Hair-Growth-Promoting Effects of Fermented Red Ginseng Marc and Traditional Polyherb Formula in C57BL/6 Mice

Round 1

Reviewer 1 Report

In the work “Hair growth promoting effects of fermented red ginseng marc and traditional polyherb formula in  C57BL/6 mice” the authors analyzed the effect of several concentrations of fermented RGM (fRGM) and traditional polyherb formula (PH) and a combination of these on hair growth and hair quality. Despite the authors found beneficial effects for the higher doses of fRGM and PH and the Combi when compared with the control, only treated with the vehicle, distilled water, I have a great concern since no synchronization of the hair growth phases was performed.

 

Please see the comments below:

Page 1 Lines 21-23 “Red ginseng marc (RGM) 21 is a byproduct remained after extracting red ginseng, but several studies have 22 shown that the RGM has still bioactive components.” – Please revise the sentence

Page 3 Lines 125-126 – Please give more details regarding the methodology used to characterize the fRGM.

Page 6 line 217 – authors described the fRGM as containing antioxidant and anti-inflammatory properties; two key parameters on hair disorders. Why the authors did not characterize the antioxidant and anti-inflammatory properties of fRGM, PH, and the mixture?

Pag 6 line 225 – “hair growth seemed to be faster in the treatment groups of fRGM….” Authors should revise. A scientific result should not be based on weak evidence. Please avoid using an expression as   “seemed”

Figure 1 -  How do the authors explain the great variation observed between the individuals in each test group? Can these differences be related to the fact that the authors did not synchronize the hair cycle stages? For the FRGM400 test group, how do the authors explain the changes in the growth pattern observed between Day 12 and Day 14 for the first mice?

Page 7 Line 248 – As for the hair growth, the authors support their claims regarding the effect of fRGM on hair length and thickness on 'Suggestive' evidence (“ …hair length and thickness were seemed to be increased…”

Figure 3 – If the authors did not match the hair cycle stages how they compare the length and thickness of hairs that might be in different cycles?

Figure 4 – The cross-sections of fRGM400 and PH200 seem to be the same. Can the authors comment on this?

Page 9 Line 286 – anlagen-phase should be anagen-phase. Please indicate in Figure 4 some of the follicles that are in the anagen phase

Figure 5 – analyzing the Ki-67 stain for the DW sample is not clear that any of the follicles is positive. How do authors explain that for this sample the follicles are mainly in other phases?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

1) Lines 50-51: “The other factors include heredity, aging, medication and psychological stress”.

Please add bibliographic reference.

 

2) Lines 56-59: “Finasteride (as a 5-α reductase type II inhibitor) and minoxidil (as an antihypertensive vasodilator) have been currently approved by the Food and Drug administration (FDA) of the United States for hair growth [6]”.

 

Be careful, because it is no longer very current (since 2008).

 

 

3) Please indicate the maximum concentration used (%) of thioglycollate.

 

4) Are you aware of obtaining the same results of hair regeneration, by proliferation of cells of the hair follicle, with other removal procedures that destroy the most distal germ cells? Such as, for example, deep burns (third degree burns) with loss of the dermis (and subcutaneous tissue) and the appearance of scarring alopecia. Or also, the inability to produce new hair by heating the germ cells with heat energy (laser) of maximum intensity.

 

5) Lines 94-104: “RGM or fermented RGM has shown antioxidant, immunomodulatory and anticancer effects [20,21]. It is believed that the bioactive components including ginsenosides and polysaccharides contained in RGM are still effective despite small amounts [20,22]. Moreover, fermentation process of RGM enhances the beneficial effects, especially strong antioxidant and anti-inflammatory effects [23]. Effective microorganism (EM) is a complex of microorganisms including photosynthetic bacteria, lactic acid bacteria and yeast, and various natural products fermented with EM enhance the antioxidant, anti-inflammatory, anticancer, anti-stress and neuroprotective effects [24-26]”.

 

Previous results already suggested that red ginseng oil (RGO) was a novel and powerful natural therapeutic product for the treatment of androgenic alopecia, possibly through the hair regrowth activity of its main components, such as linoleic acid and β-sitosterol. RGO and its main components were capable of reducing TGF-β protein levels, but improved the expression of the anti-apoptotic protein Bcl-2. So, is it likely that the benefits of fRGM's antioxidant capacity are contributed by effective microorganisms (EM) and are completely different from RGM's own natural antioxidants? Is the final RGM extract (without fermentation process) likely to be originally antioxidant deficient?

Could this indicate that the efficiency of fRGM is the same as that of polyherb extracts, and thus that fermented ginsenosides extracts of RGM is not the specific cause of the benefit?

 

Do you know if the supply of testosterone in these animals would block the beneficial process of fRGM on anagen stimulation?

 

6) I miss a final concluding section with a small paragraph.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

All the questions have been addressed by the authors.

Author Response

We appreciate you for a kind comment. 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The article investigates the impact of red ginseng on mouse hair follicle growth as a potential treatment for hair growth disorders. While the concept of using such a product on hair growth is interesting, I do not think the methodology used and the interpretations are correct or sufficient. 

 

Point by point

(1.) Grammar problems occur throughout the manuscript. It needs to be rewritten and rechecked. Just some examples below. 

'which is occurred' -> 14

'mice model' -> line 18

'the altered growth cycle' ->37

'to now, finasteride' -> Line 41

 

Results

(2.) 3.1 components of fRGM.  Did you analyse this yourself? Where is the data? I don't really understand what this results adds.

 

(3.)   3.2

- these results are poorly described and are hard to follow. 'Markedly grown' is not correct, it was not significant. 

-What your results are showing is entry and progression into anagen, not just hair growth.

-Some mice show no hair growth over 2 weeks e.g. in the DW group at day 14. If the mice were correctly synchronised with Wax (as described), I do not understand why the mice are showing such substantial differences in hair cycle. Moreover, one of the DW mice has no hair growth at day 14, This does not seem to be captured in figure 1b or c. Please can you discuss this. 

As in almost all groups, there is  quite dramatic differences between each mouse, more mice are needed for these results. 

 

(4.)  3.3 

How exactly were '10 hairs collected', weighed and measured. Were they plucked or micro-dissected out of the skin?  If plucking you cannot calculate the hair growth as there is no way of standardising how much of the hair is successfully plucked out of the skin.  If you have micro dissected them, what was the reference point of measurements of length?  There is huge amount of variability and this needs to be standardised. 

On this basis, how did you pick the region for hair sampling? Your mice as mentioned have huge differences in patterning and therefore hair cycle stage and hair length. What you could be measuring is hair cycle differences not hair growth differences. you region should be clearly documents and in my opinion more than 10 hairs is needed. 

 

(5.)   3.4 

Figure 3 is too small. 

I have quite a few concerns with the hair cycle data. This is methodologically incorrect and with this in mind, as you synchronised the mouse hair cycle with wax, it is very unlikely you have telogen and catagen hair follicles. The majority will be entering and progressing through anagen (as evident in your figure 1). 

Distinguishing catagen/telogen/anagen as you have done is completely incorrect. e.g. as late as catagen stage 6, the hair follicle will be in the adipose layer so merely drawing a cut off line is not sufficient. Moreover, the images shown are poorly aligned and you have cross sections of most hair follicles. These are extremely difficult to stage. On this basis, those which would be classed as telogen by your criteria have pigmentation, which would not be present in this stage. Most likely you are counting the same hair follicle multiple times as the sections are not good. To stage hair follicles correctly, you must systematically and accurately stage them following published criteria by 'Muller-Rover, 2001'. 

 

(6).  3.5 

The images for figure 4 are too small. 

Again I have concerns with this experiment. From what I can see, you have clearly anagen stage 6 hair follicles which are completely negative for ki-67. Moreover, I do not see any positive staining within the epithelial hair follicles. You need to redo these experiments with an appropriate positive control. 

 

(7).  Discussion

Some of your conclusions drawn here are not supported by the data you show. For example you do no show an increased ratio of anagen hair follicles or the increase in total hair follicles. 

 

Moreover, I'm confused by the statement 'increased ki-67 positive follicles in the dermal papilla'. You do not show this. The dermal papilla is the mesenchymal component of the hair follicle.

 

 

Taken together, I think this manuscript needs substantial work on the methodology. From here the discussion would need to be rewritten with the correct results in mind. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript entitled "Promoting effects of fermented red ginseng marc and traditional polyherb formula on hair growth in C57BL/6 mice" describes the effect of red ginseng on hair growth.

The manuscript is well written and the results are suported by literature.

In my opinion, this paper is suitable to be published after minor revisions.

Comments:

- Abbreviations should be described when used for the first time

- The authors should pay attention to significant numbers

 

Author Response

Comments and Suggestions for Authors

The manuscript entitled "Promoting effects of fermented red ginseng marc and traditional polyherb formula on hair growth in C57BL/6 mice" describes the effect of red ginseng on hair growth. The manuscript is well written and the results are suported by literature. In my opinion, this paper is suitable to be published after minor revisions.

Comments:

- Abbreviations should be described when used for the first time

- The authors should pay attention to significant numbers

Response: We appreciate you for a kind comment. We re-checked all of the abbreviation to be the first expressions as the full name, and numbers in our manuscript. The changed parts were expressed in red for responses to reviewers and in blue for better expressions. Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

In the work “Promoting effects of fermented red ginseng marc and 2 traditional polyherb formula on hair growth in 3 C57BL/6 mice” the authors analyzed the effect of several concentrations of fermented RGM (fRGM) and traditional polyherb formula (PH) and a combination of these on hair growth and hair quality. The authors found beneficial effects for the higher doses of fRGM and PH and the Combi when compared with the control, only treated with the vehicle, distilled water. The manuscript is well written and with very good and interesting results. However, some minor issues should be addressed:

• As a suggestion in Lines 15-16: “Red ginseng marc (RGM) is a byproduct after extracting red ginseng, but several studies have shown the RGM has still bioactive ginsenosides.”- consider to revise this sentence
• Please increase the size of Figures 1a, b and c.
• Figure 1 b is very hard to read
• Please increase the size of Figure 3 a or divide into two panels for a better reading
• Please increase the size and quality of Figure 4 a, it is very hard to read and analyze

Author Response

Response to Reviewer 3 Comments

Point 1: As a suggestion in Lines 15-16: “Red ginseng marc (RGM) is a byproduct after extracting red ginseng, but several studies have shown the RGM has still bioactive ginsenosides.”- consider to revise this sentence

Response 1: We made small changes on line 17-18 in page 1 (Please see the attachment).

Point 2: Please increase the size of Figures 1a, b and c. Figure 1 b is very hard to read

Response 2: We increased the image size of figure 1a, and separated by two figures (Fig. 1a to Fig. 1; Fig. 1b-c to Fig. 2a-b, in the revised file). We also updated Fig 1b with changing the colors of marks for each group. However, because the results had no significances, many parts were overlapped. Please understand that we did our best to improve the graphs (Please see the attachment).

Point 3: Please increase the size of Figure 3 a or divide into two panels for a better reading

Point 4: Please increase the size and quality of Figure 4 a, it is very hard to read and analyze

Response 3 and 4: We increased image sizes of Figs 3 and 4 (Figs 4 and 5, respectively, in the revised file) and their qualities, and we added more information on lines 185-188 in page 7 and the figure legend in page 8 (Please see the attachment).

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

While I appreciate the effort to improve the English, I am disappointed that no effort has been made to improve the serious problems with the methodology. 

 

Response 3-3. You use these papers multiple times to describe inconsistencies in your observations. I am concerned with these experiments, because despite what these 2 papers say, the research into the hair cycles of these mice has been documented for decades to be consistent and reproducible. If these really are consistent observations, a higher number of mice is required to fully document these differences. 

 

Response 3-4.  As you have suggested, increasing the sample size may make your results significant, so I fail to see why you have not done them. 'Suggestive' evidence is not sufficient for a published scientific article. 

 

Response 4:

The small hair follicles you see in the ‘no hair’ areas are most likely early anagen hair follicles. You are therefore comparing early anagen with late anagen which should not be done. For accurate measurements, hair cycle stages MUST be matched.

 

Response 5:

I am extremely concerned with your hair cycle data. The papers you reference specifically ‘Junlatat’, ‘Shin’ and ‘Sumikawa’ use the ‘Muller Rover’ paper I recommended as their guidance for hair cycle stage. They do not divide the skin into regions as you have done, they use the hair follicle morphology and position in the dermis to aid staging. Even catagen hair follicles are in the adipose layer and do not retreat to the upper dermis until very late catagen. Moreover, the hair cycle was assessed on follicles where the dermal papilla was clearly visible. I did not ask for a detailed hair cycle stage analysis, but reference to the morphological criteria for anagen, catagen and telogen must be followed.

 

In the images you show in your paper, the hair follicles are in anagen as evident by the pigmentation, shape and the fact that the adipose layer is large. Even the examples in your ‘response letter’ that you label as telogen, are anagen , which can be determined by the fact that there is heavy  pigmentation. Your methodology is flawed, you Cannot use cross sections to stage hair follicles. The dermal papilla must be visible and the position in the dermis must be observable.  In your methodology, you are classing the upper parts of anagen hair follicles (infundibulum and isthmus) as telogen therefore your results are wrong. The sections must be recut and reanalysed following well accepted publications as mentioned previously. 

 

 

Response 6:

If there are many reports suggesting that these mice, which have been well characterized, are indeed showing differences in hair cycle, even when synchronised then this needs to be published. Anecdotal evidence is not sufficient to justify a publication.

Despite this, I would be more willing to accept your argument if you had staged your hair follicles correctly. From all the data you have shown, you are merely showing anagen progression. I have not seen any convincing evidence to suggest there is a change in hair cycle stage.

 

 

Other points:

 

Line 122-124. How did you standadise your weight measurements. Plucking removes epithelial tissue as well as the hair shaft and is difficult to standardise. The differences may be due to the inconsistencies with plucking.

 

Figure 2 : you must show the plucked hair follicles as images with the length changes  evident.

199-202: Proliferated root sheath and mature root sheath does not make sense. What are you referring to?

In addition, as discussed, the location in the skin is not sufficient to stage hair follicles, especially with cross sections. In addition, you have a thick adipose layer which is not seen in telogen. Either the follicles are not in telogen, which seems likely from the images shown, or your follicles are not synchronised successfully (figure 4).

Author Response

Please see the attachment.

Response to Reviewer 1 Comments
Point 1. Response 3-3. You use these papers multiple times to describe inconsistencies in your observations. I am concerned with these experiments, because despite what these 2 papers say, the research into the hair cycles of these mice has been documented for decades to be consistent and reproducible. If these really are consistent observations, a higher number of mice is required to fully document these differences.
Response 3-4. As you have suggested, increasing the sample size may make your results significant, so I fail to see why you have not done them. 'Suggestive' evidence is not sufficient for a published scientific article.
Response 1: In our experimental design, the male C57BL/6 mice (not female) were anagen-synchronized via cliffing and waxing in a same manner. And the mice were divided by eight groups, based on the body weights. You mentioned that the DW group should show almost similar hair growth, however, the Fig. 1a is actual our result. We previously observed that the hair was completely grown around 3 weeks after the hair-removal, and it was difficult to detect differences among the groups at the time-point for earlier promoting effects of hair growth. If we took images later than the second week post-waxing, the DW group could show the similar images. We revised these with other two studies in Round 1, but we could show more references with similar results for the various hair growth on the middle or the later time-point of hair growth. We made small changes for the randomized grouping on lines 108-110 in page 3 (Highlighted).
Although kinetic change of the hair growth was not significant (Fig. 2a), the total hair growth from the initial to the last treatments was significantly increased in the high doses of the fRGM and PH, and the Combi groups compared with the DW group (Fig. 2b). We could increase the sample sizes, but we think that it is still difficult to get significant differences on specific days post-treatment until the last treatment, and it is not meaningful for our objectives in this study. The final hair growth was significantly improved in the treatments compared with the DW group, so we don’t think that these are insufficient evidences.
Point 2. Response 4: The small hair follicles you see in the ‘no hair’ areas are most likely early anagen hair follicles. You are therefore comparing early anagen with late anagen which should not be done. For accurate measurements, hair cycle stages MUST be matched.
Response 2: We compared the hair length and thickness at a same time-point after treatments for 2 weeks, however, we didn’t compare the hair cycle stages among groups. We measured them in 10 hairs showing similar shapes, and we think that it can be representative results for the regrown hairs.
Point 3. Response 5: I am extremely concerned with your hair cycle data. The papers you reference specifically ‘Junlatat’, ‘Shin’ and ‘Sumikawa’ use the ‘Muller Rover’ paper I recommended as their guidance for hair cycle stage. They do not divide the skin into regions as you have done, they use the hair follicle morphology and position in the dermis to aid staging. Even catagen hair follicles are in the adipose layer and do not retreat to the upper dermis until very late catagen. Moreover, the hair cycle was assessed on follicles where the dermal papilla was clearly visible. I did not ask for a detailed hair cycle stage analysis, but reference to the morphological criteria for anagen, catagen and telogen must be followed.
In the images you show in your paper, the hair follicles are in anagen as evident by the pigmentation, shape and the fact that the adipose layer is large. Even the examples in your ‘response letter’ that you label as telogen, are anagen , which can be determined by the fact that there is heavy pigmentation. Your methodology is flawed, you Cannot use cross sections to stage hair follicles. The dermal papilla must be visible and the position in the dermis must be observable. In your methodology, you are classing the upper parts of anagen hair follicles (infundibulum and isthmus) as telogen therefore your results are wrong. The sections must be recut and reanalysed following well accepted publications as mentioned previously.
Point 4. Response 6: If there are many reports suggesting that these mice, which have been well characterized, are indeed showing differences in hair cycle, even when synchronised then this needs to be published. Anecdotal evidence is not sufficient to justify a publication. Despite this, I would be more willing to accept your argument if you had staged your hair follicles correctly. From all the data you have shown, you are merely showing anagen progression. I have not seen any convincing evidence to suggest there is a change in hair cycle stage. 

Response 3 and 4: As you mentioned, we adapted the data for the hair growth cycle in Fig. 4a and the figure legend, Table 2 and the relevant descriptions on line 129 in page 3, lines 187-190 in page 7, lines 200-209 in page 8 and lines 246-248 in page 10 (Highlighted). According to the reference of Muller Rover et al. (2001), the anagen-synchronized hair follicles can be placed in the dermis (anagen I and II, as well as a telogen stage) and mostly in the subcutis (anagen III to VI and catagen stages), with the characteristic morphologies. We could observe the catagen- and telogen-phased follicles in H-E stains (We agree that the arrows and arrowheads were wrong in Fig. 4a, and deleted them), however, we also agree that it can’t be exact to discriminate the hair growth cycle in the cross sections. We think that the anagen-synchronized follicles are mostly going on the growth development to the anagen VI stages in deeper subcutis. So, we re-examined the follicles in the dermis and subcutis, because the data suggest promoting effects on development of the anagen-phased follicles.
Point 5. Other points:
Point 5-1. Line 122-124. How did you standadise your weight measurements. Plucking removes epithelial tissue as well as the hair shaft and is difficult to standardise. The differences may be due to the inconsistencies with plucking.
Response 5-1: For the hair weights, we plucked the hairs carefully not to injure the epithelial tissues in a same manner for all of the samples, and we were blinded to the groups. Although the method is rough, however, the result showed significant differences in the hair weights.
Point 5-2. Figure 2 : you must show the plucked hair follicles as images with the length changes evident.
Response 5-2: We are so sorry that we don’t have good high-magnified images for showing as one of the figures. The image analyses were performed under microscopy in slides placing many hairs (but separated).
Point 5-3. 199-202: Proliferated root sheath and mature root sheath does not make sense. What are you referring to?
Response 5-3: We deleted the expressions.

Point 5-4. In addition, as discussed, the location in the skin is not sufficient to stage hair follicles, especially with cross sections. In addition, you have a thick adipose layer which is not seen in telogen. Either the follicles are not in telogen, which seems likely from the images shown, or your follicles are not synchronised successfully (figure 4).
Response 5-4: We think that the time for waxing was enough for the synchronization, because the longer treatment of NiClean showed redness or some skin rash. As you mentioned in point 3, most of the follicles were in anagen stages. It means the synchronization was not wrong. The anagen-phased follicles are located to the deeper subcutis depending on the development. We just counted the follicles in the dermis and subcutis, and re-examined them. We expected that the follicles in the subcutis can be in the later stages of anagen or catagen stages. We fixed mis-spelling and grammar on line 142 in page 4 and line 173 in page 6 (Highlighted).

Author Response File: Author Response.pdf