Development of a Simple High-Performance Liquid Chromatography-Based Method to Quantify Synergistic Compounds and Their Composition in Dried Leaf Extracts of Piper Sarmentosum Roxb.
Round 1
Reviewer 1 Report
The presented study described standardized analytical HPLC method to identify and quantify tannin, flavonoid, cinnamic acid, essential oil, and vitamin extracted from P. sarmentosum leaves using methanol, chloroform, and hexane and comprised different drying treatments of leaves and their influence on the content of active substances.
I am impressed with the hard and detailed work that the Authors have done. It is extremely important to describe a good method for the analysis of substances with pharmacological potential. The authors did a very good job.
The article is written very carefully, detailing the methodology and results. I believe it can be published as it is.
Author Response
The author's note file is attached.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This is a generally well written manuscript with a good development of analytical method for the phytochemical composition of extracts from piper sarmentosum. My big concern is a lack of agreement of the phytochemical composition in another report and the lack of identification in this manuscript of the glycosylated flavonoids that are normally found predominately in plant tissue extracts. The aglycone forms (myricetin, quercetin, apiginin, kaempferol) or usually found in intact tissues in the glycosylated form. Granted this series of extractions would emphasize the extraction of the aglycones especially with chloroform and hexane, but the methanol extract should have more of the glycosylated forms. In figure 3 I see one large peak at RT 15.2 is not identified. A quick search of the literature on line resulted a few recent papers on P. sarmentosum phenolics, including this paper:
Cutt Fazzlieanie Bactiar, Nur Azlina Mohd Fahami. LC-MS Analysis of Phytocomponents in the Methanol Extract of Piper Sarmentosum Leaves. Pharmacogn. J. (2019) 11(5) 1071-1076.
They identified by LC-MS a number of compounds, some quite prominent in the methanol extrt of the leaves including compounds not noted in this manuscript including the flavonoids amurensin (a acyl substituted flavonoid glucoside), hesperidin, and didymin as well as a number of other phenolic compounds (Brachaminde B, methyl piperate, piperitol, Guineensine) were noted, but none of these compounds were identified in this manuscript. Is there a problem with species identification or varietal variation here? Could one of these compounds be the unidentified peak at 15.2? I realize the Bactiar paper may be a outlier, but it is of a concern to me.
While the analytical work is fairly comprehenisive I am concerned that 1) not all the major flavonoids and phenolics have been identified and 2) that a proper search of the literature for the phytochemical composition of P. sarmentosum has not been included in this manuscript. For example Reference 2 notes the presence of vitexin in all parts of the plant, but vitexin is not noted here. The authors note that they tentatively identified eugenol in their extracts, the ID was based on two literature reports. Could this be one of the other compounds noted in the Bactiar reference? The authors make no mention of any of the piperine-type compounds, yet they have also been reported in this plant. Also not addressed from reference 2 is the presence of the sarmentosumins A, B, C, D which I would think should be seen here.
I am going to suggest a major revision just based on how these particular plant specimens phytochemical compositions compare with that found in the review reference 2. It seems pretty incomplete or at least a better coverage of why these analytical results vary from that found in reference 2 and the Bactiar paper.
Author Response
The author's note file is attached.
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript presented an analytical method quantifying 14 ingredients in the extracts of Piper Sarmentosum leaves. The method has been validated and used to compare effects of different leaf drying methods on the final chemical composition of the extracts. A method capable of analyzing so many compounds in a single chromatgraphic run is certainly good and important for quality control of the materials. The study described appear to be purely chemical analysis and I am not sure how the authors could concluded that the 14 ingredients being analyzed are "synergistic". In the literature, there are some more characteristics compounds have been isolated from P. Sarmentosum. It seems that these compounds (sarmentosine, sarmentine, piperine and pellitorine) may also present in the leaves of the plant (DOI: 10.1016/j.indcrop.2019.02.020). Perhaps, the authors may consider whether these compounds should be included or give some rationale why these 14 compounds are selected for this herb. The description of the preparation of spiked samples sounds strange. (Line 279). What are the reasons for the standards beign added and "incubated" at room temperature? Do the authors expect the standards may react or change in some ways? If so, would that affect the calculation of the recovery? If not, why did they wait for 24 hours before they analysis the spiked samples?
The chromatgraphic condition was one of the major results of this manuscript. However, the presentation is not clear. In line 169 -171, it seems that the gradient is from 10 - 40% acetonitrile (0 - 28 min), then from 40 to 60% acetonitrile (28 - 67 min), then 60 - 90% in (67 - 67+50 min) followed by re-equilibrtation. However, from the later description, I guess they refered to 10 - 40% acetonitile in 0-28 min, 40 to 60% A in 28-39 min and 60 to 90% A in 39 to 50 min. In the results, appartently the third stage (60 - 90% A) does not affect the separation. In that case, does that mena 60 to 90 min in 10 min is arbitary set or that has been optimzed? Why does the method require 26 min re-ealibrated before next run? I believe the manuscript is interesting, but there are questions needs to be addressed.
Author Response
The author's note file is attached.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Much improved, thank you for going over the paper more carefully
Author Response
Reviewer(s)' Comments to Author:
Reviewer #2
General:
Much improved, thank you for going over the paper more carefully
Reply: We highly appreciate the reviewer’s qualification that the paper is much improved.
Sincerely,
Rayudika Aprilia Patindra Purba
Pramote Paengkoum
Corresponding authors
Reviewer 3 Report
I do not consider the responses of the authors adequately addressed the issues raised in the previous comments.
1. About the word "synergistic" in the title. The manscript provided no new evidence that some these compounds worked synetically to address a disease, although, we could infer that may be the case, but there is no proof. Thus, I would suggest remove such word from the title.
2. About the justification of the choice of the compounds for quality control. As stated in previous comments, there are some more characteristics compounds have been isolated from P. Sarmentosum. It seems that these compounds (sarmentosine, sarmentine, piperine and pellitorine) may also present in the leaves of the plant. I would think a better justification about the choice of the markers would be important. Why there four compounds in the previous paper were not included?
3 The The description of the preparation of spiked samples sounds strange. (Line 279). What are the reasons for the standards beign added and "incubated" at room temperature? Do the authors expect the standards may react or change in some ways? If so, would that affect the calculation of the recovery? If not, why did they wait for 24 hours before they analysis the spiked samples? The authors replied that "According to our best knowledge, this procedure is regularly method for preparing stock solution. A quick search of the literature on line resulted a few recent papers on the procedure, e.g., this paper (Ying et al., 2009, https://pubmed.ncbi.nlm.nih.gov/19298706/)."
I read throught the cited paper, they prepared standard solutions and compare instrumental response when the standard solutions were stored for different period of time to examine the stability of the standards in solvent used. I agree that this is regular for preparing stock solution and check stability of the standards. However, the authors incubated spiked samples (line 281 - 283) for recovery study which is not common and I did not see the rationale behind.
4. I am surprised to see the author replied that the last part of the gradient program (line 329-311 was set arbitary. Has the gradient program been optimized? Would it be better to step to 90% after 39 and flush the column for 6 min before re-equlibrate for the next run?
Author Response
Please find the Author's note in attachment.
Author Response File: Author Response.pdf
