Isolation, Characterization, and Breast Cancer Cytotoxic Activity of Gyrophoric Acid from the Lichen Umbilicaria muhlenbergii
Round 1
Reviewer 1 Report
Article: "The pharmaceutical potential of the lichen-derived secondary metabolite gyrophoric acid as a potent therapeutic agent".
Gyrophoric acid is well known as constituent of Umbilicaria muhlenbergii and it is well known for anti-cancer efficacy against different cancer cell lines including MCF-7 cell lines. What is new in this manuscript?
Mohammadi, M., Bagheri, L., Badreldin, A., Fatehi, P., Pakzad, L., Suntres, Z. and van Wijnen, A.J., 2022. Biological Effects of Gyrophoric Acid and Other Lichen Derived Metabolites, on Cell Proliferation, Apoptosis and Cell Signaling Pathways. Chemico-Biological Interactions, 351, p.109768.
Letwin, L., Malek, L., Suntres, Z. and Christopher, L., 2020. Cytotoxic and Antibiotic Potential of Secondary Metabolites from the Lichen Umbilicaria muhlenbergii. Current Pharmaceutical Biotechnology, 21(14), pp.1516-1527.
Author Response
Novelty has been discussed with the paragraph below. Two new references were provided (38,41).
Lines 84-90:
The current work is a continuation of our previous studies on U. muhlenbergii (38,41) and provides new evidence for the effects of purified GA on cancer cells. Although GA is known for its anti-tumor activity, reports on its isolation methods are scant. Our objective was to present a systematic study that describes an efficient extraction and purification protocol for isolation of GA from U. muhlenbergii. The protocol that we have developed may be used as an alternative route for production of GA acid as well as isolation and purification of other polyphenolic depside molecules.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Overall, this is a great systematic study that included the purification, chemical characterization and potency characterization of GA. This study would provide a new way for GA production, and also showed great evidence on GA’s effect for cancer cells. However, there are some details that may need the author’s further clarification.
Introduction section
(1). Page2, line 55-64 (second paragraph of the introduction section) : The author mentioned lichen crude extracts and lichen secondary metabolites have cytotoxic effects on cancer cells, and also cited previous studies.
Is there any hypothesis or existing studies on why lichen crude and its secondary metabolites could have these effects on cancer cells? If so, it would be nice if the author could add some discussion on this.
(2). Page2, line 66-75 (last paragraph of the introduction section: What’s the novelty and importance of this study comparing with previous studies? Could the authors highlight the novelty and importance of this study?
Results and Discussion Section
(3). Page4, line 181-185: based on the data, it seems the purification process yield is very sensitive to solvent type, does the author have any hypothesis on the cause? If so, could the author add some discussions on it.
(4). Page5, Table 1: did the author characterized the purity of F2-F4? If so, it would be very beneficial if the author could add the purity data to Table 1.
(5). Page6, Figure S1, line 217-219: the author mentioned absorption at λmax 270nm and 304nm to indicate the presence of carbonyl group and aromatic group. However, from figure S1, it seems not very clear on 270nm and 304nm absorption. Could the author add more information and discussion to clarify.
(6). Page6, Figure S2, line 221-224: the x axis of figure S2 seems ended at 3600cm-1, can author extend it >3700cm-1 since the author also used data at 3665cm-1. Also, there is a typo in x-axis label of S2 (“wavelenght”), could the author fix the typo and add the unit in x-axis label.
(7). Page8, section 3.4: from Figure5, it showed the effect of GA on MTT cell viability first increase then decrease as the dose increase.This trend seems very unique, as from general understanding, the cell viability would decrease as dose increase if the treatment is effective. Could the author add some discussions on what may cause this unique trend?
(8). Page8, section 3.4: the author mentioned “As evident from Figure 5 of this 268 study, GA isolated from U. muhlenbergii crude extracts decreased the cell viability of MCF- 269 7 cells in a time- and dose-dependent manner”, Figure 5 showed the “dose-dependent manner”, is there any data that could show “time-dependent manner”
Author Response
Article: "The pharmaceutical potential of the lichen-derived secondary metabolite gyrophoric acid as a potent therapeutic agent".
Reviewer-2
Overall, this is a great systematic study that included the purification, chemical characterization, and potency characterization of GA. This study would provide a new way for GA production, and also showed great evidence on GA’s effect for cancer cells. However, there are some details that may need the author’s further clarification.
Introduction section
Comment 1. Page2, line 55-64 (second paragraph of the introduction section) : The author mentioned lichen crude extracts and lichen secondary metabolites have cytotoxic effects on cancer cells, and also cited previous studies. Is there any hypothesis or existing studies on why lichen crude and its secondary metabolites could have these effects on cancer cells? If so, it would be nice if the author could add some discussion on this.
Answer: Discussion provided with two paragraphs (see below), and supported by inclusion of three new references (13,37,38):
Lines 55-57:
More than 1,000 secondary metabolites have been identified in different lichens with various biological and pharmacological activities due to the presence of numerous aliphatic, cycloaliphatic, aromatic, and terpenoid compounds [13,26,27].
Lines 66-74:
Overall, research on lichen-derived secondary metabolites to date has demonstrated their great potential for use as therapeutic agents. Strong evidence has been obtained for their mechanism of action on cancer cells that includes apoptosis, necrosis or autophagy with the cell cycle arrest at G0/G1 phases, cell cycle regulation associated with cyclin-dependent kinases (CDK4, CDK6) or cyclin D1, oxidative stress due to superoxide dismutase (SOD) or malondialdehyde (MDA), modulation of inflammatory responses via TNF-α, IL-1β, IL-6, and TGF-β1 and targeting of microRNA molecules, modulation of anti-proliferative effects by regulating signaling pathways (ERK1/2 and AKT) or proliferating protein marker Ki-67 [37,38].
Comment 2. Page2, line 66-75 (last paragraph of the introduction section: What’s the novelty and importance of this study comparing with previous studies? Could the authors highlight the novelty and importance of this study?
Answer: Novelty was addressed with the following new paragraph. We also cited two new references (38, 41).
Lines 84-90:
The current work is a continuation of our previous studies on U. muhlenbergii (38,41) and provides new evidence for the effects of purified GA on cancer cells. Although GA is known for its anti-tumor activity, reports on its isolation methods are scant. Our objective was to present a systematic study that describes an efficient extraction and purification protocol for isolation of GA from U. muhlenbergii. The protocol that we have developed may be used as an alternative route for production of GA acid as well as isolation and purification of other polyphenolic depside molecules.
Results and Discussion Section
Comment 3. Page4, line 181-185: based on the data, it seems the purification process yield is very sensitive to solvent type, does the author have any hypothesis on the cause? If so, could the author add some discussions on it.
Answer: The following paragraph and a new reference (50) were added:
Lines 199-206:
In many cases, purification of secondary metabolites from lichens is tedious and complex. The product yield is affected by the type of solvent used, presence of other compounds with similar structures and colored substances (pigments), possible interactions and/or formation of hydrogen bonding between the targeted molecule, the solvent, and other molecules. All that may create a masking effect due to overlapping of compounds of different solubility and polarity with the molecule of interest, gyrophoric acid in our case, thereby altering molecule’s polarity and reducing its purification yield [50].
Comment 4. Page5, Table 1: did the author characterized the purity of F2-F4? If so, it would be very beneficial if the author could add the purity data to Table 1.
Answer: We do appreciate this comment. As our main objective in this study was to isolate and characterize gyrophoric acid, we decided to work with one (the most promising) fraction only (F1). Hence, we did not characterize the other fractions (F2-F4).
Comment 5. Page6, Figure S1, line 217-219: the author mentioned absorption at λmax 270nm and 304nm to indicate the presence of carbonyl group and aromatic group. However, from figure S1, it seems not very clear on 270nm and 304nm absorption. Could the author add more information and discussion to clarify.
Answer: A short paragraph added, and a new reference (60) cited:
Lines 232-235:
The UV absorbance of F1 (Figure S1) was measured in the wavelength range of 240-340 nm. The UV absorbance peaks for gyrophoric acid were detected at 210, 240, 310, and 340 nm, with high similarity for the depside groups [48] and in good agreement with previous findings [58,59,60].
Comment 6. Page6, Figure S2, line 221-224: the x axis of figure S2 seems ended at 3600cm-1, can author extend it >3700cm-1 since the author also used data at 3665cm-1. Also, there is a typo in x-axis label of S2 (“wavelenght”), could the author fix the typo and add the unit in x-axis label.
Answer: We thank the reviewer for these comments. The x-axis of Figure S2 was extended to 4000 cm-1, and the x-axis label was corrected to “Wavelength”. Figure S2 was updated in the Supplementary file.
Comment 7. Page8, section 3.4: from Figure5, it showed the effect of GA on MTT cell viability first increase then decrease as the dose increase. This trend seems very unique, as from general understanding, the cell viability would decrease as dose increase if the treatment is effective. Could the author add some discussions on what may cause this unique trend?
Answer: The following paragraph was added:
Lines 285-294:
At 300 µg/mL GA, the cell proliferation was inhibited by approximately 45% and thereafter, with a further increase in the GA dose up to 500 µg/mL, reached a plateau. Keeping in mind the SD of the data presented in Figure 5, no significant difference in the cell viability was observed in the 300-500 µg/mL GA concentration range. This might be due to the fact that the MCF-7 cancer cells were exposed to GA for only 4 h. The low exposure time did not allow to develop a clear dose-dependent correlation between cell viability and GA concentration using the MTT assay. Similar observations were reported for the impact of GA dose on the inhibition of cell proliferation for HDF cells after their incubation with GA for 24 h [19]. Increasing the incubation time to 72 h produced a well-pronounced dose-dependent effect of GA on cell proliferation.
Comment 8. Page8, section 3.4: the author mentioned “As evident from Figure 5 of this 268 study, GA isolated from U. muhlenbergii crude extracts decreased the cell viability of MCF- 269 7 cells in a time- and dose-dependent manner”, Figure 5 showed the “dose-dependent manner”, is there any data that could show “time-dependent manner”
Answer: Thank you for this comment. Unfortunately, we have no data to support a statement on the time dependency of cell viability on the GA dose, although such evidence can be found elsewhere in the literature. We have removed “time-dependent” from the above sentence.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The title does not reflect essential contents, because the work is about isolation of gyrophoric acid (GA) from the lichen Umbilicaria muhlenbergii.
Author Response
Yes, we agree the title could be more specific to closely reflect the work done in this paper, and have no objection to modify it. Here we propose a new title:
Isolation, characterization, and breast cancer cytotoxic activity of gyrophoric acid from the lichen Umbilicaria muhlenbergii
