Dear Reviewer
Thank you very much for useful comments which I revised them already. I hope that
it is correct and I will not back to me again.
Reviewer:MARCIO FRONZA
Reviewer's report:
Major Compulsory Revisions
COMENTS TO AUTHORS

Authors have made substantial changes in the manuscript. Specially in the
statistical analysis and in the variations in the figures that are much lower now. How it
happens?
-
Because we used standard error of mean (SEM) instead standard deviation
(SD) for represented the variability of our data. The SEM describes the
variation of all possible sample means and as calculated by the SD of the
sample data divided by the square root of the sample size. So, the SEM value
will always be smaller than the SD of the sample. And I added the sentence
which Prof. David Senchina suggested to describe your understanding in the
statistical analysis. ( I paste this sentence as yellow line)

Some important questions which I have address before have not been answered
and they are pertinent:

In abstract, dioscorealide B are described as the main bioactive constituent in the
ethanolic and aqueous crude extracts from D. membranacea Pierre. However no data
confirm this information. At least, the HPLC profile is necessary. Moreover, the
unknown compounds must be shown on the chromatogram. I think authors cannot
identify the major compounds. The 1H and 13C NMR data should be added in the
manuscript or in the supplementary information.
- In abstract, we change this sentence “The aims of this study were to investigate the
effects of ethanolic and aqueous crude extracts from D. membranacea Pierre, and its main
bioactive constituent dioscorealide B” to “The aims of this study were to investigate the
effects of ethanolic and aqueous crude extracts from D. membranacea Pierre, and pure
compound from D. membranacea Pierre, dioscorealide B” in page 2, line 7.
- We added the HPLC profile (Figure 6) and inserted the sentence “The HPLC profile
of dioscorealide B from D. membranacea Pierre extract as shown in Figure 6.” In page 10,
Line 23.
- We added the 1H and 13C NMR data in Table 1

Moreover now authors have now add the information in the Results and
Discussion (page 11, paragraph 1, line 5): “Dioscorealide B is a naphthofuranoxepin
which found in the rhizome of D. membranacea as shown its structure in Figure 5.
Dioscorealide B content is 6.14 + 0.13 mg/g of ethanolic extract but not found in the
water extract” . How they did the quantification? 6.14 mg/g represents only 0,614% in
the extract. Authors should better explain this fact.
-
We added this sentence “The quantitation of Dioscorealide B in the rhizome of D.
membranacea Pierre was analysed by High Performance Liquid Chromatography-UV
detection followed by the same condition in Sukkan [20]. Dioscorealide B content
was calculated for standard curve which was standard curve between area under the
curve (AUC) of peak on HPLC and concentration, it was determined as yield with
6.14 ± 0.13 mg/g of ethanolic extract. However Dioscorealide B was not found in the
water extract. The HPLC profile of Dioscorealide B from D. membranacea Pierre
extract as shown in Figure 6.” In page 10.
-
It is in extract for 0.614% but it is active compound which it is effect for only cancer
cell but less to normal cell so I need to know that it is effect for immunomodulatory ,
isn’t it?. Thus I have to do experiment and compare with crude extract. I described in
introduction that this compound showed many activity. Sometime the active
compound showed less amount but it make the extract showed activity.
David Senchina
Reviewer's report
This report relates to the revised version of the manuscript. The authors have
addressed most of the criticisms from both reviewers. With the authors'
explanation I better understood their rationale for the statistical method chosen;
however, without the author response (i.e., if I only had the manuscript), I'm still
not sure I would've understood the entire experiment.
One Minor Essential Revision:
If I am understanding the experimental design correctly based off the authors'
response, then the third sentence of "Statistical Analysis" might be more clear if it
was written along these lines:
"Data in the figures represent the combined results from twelve human subjects,
with each condition performed in triplicate for each subject."
I am making this request solely because I think it will help readers understand the
statistics. The current sentence could be interpreted multiple ways.
Some of the additions to the manuscript could use some minor English editing,
but they are understandable.
Answer by author
Thank you very much for your suggestion and I added your sentence already.
-
Author added the sentence of “ Data in the figures represent the combined results
from twelve human subjects, with each concentration performed in triplicate for each
subject." in part of statistic analysis instead the sentence “ All data are for three
separated experiments, with triplicates in each individual experiment for each
compound.”.