract has the most significant antibacterial effect, the paper will be more convincing for the readership. 

 

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled ”Studies Regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb”, co-authored by Erdogan Elvis Șachir, Cristina Gabriela Pușcașu, Aureliana Caraiane, Gheorghe Raftu, Florin Ciprian Badea, Mihaela Mociu, Claudia Maria Albu,Liliana Sachelarie, Loredana Liliana Hurjui, Cristina Bartok-Nicolae.

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

1. Materials: If the Epilobium parviflorum Schreb has already been dried for one month after the extracting method applied using the ethanol with relatively high concentration solvent, how can you differentiate the antibacterial effect of the ethanol and the Schreb, as both of them may reduce the adhesive of the bacteria, and the Schreb supposed to be dead also.

We have used dry aerial parts of the Epilobium parviflorum Schreb in the extracting method, the vegetable extract was used fresh not stored for one month in accordance with the process for extraction technologies formedicinal and aromatic parts(https://www.unido.org/sites/default/files/2009-10/Extraction_technologies_for_medicinal_and_aromatic_plants_0.pdf, page 28) and in according to European Pharmacopoeia.

The hydroalcoholic plant extracts were placed in a rotary evaporator (IKA-RV 10 digital V, Staufen Baden-Wurtemberg, Germany) to evaporate the solvent, at temper-atures lower than 40 °C and under reduced pressure. Finally, the remaining ethanol was evaporated placing the flask on the kiln drier until obtaining a consistent weight (three days) [26].

2. Methods: Please provide the reference for the bacterial test, as normally we may use CDC imaging to obtain the CFU/ml for the bacterial characterization. As for the human test, how did you control many other factors? the sample seems not large enough as well.

The bacterial test was made by adapting the Kirby-Bauer method in accordance with the protocol described by Romanian Microbiology Society in the Clinical microbiology guide(Dumitru Buiuc, Marian Neguţ, Tratat de microbiologieclinică, Ediţia a III-a, 2017, EdituraMedicală).

At the begging infected dentine was collected from the root canal and stored on medium culture, from all the infected dentine we identified using API the bacterial speciesafter that, we have choose the most common bacterial developed in endodontic pathology and we have spent successively the bacterial to obtain pure cultures of the identified species on which the test was performed.

3. Format & template: Please pay attention to the alignment of the format, e.g., Section 2.2 and 2.3 are not aligned properly.

We have changed the alignment

2.2. Determination of the content of the bioactive compound in plant extracts

Ethanol as a solvent extraction in analytical grade was obtained from Merck in Darmstadt, Germany; pyrogallol and quercetin were obtained from Sigma-Aldrich Co. in St. Louis, the US.

The Folin-Ciocalteau test method is the simplest method available for measuring the phenolic content of organic products [27]. The basic mechanism is an oxidation / reduction reaction with the phenolic group being oxidized and the reduction of the metal ion [28]. The principle of the method is based on the reaction between Fo-lin-Ciocalteu reagent and phosphomolybdic acid with the phenolic compounds in the sample, resulting in a mixture of blue oxides [29]. The spectrophotometric measure-ments were performed with a Specord M400 spectrophotometer, Carl Zeiss Yena using a 1 cm quartz cell.

2.3. Bacteriological tests

• Study group

4. Results: Regarding the ANOVA statistical analysis, the standard deviation (SD) result seems too big comparing with the average, which means such results may not be valid to support the conclusion that drawn from the experiment.

We are sorry to inform you that there was a communication error between the statistician and the person who created the table, we have modified this text error.

Vegetable extract	Determinations no.	Absorbance	EqQr/mL
Hydroalcoholic	1	2,3569	2,87
	2	2,3411	2,84
	3	2,3779	2,90
		Mean/SD	2,87±0,02
Aqueous	1	1,1332	0,9
	2	1,0619	0,78
	3	1,1341	0,9
		Mean/SD	0,86±0,06

 

5. Discussion: So many experimental factors which may affect the final result, although it is able to prove that such scherb has an antibacterial effect, but the supportive evidence is too weak. Even there is no microscopy observation.

The highest antibacterial activity had the hydroalcoholic plant extract, which managed to inhibit the growth of the following bacterial strains White Staphylococcus, Streptococcus mitis, Streptococcus sanguis, Enterococcus faecalis having the areas of inhibition expressed in mm 19, 26, 26, 12. The aqueous plant extract had no antibacterial activity, all bacterial strains developed on the tested disc.      

6. If you can narrow down which component within the extract has the most significant antibacterial effect, the paper will be more convincing for the readership.

5.Conclusions

Hydroalcoholic plant extract had the highest quantity of polyphenols and flavonoids.The hydroalcoholic plant extract obtained from Epilobium parviflorum Schreb had the best antibacterial activity.

We are very grateful to you for the review report and for the extremely useful suggestions!

Sincerely,

Dr. Erdogan Elvis Șachir 

 

 

Reviewer 2 Report

Dear Authors

I read the article with subject Studies Regarding the Antibacterial Effect of Plant Extracts

Obtained from Epilobium parviflorum Schreb. It is an interesting subject and research.

I have just some comments on this article.

The table's title must be the top of the table and the title of the figure below the figure.

Please change the type of Graph 3 (if possible).

Some comments include:

Line 15 trend in using

Line 17. This problem dominated

Line 18 space The aim

Line 26 theology

Lien 37 most of

Line 38 type of removing

Line 43 aside from

Line 46 intraarticular

Line 50 Candida albicans. In contrast,

Line 81 It seems that you have an unnecessary comma.

Line 104 It will contact the rest of the solvent, thus obtaining

 Line 109 space The method

Line 119 check the English grammar

Line 137 To verify the absence of microorganisms

Line 142 The result came as a sterile sample

Line 145 Determination of the content of the bioactive compound in plant extracts

Line 160 was revised in 2013, evaluation took place for two years

Line 175, the contributory

Line 176 previous 6 months.

Line 178 for inclusion

Line 206 The method's adaptation consisted of replacing the antibiotic discs with sterile.

Line 221-225 it is not clear

Line 254 ultrasonicated hydroalcoholic

Line 282 on the Escherichia coli strain, none of the plant extracts have

Line 296 except for Escherichia

Line 351 hydrodistillation was determined

Line 363 competing with those

Line 382 to overcome these limitations

Line 385-394 check the space after the dot (.)

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled ”Studies Regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb”, co-authored by Erdogan Elvis Șachir, Cristina Gabriela Pușcașu, Aureliana Caraiane, Gheorghe Raftu, Florin Ciprian Badea, Mihaela Mociu, Claudia Maria Albu,Liliana Sachelarie, Loredana Liliana Hurjui, Cristina Bartok-Nicolae.

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

The table's title must be the top of the table and the title of the figure below the figure.

We have changed that.

Please change the type of Graph 3 (if possible).

We have changed that.

Some comments include:

 

Line 15 trend in using

We have changed that.

Line 17. This problem dominated

We have changed that.

Line 18 space The aim

We have changed that.

Line 26 theology

We have changed that

Lien 37 most of

We have changed that.

Line 38 type of removing

We have changed that

Line 43 aside from

We have changed that

Line 46 intraarticular

We have used the term intraradicular to refer of the endodontic space of a tooth (inside the root canal)

Line 50 Candida albicans. In contrast,

We have changed that

Line 81 It seems that you have an unnecessary comma.

We have changed that

Line 104 It will contact the rest of the solvent, thus obtaining

We have changed that

 Line 109 space The method

We have changed that

Line 119 check the English grammar

We have checked it.

Line 137 To verify the absence of microorganisms

We have changed that

Line 142 The result came as a sterile sample

We have changed that

Line 145 Determination of the content of the bioactive compound in plant extracts

We have changed that

Line 160 was revised in 2013, evaluation took place for two years

We have changed that

Line 175, the contributory

We have changed that

Line 176 previous 6 months.

We have changed that

Line 178 for inclusion

We have changed that

Line 206 The method's adaptation consisted of replacing the antibiotic discs with sterile.

We have changed that

Line 221-225 it is not clear

We have changed that

Line 254 ultrasonicated hydroalcoholic

We have changed that

Line 282 on the Escherichia coli strain, none of the plant extracts have

We have changed that

Line 296 except for Escherichia

We have changed that

Line 351 hydrodistillation was determined

We have changed that

Line 363 competing with those

We have changed that

Line 382 to overcome these limitations

We have changed that

Line 385-394 check the space after the dot (.)

 

We are very grateful to you for the review report and for the extremely useful suggestions!

Sincerely,

Dr. Erdogan Elvis Șachir 

 

 

Reviewer 3 Report

see the attach file

Comments for author File: Comments.docx

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled ”Studies Regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb”, co-authored by Erdogan Elvis Șachir, Cristina Gabriela Pușcașu, Aureliana Caraiane, Gheorghe Raftu, Florin Ciprian Badea, Mihaela Mociu, Claudia Maria Albu,LilianaSachelarie, Loredana Liliana Hurjui, Cristina Bartok-Nicolae.

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

1. All abbreviations should be first identified before use them even if they were in abstract or another part of the manuscript

We have changed that.

Polyphenols and flavonoids were present in all plant extract; the hydroalcoholic extract had the highest number of polyphenols-17,44 pyrogallol equivalent (Eq Pir) /mL and flavonoids-3,13 quercetin equivalent (Eq Qr) /mL.

2. The abstract does not report the main findings of the study in a clear manner. For example general expressions are used which do not provide useful information to the readers. Information that is more specific is required in the abstract.

We have changed that

Abstract: The present study was carried out to develop an experimental endodontic irrigant solu-tion based on plant extracts obtained from Epilobium parviflorum Schreb that largely replenish the properties of the usual antiseptics used in dentistry is the aim of this study. Experts recommend the use of natural remedies as an adjunctive to classical therapy, all the more so as the antibacterial activity of plant extracts comes up against antibiotic resistance, this problem dominated the second millennium and seems to persist in an upward trend in the third millennium. Background: This study investigated the phytochemical contents of plant extracts obtained from Epilobium parvi-florum Schreb and their potential antibacterial activity. Methods: Identification and quantification of biologically active compounds were made by UV field photo spectrometry adapting the Fo-lin-Ciocalteau test method. Antibacterial activity was tested on bacterial cultures collected from tooth with endodontic pathology using modified Kirby-Bauer diffuse metric method. Results: Pol-yphenols and flavonoids were present in all plant extract; the hydroalcoholic extract had the highest amount of polyphenols-17,44 pyrogallol equivalent (Eq Pir) /mL and flavonoids-3,13 quercetin equivalent (Eq Qr) /mL. Plant extracts had antibacterial activity among the tested bacterial species with the following inhibition diameter: White staphylococcus (16.5 mm), Streptococcus mitis (25 mm), Streptococcus sanguis (27 mm), Enterococcus faecalis (10 mm). Conclusions: Hydroalcoholic plant extract obtained from Epilobium parviflorum Schreb had the highest quantity of polyphenols, flavonoids and antibacterial activity.

3. The introduction does not point out the gap of the literature. The study seeks to fill and novelty of the study over the existing literature. This point showed be further elaborated.

We have changed that

1Introduction

Epilobium is a genus of perennial herbaceous plant (Onagraceae family). The most common species include Epilobium parviflorum Schreb., Epilobium hirsutum L., Epilobium rosmarinifolium (Epilobium dodonaeiVill.), Epilobium roseumSchreb., and Epilobium an-gustifolium L. [1].

The anti-inflammatory and antiproliferative activity of Epilobium parviflorum Schreb extracts was initially studied on cells in the context of benign prostatic hyper-plasia in parallel the analgesic, antioxidant, antibacterial and antifungal action were studied [2-4]. Plant polyphenols represent a group of chemical substances ubiquitously distributed in all higher plants. These secondary metabolites possess free radical scav-enging and antimicrobial activity. These properties can be advantageously exploited, especially because of the abundance of polyphenols and their derivatives in various agricultural and food industry waste and by‐products and the possibility of convenient extraction by either organic or aqueous solvents [5-7].

In the oral cavity, nearly 700 species of bacteria can be found, most of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria [8]. There are two types of intra-radicular infections: primary and secondary, each with specific microbiology. The primary infection of the root canal system results from colonization with a hetero-genous group of microbes that have entered the pulp tissue, when exposed during car-ies or traumas; this group is dominated by Gram-negative oral anaerobic bacteria (Prevotella spp., Porphyromonas spp., Fusobacterium spp., Veillonella spp.), aside from bacterial species which are also part of the commensal oral microflora: Gram-positive anaerobic bacteria (Propionibacterium spp., Bifidobacterium spp., anaerobic strepto-cocci), treponemas or Campylobacter spp.[8,9]. The secondary intraradicular infection appears inside the root canal system, after the treatment of the affected tooth has been initiated. Secondary intraradicular infections harbor a limited number of bacterial species, with predominance of Gram-positive bacteria, namely Enterococcus faecalis (E. faecalis), oral streptococci, lactobacilli or Candida albicans, in contrast Gram-negative bacteria are involved to a lesser extent [10]. The most common micro-organism found in asymptomatic, persistent endodontic infections is E. faecalis. Its in-cidence in this type of infection varies from 24% to 77% [8,10]. E. faecalis possesses enzymes and constitutive structures that are able to suppress the action of lympho-cytes and promote inflammation, which contributes to progression of endodontic in-fections [10,11]. Also, this microorganism has the ability to attach to dentin due to an inner component which is the collagen-binding protein [9,11,12].

Several studies have evaluated the antimicrobial efficacy of endodontic irrigants such as sodium hypochlorite (NaOCl), ethylene diamine tetra acetic acid (EDTA) and chlorhexidine (CHX) against E. faecalis biofilms [13]. In general, the aim of any disin-fection strategy is to reduce the bacterial load to a subcritical level so that the patient’s immune response allows healing by itself [14]. Endodontic research has always been focused on developing methods or endodontic irrigants that can completely remove the bacterial biofilm with minimum side-effects. Several endodontic irrigants are being widely used in the treatment of biofilms with varied effectiveness. Although 2% CHX has been proven to possess a high antimicrobial property, it has failed to disrupt the biofilm. However, NaOCl can disrupt the biofilm, disintegrating the dental pulp which is a built-in organ, but it is a well-known irritant to periapical tissues. Therefore, iden-tification of natural products in the disinfection of root canals can be interesting [13,15,16].

In unconventional modern practice, endodontists seek the use of natural remedies as an adjunct to classical therapy, especially since the antibacterial action of plant ex-tracts meets the phenomenon of antibiotic resistance, a problem that dominated med-ical practice in the second millennium and which seems to persist in an upward trending the third millennium [17].

Plant extracts are fluid, soft or dry pharmaceutical / phytopharmaceutical prepa-rations obtained by extracting plant products with different solvents [18].

In recent years, emphasis has been placed on the pharmaceutical and therapeutic revaluation of herbal preparations through a good knowledge of the physio-chemical and therapeutic properties of the active ingredients in medicinal plants and the de-velopment of extraction techniques and quality control means. Extractive solutions are pharmaceutical forms that contain smaller or larger proportions of active substances together with less active ones and ballast, extracted with the help of solvents. The ex-traction process involves the separation of the medically active portions of the plant tissues from the inactive or inert components by the use of selective solvents in stand-ardized procedures [19].

Aqueous extractive solutions are prepared from different parts of the plant or mixtures of medicinal plants, using water as a solvent, preferably distilled or softened [20]. Description of the double maceration technique: the extracted product is first mixed with 1/2-2/3 of the total amount of solvent, after which the liquid is separated and the residue is pressed. It will contact the rest of the solvent, thus obtaining a new amount of extractive solution. The two extractive liquids will combine and filter after a 24-hour rest [21].

Ultrasound-assisted extraction (UAE) is one of the most important techniques used for the extraction of valuable compounds from plant materials and is quite adaptable in the laboratory or on an industrial scale [22,23]. The method involves the use of ultrasound, with frequencies ranging from 20 kHz to 2000 kHz, which increase the permeability of cell walls and produce cell lysis, thus promoting the extraction of biologically active compounds [24].

The aim of the study is the use of plant extracts, obtained from Epilobium parviflo-rum Schreb, as a basis for alternative use in perspective in the treatment of endodontic pathology.

The objectives of this study were:

1) Identification and quantification of phenols and flavonoids found in vegetable extracts obtained from Epilobium parviflorum Schreb;

2) Testing the antibacterial activity of the vegetable extracts.

4. The abstract should be comprehensive

We have changed that

Abstract: The present study was carried out to develop an experimental endodontic irrigant solu-tion based on plant extracts obtained from Epilobium parviflorum Schreb that largely replenish the properties of the usual antiseptics used in dentistry is the aim of this study. Experts recommend the use of natural remedies as an adjunctive to classical therapy, all the more so as the antibacterial activity of plant extracts comes up against antibiotic resistance, this problem dominated the second millennium and seems to persist in an upward trend in the third millennium. Background: This study investigated the phytochemical contents of plant extracts obtained from Epilobium parvi-florum Schreb and their potential antibacterial activity. Methods: Identification and quantification of biologically active compounds were made by UV field photo spectrometry adapting the Fo-lin-Ciocalteau test method. Antibacterial activity was tested on bacterial cultures collected from tooth with endodontic pathology using modified Kirby-Bauer diffuse metric method. Results: Pol-yphenols and flavonoids were present in all plant extract; the hydroalcoholic extract had the highest amount of polyphenols-17,44 pyrogallol equivalent (Eq Pir) /mL and flavonoids-3,13 quercetin equivalent (Eq Qr) /mL. Plant extracts had antibacterial activity among the tested bacterial species with the following inhibition diameter: White staphylococcus (16.5 mm), Streptococcus mitis (25 mm), Streptococcus sanguis (27 mm), Enterococcus faecalis (10 mm). Conclusions: Hydroalcoholic plant extract obtained from Epilobium parviflorum Schreb had the highest quantity of polyphenols, flavonoids and antibacterial activity.

5. Key words must be arranged alphabetically

We have changed that

antibacterial activity; Epilobium parviflorum Schreb; vegetable extract; flavonoids; phenols;

6. The objectives and conclusion of the study are not clear and need to re-write

We have changed that

The objectives of this study were:

1) Identification and quantification of phenols and flavonoids found in vegetable extracts obtained from Epilobium parviflorum Schreb;

2) Testing the antibacterial activity of the vegetable extracts.

Conclusions

Hydroalcoholic plant extract obtained from Epilobium parviflorum Schreb had the highest quantity of polyphenols, flavonoids and antibacterial activity.

7. A relevant hypothesis for the study is missing from the introduction. A true scientific question should be formed

We have changed that

1. Introduction

This study investigated the phytochemical contents of plant extracts obtained from Epilobium parviflorum Schreb and their potential antibacterial activity.

8. Simplify the statement in the paper. Please combine and condense the discussion and conclusion

We have changed that

Conclusions

Hydroalcoholic plant extract obtained from Epilobium parviflorum Schreb had the highest quantity of polyphenols, flavonoids and antibacterial activity.

We are very grateful to you for the review report and for the extremely useful suggestions!

Sincerely,

Dr. Erdogan Elvis Șachir 

 

 

Reviewer 4 Report

Dear authors,

Please, check again your manuscript - need to be improved: decimal comma..

citation in the text assign the year, English as well.

 

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled ”Studies Regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb”, co-authored by Erdogan Elvis Șachir, Cristina Gabriela Pușcașu, Aureliana Caraiane, Gheorghe Raftu, Florin Ciprian Badea, Mihaela Mociu, Claudia Maria Albu,LilianaSachelarie, Loredana Liliana Hurjui, Cristina Bartok-Nicolae.

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

Dear authors,

 

Please, check again your manuscript - need to be improved: decimal comma..

 

citation in the text assign the year, English as well.

We have assigned the year in the text citation.

We have checked the English grammar and also the typing mistakes were removed. 

 

We are very grateful to you for the review report and for the extremely useful suggestions!

Sincerely,

Dr. Erdogan Elvis Șachir 

Reviewer 5 Report

Authors explain the studies on the antibacterial effect of vegetable extracts from Epilobium parviflorum Schreb. The scientific concept is sound and applicable. However, the manuscript needs some major revisions for an accepted quality publication.

Abstract:

Line 28: highest number or amount?

Introduction:

The introduction needs to be improved. There is a gap between the explanation of the oral cavity microbes and Epilobium parviflorum plant extracts. The connection to oral cavity microbes is somewhat misleading compared with the title at the beginning of the introduction. I suggest to start with the introduction of  Epilobium parviflorum plant, bioactive composition, its potential extraction methods,  and the potential use/role as an antibacterial agent to use as an endodontic irrigate based on the supporting literature.

Materials and methods :

All the used equipment need to have the manufacturer name, city and /the country.

Line 188: incomplete sentence

Line 190-192: belong to results section?

Results:

Figure 1-3: labelling of a-f in plates is messed up. Explain the results more. It would be great if the bioactive phenolic composition of the plant extract was analyzed and shown (may be using HPLC) under this section.

Discussion:

It is necessary to re-write with a starting paragraph. Some contents belong to other sections of the manuscript. Eg: Line 320-323: objectives should be described in the introduction section. Explanations from line324-328 belong to results section.

Conclusion:

This needs to be clarified and re-written. The meanings of the sentences are not clear.  For example Line 386: All vegetable extracts (does this mean tested extracts) and hydrochloric extract (of what plant) etc.

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled ”Studies Regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb”, co-authored by Erdogan Elvis Șachir, Cristina Gabriela Pușcașu, Aureliana Caraiane, Gheorghe Raftu, Florin Ciprian Badea, Mihaela Mociu, Claudia Maria Albu,LilianaSachelarie, Loredana Liliana Hurjui, Cristina Bartok-Nicolae.

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

Abstract:

 

Line 28: highest number or amount?

We have changed that in highest amount

Introduction:

 

The introduction needs to be improved. There is a gap between the explanation of the oral cavity microbes and Epilobium parviflorum plant extracts. The connection to oral cavity microbes is somewhat misleading compared with the title at the beginning of the introduction. I suggest to start with the introduction of  Epilobium parviflorum plant, bioactive composition, its potential extraction methods,  and the potential use/role as an antibacterial agent to use as an endodontic irrigate based on the supporting literature.

Introduction

Epilobium is a genus of perennial herbaceous plant (Onagraceae family). The most common species include Epilobium parviflorum Schreb., Epilobium hirsutum L., Epilobium rosmarinifolium (Epilobium dodonaeiVill.), Epilobium roseumSchreb., and Epilobium an-gustifolium L. [1].

The anti-inflammatory and antiproliferative activity of Epilobium parviflorum Schreb extracts was initially studied on cells in the context of benign prostatic hyper-plasia in parallel the analgesic, antioxidant, antibacterial and antifungal action were studied [2-4]. Plant polyphenols represent a group of chemical substances ubiquitously distributed in all higher plants. These secondary metabolites possess free radical scav-enging and antimicrobial activity. These properties can be advantageously exploited, especially because of the abundance of polyphenols and their derivatives in various agricultural and food industry waste and by‐products and the possibility of convenient extraction by either organic or aqueous solvents [5-7].

In the oral cavity, nearly 700 species of bacteria can be found, most of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria [8]. There are two types of intra-radicular infections: primary and secondary, each with specific microbiology. The primary infection of the root canal system results from colonization with a hetero-genous group of microbes that have entered the pulp tissue, when exposed during car-ies or traumas; this group is dominated by Gram-negative oral anaerobic bacteria (Prevotella spp., Porphyromonas spp., Fusobacterium spp., Veillonella spp.), aside from bacterial species which are also part of the commensal oral microflora: Gram-positive anaerobic bacteria (Propionibacterium spp., Bifidobacterium spp., anaerobic strepto-cocci), treponemas or Campylobacter spp.[8,9]. The secondary intraradicular infection appears inside the root canal system, after the treatment of the affected tooth has been initiated. Secondary intraradicular infections harbor a limited number of bacterial species, with predominance of Gram-positive bacteria, namely Enterococcus faecalis (E. faecalis), oral streptococci, lactobacilli or Candida albicans, in contrast Gram-negative bacteria are involved to a lesser extent [10]. The most common micro-organism found in asymptomatic, persistent endodontic infections is E. faecalis. Its in-cidence in this type of infection varies from 24% to 77% [8,10]. E. faecalis possesses enzymes and constitutive structures that are able to suppress the action of lympho-cytes and promote inflammation, which contributes to progression of endodontic in-fections [10,11]. Also, this microorganism has the ability to attach to dentin due to an inner component which is the collagen-binding protein [9,11,12].

Several studies have evaluated the antimicrobial efficacy of endodontic irrigants such as sodium hypochlorite (NaOCl), ethylene diamine tetra acetic acid (EDTA) and chlorhexidine (CHX) against E. faecalis biofilms [13]. In general, the aim of any disin-fection strategy is to reduce the bacterial load to a subcritical level so that the patient’s immune response allows healing by itself [14]. Endodontic research has always been focused on developing methods or endodontic irrigants that can completely remove the bacterial biofilm with minimum side-effects. Several endodontic irrigants are being widely used in the treatment of biofilms with varied effectiveness. Although 2% CHX has been proven to possess a high antimicrobial property, it has failed to disrupt the biofilm. However, NaOCl can disrupt the biofilm, disintegrating the dental pulp which is a built-in organ, but it is a well-known irritant to periapical tissues. Therefore, iden-tification of natural products in the disinfection of root canals can be interesting [13,15,16].

In unconventional modern practice, endodontists seek the use of natural remedies as an adjunct to classical therapy, especially since the antibacterial action of plant ex-tracts meets the phenomenon of antibiotic resistance, a problem that dominated med-ical practice in the second millennium and which seems to persist in an upward trending the third millennium [17].

Plant extracts are fluid, soft or dry pharmaceutical / phytopharmaceutical prepa-rations obtained by extracting plant products with different solvents [18].

In recent years, emphasis has been placed on the pharmaceutical and therapeutic revaluation of herbal preparations through a good knowledge of the physio-chemical and therapeutic properties of the active ingredients in medicinal plants and the de-velopment of extraction techniques and quality control means. Extractive solutions are pharmaceutical forms that contain smaller or larger proportions of active substances together with less active ones and ballast, extracted with the help of solvents. The ex-traction process involves the separation of the medically active portions of the plant tissues from the inactive or inert components by the use of selective solvents in stand-ardized procedures [19].

Aqueous extractive solutions are prepared from different parts of the plant or mixtures of medicinal plants, using water as a solvent, preferably distilled or softened [20]. Description of the double maceration technique: the extracted product is first mixed with 1/2-2/3 of the total amount of solvent, after which the liquid is separated and the residue is pressed. It will contact the rest of the solvent, thus obtaining a new amount of extractive solution. The two extractive liquids will combine and filter after a 24-hour rest [21].

Ultrasound-assisted extraction (UAE) is one of the most important techniques used for the extraction of valuable compounds from plant materials and is quite adaptable in the laboratory or on an industrial scale [22,23]. The method involves the use of ultrasound, with frequencies ranging from 20 kHz to 2000 kHz, which increase the permeability of cell walls and produce cell lysis, thus promoting the extraction of biologically active compounds [24].

The aim of the study is the use of plant extracts, obtained from Epilobium parviflo-rum Schreb, as a basis for alternative use in perspective in the treatment of endodontic pathology.

The objectives of this study were:

1) Identification and quantification of phenols and flavonoids found in vegetable extracts obtained from Epilobium parviflorum Schreb;

2) Testing the antibacterial activity of the vegetable extracts.

Materials and methods :

 

All the used equipment need to have the manufacturer name, city and /the country.

We have changed that

Ethanol as a solvent extraction in analytical grade was obtained from Merck in Darmstadt, Germany; pyrogallol and quercetinwere obtained from Sigma-Aldrich Co. in St. Louis, the US.

Line 188: incomplete sentence

We have changed that

Line 190-192: belong to results section?

We have changed that

3.3. Test results for antibacterial activity of plant extracts

Among the bacterial species, identified from the pathological products taken, are: White Staphylococcus, Streptococcus mitis, Streptococcus sanguis, Enterococcus fae-calis and Escherichia coli.

Results:

 

Figure 1-3: labelling of a-f in plates is messed up. Explain the results more. It would be great if the bioactive phenolic composition of the plant extract was analyzed and shown (may be using HPLC) under this section.

We have changed that

3.5. Antibacterial effect of experimental vs. commercial irrigants

As can be seen in Figure 1, Figure 2, Figure 3, the best parallelism regarding the antibacterial effect was shown to be between the test solutions and the antiseptics mentioned on the strains of White Staphylococcus, Streptococcus mitis, Streptococcus sanguis. The highest antibacterial activity had the hydroalcoholic plant extract, which managed to inhibit the growth of the following bacterial strains White Staphylococcus, Streptococcus mitis, Streptococcus sanguis, Enterococcus faecalis having the areas of inhibition (expressed in mm) 19, 26, 26, 12. The aqueous plant extract had no antibacterial activity, all bacterial strains developed on the disc saturated with the test solution

Discussion:

 

It is necessary to re-write with a starting paragraph. Some contents belong to other sections of the manuscript. Eg: Line 320-323: objectives should be described in the introduction section. Explanations from line324-328 belong to results section.

We have changed that

The total phenolic content of the aqueous plant extract obtained from Epilobium parviflorum Schreb was determined from the regression equation (Graph 1) of the cali-bration curve, ranging from 5.62 to 5.65 mg EqPir / g plant powder with an arithmetic mean of 5.63 mg EqPir / g vegetable powder (Table 2).

 The highest polyphenol content was the hydroalcoholic plant extract with a mean of 17,44 Eq Pir/mL, at the opposite pole the lowest polyphenol content was the aqueous plant extract with a mean of 5,63 Eq Pir/mL.

The total flavonoid content of the aqueous plant extract obtained from Epilobium parviflorum Schrebwas determined from the regression equation (Graph 2) of the cali-bration curve, ranging from 0.78 to 0.9 mg EqQr / g plant powder with an arithmetic mean. of 0.86 mg EqQr / g vegetable powder (Table 4). The hydroalcoholic plant ex-tract had the highest flavonoid content with a mean of 2,87 Eq Qr/mL, and the aque-ous plant extract had the lowest level with a mean of 0,86 Eq Qr/mL.  

 

Conclusion:

 

This needs to be clarified and re-written. The meanings of the sentences are not clear.  For example Line 386: All vegetable extracts (does this mean tested extracts) and hydrochloric extract (of what plant) etc.

We have changed that

5. Conclusions

Hydroalcoholic plant extract obtained from Epilobium parviflorum Schreb had the highest quantity of polyphenols, flavonoids and antibacterial activity.

 

We are very grateful to you for the review report and for the extremely useful suggestions!

Sincerely,

Dr. Erdogan Elvis Șachir 

Round 2

Reviewer 1 Report

The authors still do not improve the aspects from scientific soundness of bioassays design, and more biotest shall be included to support the conclusion that the authors have drawn. Besides, the tables and figures cannot consider as clearly presented. Hence, i will not suggest to publish under the current form.

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled ”Studies Regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb”, co-authored by Erdogan Elvis Șachir, Cristina Gabriela Pușcașu, Aureliana Caraiane, Gheorghe Raftu, Florin Ciprian Badea, Mihaela Mociu, Claudia Maria Albu,Liliana Sachelarie, Loredana Liliana Hurjui, Cristina Bartok-Nicolae.

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

We have added more figures describing the entire process from the bacterial culture sampling up to modified Kirby-Bauer test used for antibacterial activity.

The pathological product, infected dentin, was harvested using a sterile Kerr file (20 ISO, Dentsply Sirona), which was then placed in a container with culture medium (Bio-Merieux, France) and transported to the Microbiology Laboratory of the Faculty of Dentistry of the "Ovidius" University of Constanța. The products were seeded on culture medium (Columbia agar + 5% sheep blood, Bio-Merieux, France) which were then thermostated at 37 ° C for 24 hours (Figure 1.a and Figure 1.b).

 

b

a

Figure 1a., Figure 1.b. - Bacterial cultures developed from pathological products after thermostating at 37 ° C for 24 hours

 

a

• Bacteriological identification technique

After seeding on culture media and thermostating, bacterial identification was made using the API (Bio-Merieux, France) method (Figure 2.a and Figure 2.b).

b

a

Figure 2.a., Figure 2.b. API kit (Bio-Merieux, France) for the identification of bacterial cultures

Among the bacterial species, identified from the pathological products taken, are: White Staphylococcus, Streptococcus mitis, Streptococcus sanguis, Enterococcus faecalis and Escherichia coli (Figure 3, Figure 4).

b

b

a

a

Figure 3. Bacterial cultures                     Figure 4. Bacterial cultures              

a-Escherichia coli; b-Enterococcus faecalis      a-Streptococcus mitis; b-Streptococcus sanguis

• Antibacterial effect testing technique of the vegetable extracts

The antibacterial properties were tested on the following bacterial species: White staphylococcus, Streptococcus mitis, Streptococcus sanguis, Escherichia coli, Enterococcus faecalis. They were sown in Mueller-Hinton and Columbia culture medium (Bio-Merieux, France). The bacterial suspension used corresponded to a concentration of 10⁷ colonies per training unit / ml micro-test, which correspond to a turbidity of the 0.5 Mac Farland tube (Figure 5, Figure 6) [30].

 

Figure 5. Sampling of bacterial colonies for        Figure 6. Measurement the turbidity of the

 later introduction into isotonic sodium              microorganism suspension solution

          chloride suspension

The Kirby-Bauer test for antibiotic susceptibility, known as the disc diffusion test, is a standard that has been used for years. This test determines the sensitivity or resistance of bacterial strains to antibiotics. The principle of the method relies on the direct proportionality relationship between the level of sensitivity and the size of the inhibition area of germs colonies developed around the tested substance. Bacterial sensitivity test was realized by adapting the Kirby-Bauer diffusion method in accordance with the protocol described by Romanian Microbiology Society in the Clinical microbiology guide [31,32].

The method’s adaptation consisted of replacing the antibiotic discs with sterile filter paper discs, with the same size as those used in antibiograms; discs were saturated with 50 μl of the test solution. These discs were applied on the culture medium previously seeded with the bacterial strains to be tested (Figure 7); the incubation time was 24 hours at 37 ° C. For comparison, similar to the test solutions we applied filter paper discs impregnated with commercial irrigants used in the practice of endodontics NaOCl 5.25% (Cerkamed, Stalowa Wola, Poland) and CHX 2% (Cerkamed, Stalowa Wola, Poland) on the surface of the sown medium (Figure 8).

 

   Figure 7. Applying the filter paper disc            Figure 8. Commercial endodontic irrigants

   saturated with 50 μl of the test solution

We are very grateful to you for the review report and for the extremely useful suggestions!

Sincerely,

Dr. Erdogan Elvis Șachir 

 

 

Reviewer 3 Report

see the attache file

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled ”Studies Regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb”, co-authored by Erdogan Elvis Șachir, Cristina Gabriela Pușcașu, Aureliana Caraiane, Gheorghe Raftu, Florin Ciprian Badea, Mihaela Mociu, Claudia Maria Albu,Liliana Sachelarie, Loredana Liliana Hurjui, Cristina Bartok-Nicolae.

1. All abbreviations should be first identified before use them even if they were in abstract or another part of the manuscript

We have changed that.

Polyphenols and flavonoids were present in all plant extract; the hydroalcoholic extract had the highest number of polyphenols-17,44 pyrogallol equivalent (Eq Pir) /mL and flavonoids-3,13 quercetin equivalent (Eq Qr) /mL.

2. The abstract does not report the main findings of the study in a clear manner. For example general expressions are used which do not provide useful information to the readers. Information that is more specific is required in the abstract.

We have changed that

Abstract: The present study was carried out to develop an experimental endodontic irrigant solu-tion based on plant extracts obtained from Epilobium parviflorum Schreb that largely replenish the properties of the usual antiseptics used in dentistry is the aim of this study. Experts recommend the use of natural remedies as an adjunctive to classical therapy, all the more so as the antibacterial activity of plant extracts comes up against antibiotic resistance, this problem dominated the second millennium and seems to persist in an upward trend in the third millennium. Background: This study investigated the phytochemical contents of plant extracts obtained from Epilobium parviflorum Schreb and their potential antibacterial activity. Methods: Identification and quantification of biologically active compounds were made by UV field photo spectrometry adapting the Folin-Ciocalteau test method. Antibacterial activity was tested on bacterial cultures collected from tooth with endodontic pathology using modified Kirby-Bauer diffuse metric method. Results: Pol-yphenols and flavonoids were present in all plant extract; the hydroalcoholic extract had the highest amount of polyphenols-17,44 pyrogallol equivalent (Eq Pir) /mL and flavonoids-3,13 quercetin equivalent (Eq Qr) /mL. Plant extracts had antibacterial activity among the tested bacterial species with the following inhibition diameter: White staphylococcus (16.5 mm), Streptococcus mitis (25 mm), Streptococcus sanguis (27 mm), Enterococcus faecalis (10 mm). Conclusions: Hydroalcoholic plant extract obtained from Epilobium parviflorum Schreb had the highest quantity of polyphenols, flavonoids and antibacterial activity.

3. The introduction does not point out the gap of the literature. The study seeks to fill and novelty of the study over the existing literature. This point showed be further elaborated.

We have changed that

1Introduction

Epilobium is a genus of perennial herbaceous plant (Onagraceae family). The most common species include Epilobium parviflorum Schreb., Epilobium hirsutum L., Epilobium rosmarinifolium (Epilobium dodonaeiVill.), Epilobium roseumSchreb., and Epilobium an-gustifolium L. [1].

The anti-inflammatory and antiproliferative activity of Epilobium parviflorum Schreb extracts was initially studied on cells in the context of benign prostatic hyper-plasia in parallel the analgesic, antioxidant, antibacterial and antifungal action were studied [2-4]. Plant polyphenols represent a group of chemical substances ubiquitously distributed in all higher plants. These secondary metabolites possess free radical scav-enging and antimicrobial activity. These properties can be advantageously exploited, especially because of the abundance of polyphenols and their derivatives in various agricultural and food industry waste and by‐products and the possibility of convenient extraction by either organic or aqueous solvents [5-7].

In the oral cavity, nearly 700 species of bacteria can be found, most of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria [8]. There are two types of intra-radicular infections: primary and secondary, each with specific microbiology. The primary infection of the root canal system results from colonization with a hetero-genous group of microbes that have entered the pulp tissue, when exposed during car-ies or traumas; this group is dominated by Gram-negative oral anaerobic bacteria (Prevotella spp., Porphyromonas spp., Fusobacterium spp., Veillonella spp.), aside from bacterial species which are also part of the commensal oral microflora: Gram-positive anaerobic bacteria (Propionibacterium spp., Bifidobacterium spp., anaerobic strepto-cocci), treponemas or Campylobacter spp.[8,9]. The secondary intraradicular infection appears inside the root canal system, after the treatment of the affected tooth has been initiated. Secondary intraradicular infections harbor a limited number of bacterial species, with predominance of Gram-positive bacteria, namely Enterococcus faecalis (E. faecalis), oral streptococci, lactobacilli or Candida albicans, in contrast Gram-negative bacteria are involved to a lesser extent [10]. The most common micro-organism found in asymptomatic, persistent endodontic infections is E. faecalis. Its in-cidence in this type of infection varies from 24% to 77% [8,10]. E. faecalis possesses enzymes and constitutive structures that are able to suppress the action of lympho-cytes and promote inflammation, which contributes to progression of endodontic in-fections [10,11]. Also, this microorganism has the ability to attach to dentin due to an inner component which is the collagen-binding protein [9,11,12].

Several studies have evaluated the antimicrobial efficacy of endodontic irrigants such as sodium hypochlorite (NaOCl), ethylene diamine tetra acetic acid (EDTA) and chlorhexidine (CHX) against E. faecalis biofilms [13]. In general, the aim of any disin-fection strategy is to reduce the bacterial load to a subcritical level so that the patient’s immune response allows healing by itself [14]. Endodontic research has always been focused on developing methods or endodontic irrigants that can completely remove the bacterial biofilm with minimum side-effects. Several endodontic irrigants are being widely used in the treatment of biofilms with varied effectiveness. Although 2% CHX has been proven to possess a high antimicrobial property, it has failed to disrupt the biofilm. However, NaOCl can disrupt the biofilm, disintegrating the dental pulp which is a built-in organ, but it is a well-known irritant to periapical tissues. Therefore, iden-tification of natural products in the disinfection of root canals can be interesting [13,15,16].

In unconventional modern practice, endodontists seek the use of natural remedies as an adjunct to classical therapy, especially since the antibacterial action of plant ex-tracts meets the phenomenon of antibiotic resistance, a problem that dominated med-ical practice in the second millennium and which seems to persist in an upward trending the third millennium [17].

Plant extracts are fluid, soft or dry pharmaceutical / phytopharmaceutical prepa-rations obtained by extracting plant products with different solvents [18].

In recent years, emphasis has been placed on the pharmaceutical and therapeutic revaluation of herbal preparations through a good knowledge of the physio-chemical and therapeutic properties of the active ingredients in medicinal plants and the de-velopment of extraction techniques and quality control means. Extractive solutions are pharmaceutical forms that contain smaller or larger proportions of active substances together with less active ones and ballast, extracted with the help of solvents. The ex-traction process involves the separation of the medically active portions of the plant tissues from the inactive or inert components by the use of selective solvents in stand-ardized procedures [19].

Aqueous extractive solutions are prepared from different parts of the plant or mixtures of medicinal plants, using water as a solvent, preferably distilled or softened [20]. Description of the double maceration technique: the extracted product is first mixed with 1/2-2/3 of the total amount of solvent, after which the liquid is separated and the residue is pressed. It will contact the rest of the solvent, thus obtaining a new amount of extractive solution. The two extractive liquids will combine and filter after a 24-hour rest [21].

Ultrasound-assisted extraction (UAE) is one of the most important techniques used for the extraction of valuable compounds from plant materials and is quite adaptable in the laboratory or on an industrial scale [22,23]. The method involves the use of ultrasound, with frequencies ranging from 20 kHz to 2000 kHz, which increase the permeability of cell walls and produce cell lysis, thus promoting the extraction of biologically active compounds [24].

The aim of the study is the use of plant extracts, obtained from Epilobium parviflo-rum Schreb, as a basis for alternative use in perspective in the treatment of endodontic pathology.

The objectives of this study were:

1) Identification and quantification of phenols and flavonoids found in vegetable extracts obtained from Epilobium parviflorum Schreb;

2) Testing the antibacterial activity of the vegetable extracts.

4. The abstract should be comprehensive

We have changed that

Abstract: The present study was carried out as a preliminary study in developing an experimental endodontic irrigant solution based on plant extracts obtained from Epilobium parviflorum Schreb that largely replenish the properties of the usual antiseptics used in dentistry. Background: This study investigated the phytochemical contents of plant extracts obtained from Epilobium parviflorum Schreb and their potential antibacterial activity. Methods: Identification and quantification of bio-logically active compounds were made by UV field photo spectrometry adapting the Fo-lin-Ciocalteau test method. Antibacterial activity was tested on pathological bacterial cultures col-lected from tooth with endodontic infections using modified Kirby-Bauer diffuse metric method. Results: Polyphenols and flavonoids were present in all plant extracts; the hydroalcoholic extract had the highest amount of polyphenols-17,44 pyrogallol equivalent (Eq Pir) /mL and flavo-noids-3,13 quercetin equivalent (Eq Qr) /mL. Plant extracts had antibacterial activity among the tested bacterial species with the following inhibition diameter: White staphylococcus (16.5 mm), Streptococcus mitis (25 mm), Streptococcus sanguis (27 mm), Enterococcus faecalis (10 mm). Con-clusions: Hydroalcoholic plant extract obtained from Epilobium parviflorum Schreb had the highest quantity of polyphenols, flavonoids and antibacterial activity.

5. Key words must be arranged alphabetically

We have changed that

antibacterial activity; Epilobium parviflorum Schreb; flavonoids; phenols; vegetable extract;

6. The objectives and conclusion of the study are not clear and need to re-write

We have changed that

There were two distinct objectives of this study:

1) Identification and quantification of phenols and flavonoids from the vegetable extracts obtained from Epilobium parviflorum Schreb;

2) Testing the antibacterial activity of the vegetable extracts.

Conclusions

All plant extracts contain polyphenols and flavonoids. Hydroalcoholic plant ex-tract obtained from Epilobium parviflorum Schreb had the highest quantity of polyphe-nols, flavonoids and antibacterial activity. The most sensitive bacterial species were those belonging to the Gram-positive group.

7. A relevant hypothesis for the study is missing from the introduction. A true scientific question should be formed

We have changed that

1. Introduction

This study investigated the phytochemical contents of plant extracts obtained from Epilobium parviflorum Schreb and their potential antibacterial activity.

8. Simplify the statement in the paper. Please combine and condense the discussion and conclusion

We have changed that

Conclusions

All plant extracts contain polyphenols and flavonoids. Hydroalcoholic plant ex-tract obtained from Epilobium parviflorum Schreb had the highest quantity of polyphe-nols, flavonoids and antibacterial activity. The most sensitive bacterial species were those belonging to the Gram-positive group.

We are very grateful to you for the review report and for the extremely useful suggestions!

Sincerely,

Dr. Erdogan Elvis Șachir 

 

 

Reviewer 5 Report

Authors have revised the manuscript and I still see some minor errors that can be fixed before publication.

Line 61, 135, 244, 268: missing space between words

Line 180: bioactive compounds (The text is about more not only one)

Line 268: the description of table can be elaborated for standards

Table 2 topic and first 3 lines are unclear to read

Table 4 legend/ description is missing.

Discussion about the role of potential bioactives is missing.

Conclusion could be elaborated a bit more.

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled ”Studies Regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb”, co-authored by Erdogan Elvis Șachir, Cristina Gabriela Pușcașu, Aureliana Caraiane, Gheorghe Raftu, Florin Ciprian Badea, Mihaela Mociu, Claudia Maria Albu,LilianaSachelarie, Loredana Liliana Hurjui, Cristina Bartok-Nicolae.

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

Authors have revised the manuscript and I still see some minor errors that can be fixed before publication.

 

Line 61, 135, 244, 268: missing space between words

 We have changed that.

Line 180: bioactive compounds (The text is about more not only one)

2.2. Identification and quantification of biologically active compounds

Line 268: the description of table can be elaborated for standards

We have elaborated the description of the table

Table 2 topic and first 3 lines are unclear to read

We have changed that.

Table 4 legend/ description is missing.

In the Table 4 is presented the total flavonoid content of the hydroalcoholic plant extract obtained from Epilobium parviflorum Schreb which was determined from the regression equation (Graph 2) of the calibration curve, ranging from 2.84 to 2.9 mg EqQr / g plant powder with an arithmetic mean of 2.87 mg EqQr / g vegetable powder; the content of the aqueous plant extract was determined from the regression equation (Graph 2) of the calibration curve, ranging from 0.78 to 0.9 mg EqQr / g plant powder with an arithmetic mean of 0.86 mg EqQr / g vegetable powder. The hydroalcoholic plant extract had the highest flavonoid content with a mean of 2.87 Eq Qr/mL, and the aqueous plant extract had the lowest level with a mean of 0.86 Eq Qr/mL.

Table 4. Results of determinations in Eq Qr / mL of total flavonoid content.
Vegetable extract	Determinations no.	Absorbance	EqQr/mL
Hydroalcoholic	1	2.3569	2.87
	2	2.3411	2.84
	3	2.3779	2.90
		Mean/SD	2.87±0.02
Aqueous	1	1.1332	0.9
	2	1.0619	0.78
	3	1.1341	0.9
		Mean/SD	0.86±0.06

 

 

 

 

SD=Standard deviation

 

Discussion about the role of potential bioactives is missing.

We have changed the order.

The constituent compounds of a plant extract obtained from Epilobium parviflorum Schreb by the method of hydrodistillation was determined in a study by Tomáš Bajera et al. (2017) among which were 40% alcohols, 13.8% polyphenols, the remaining percentages being occupied by esters, aldehydes, ketones, aromatic hydrocarbons; Although the method for determining the biologically active compounds is completely different from that of the present study, the results obtained competing with those found by Tomáš Bajera who demonstrated the existence of polyphenols in the extract of Epilobium parviflorum Schreb.

The results obtained on antibacterial susceptibility are consistent with the content of polyphenols and flavonoids, with a direct proportionality between biologically ac-tive compounds and antibacterial activity.

Although it is difficult to compare the data in our study are consistent with those in the literature, both ethanolic extracts have antibacterial activity in direct proportion to the concentration of polyphenols and flavonoids.

Conclusion could be elaborated a bit more.

All plant extracts contain polyphenols and flavonoids. Hydroalcoholic plant extract obtained from Epilobium parviflorum Schreb had the highest quantity of polyphenols, flavonoids and antibacterial activity. The most sensitive bacterial species were those belonging to the Gram-positive group.

 

We are very grateful to you for the review report and for the extremely useful suggestions!

Sincerely,

Dr. Erdogan Elvis Șachir 

Round 3

Reviewer 1 Report

There are some improvement made for the antibacterial sector for the supporting. But still the overall quality for the antibacterial test was not there, I would suggest you to further improve this sector and using more microscopy method for the observations. and exclude the experimental factors which may affect the final result with proper methodology statement within the paper.

You can take a reference from the article structure from the following articles:

1. Rapid preparation and antimicrobial activity of polyurea coatings with RE‐Doped nano‐ZnO doi : 10.1111/1751-7915.13891

2. Antimicrobial Nanomaterials and Coatings: Current Mechanisms and Future Perspectives to Control the Spread of Viruses Including SARS-CoV-2  DOI: 10.1021/acsnano.0c05937

 

 

Author Response

The authors acknowledge the useful observations and suggestions of the reviewer’s as concerns the manuscript entitled ”Studies Regarding the Antibacterial Effect of Plant Extracts Obtained from Epilobium parviflorum Schreb”, co-authored by Erdogan Elvis Șachir, Cristina Gabriela Pușcașu, Aureliana Caraiane, Gheorghe Raftu, Florin Ciprian Badea, Mihaela Mociu, Claudia Maria Albu,Liliana Sachelarie, Loredana Liliana Hurjui, Cristina Bartok-Nicolae.

According to the reviewer’s recommendations, all the suggestions were considered, as follows:

There are some improvement made for the antibacterial sector for the supporting. But still the overall quality for the antibacterial test was not there, I would suggest you to further improve this sector and using more microscopy method for the observations. and exclude the experimental factors which may affect the final result with proper methodology statement within the paper.

 

You can take a reference from the article structure from the following articles:

 

1. Rapid preparation and antimicrobial activity of polyurea coatings with RE‐Doped nano‐ZnOdoi : 10.1111/1751-7915.13891

 

2. Antimicrobial Nanomaterials and Coatings: Current Mechanisms and Future Perspectives to Control the Spread of Viruses Including SARS-CoV-2 DOI: 10.1021/acsnano.0c05937

We have entered the references indicated in limitations of the study.

The limitations of the study are given by the final number of the study group and the method for testing the antibacterial activity; to overcome these limitations for future perspective, it is necessary to conduct a study with a larger number of patients and to extend the research using microscopy method for the antibacterial activity observations with the same high standards used in the studies conducted by Sara M. Imani (2020) and Yuanzhe Li (2022) [36,37]. 

 

We are very grateful to you for the review report and for the extremely useful suggestions!

Sincerely,

Dr. Erdogan Elvis Șachir