Antioxidant and Moisturizing Effect of Camellia assamica Seed Oil and Its Development into Microemulsion

Round 1

Reviewer 1 Report

The present study is an interesting contribution to the field, however, it suffers from several serious problems, especially:

- the composition of the oil(s) tested is missing in the manuscript. This is nowadays unacceptable.

- the methods are not clearly described, the solvents used for DPPH and lipid peroxidation inhibition are missing, as well as the composition of the oil phase in the microemulsions

- the language of the manuscript is sometimes not comprehensive at all, especially in the discussion (I did not read it to the very end)

For detailed comments, see the enclosed file

Comments for author File: Comments.pdf

Author Response

Author's Reply to the Review Report (Reviewer 2)

Comment No. 1

the composition of the oil(s) tested is missing in the manuscript. This is nowadays unacceptable.

Response

The composition of tea seed oil was described in the introduction part as following; “high oleic acid, medium linoleic acid, and low linolenic acid”. Additionally, the components of tea seed oil are well-known, therefore, we did not determine the composition of the oil in the present study but focused on the formulation development.

Comment No. 2

the methods are not clearly described, the solvents used for DPPH and lipid peroxidation inhibition are missing, as well as the composition of the oil phase in the microemulsions

Response

Dimethyl sulfoxide (DMSO) was used as a solvent for DPPH and lipid peroxidation inhibition assay. More detail about the solvent has already been described in line number 91-92 as following; “test sample solution or tocoferyl acetate (TA) as standard solution in DMSO” and line number 104-105 as following; “test sample solution or quercetin (Q) as standard solution in DMSO”.

In addition, C. assamica seed oil (CA) was used as the oil phase in the microemulsions. More detail about the microemulsion formulation has already been described in line number 137-142 as following; “Microemulsions were developed by mixing CA (oil phase) with Smix and water, when Smix were the combination of Tween 85 and ethanal (4:1) or Tween 85 and propylene glycol (2:1). Three microemulsion formulations were developed, including microemulsion A, B, and C. Tween 85 and ethanal (4:1) were used as Smix in formulation A, where the CA:Smix:water ratio was 1:8:1. On the other hand, Tween 85 and propylene glycol (2:1) were used as Smix in formulation B and C, where the CA:Smix:water ratio were 1:8:1 and 1:7:2, respectively.”

Comment No. 3

the language of the manuscript is sometimes not comprehensive at all, especially in the discussion (I did not read it to the very end)

Response

We have followed the reviewer’s comment. The language has already been improved all over the manuscript.

Comment No. 4

Define CA (line 70)

Response

CA (line 71) is an abbreviation of C. assumica seed oil, which has already been described in line number 67.

Comment No. 5

what was the rationale for such a strange concentration?

Response

Since the test samples in our study were fixed oil, there were some limitation about the solubility. The concentration of 37.5 mg/mL was the highest concentration which gave the clear solution in DPPH and lipid peroxidation assay. Therefore, we used this concentration for the antioxidant activity determination.

Comment No. 6

show simple concentration, not log of it here

Response

Figure 2 was plotted by using GraphPad Prism program which was used for the calculation of IC₅₀ value. The X axis need to be log concentration because the equation of sigmoidal curve was used for the IC₅₀ value calculation.

Comment No. 7

Figure 4: what is the oil phase here? glycerin? or glycerin in mixture with the tea seed oil?

Response

The oil phase was C. assumica seed oil (CA). We have already added more detail about the oil phase in line number 126 as following; “CA was used as an oil phase.”

Comment No. 8

this is confusing as Smix seem to have various composition

Response

Smix was the mixture of surfactant and co-surfactant. Surfactants used in the present study included Tween 20, Tween 60, Tween 80, Tween 85, Span 80, and Triton X-114. Co-surfactants used in the present study included ethanol, isopropanol, and propylene glycol.

Comment No. 9

in which solvent? In Figure 1, 37,5 mg/ml 'i.e. 37.5 %, of CA showed 80% DPPH scavenging. How does this correspond with only 10% scavenging at 10%.

Response

Dimethyl sulfoxide (DMSO) was used as a solvent for DPPH assay. The results from this study noted that higher concentration of CA possessed higher antioxidant activity. Additionally, microemulsion formulation could enhance the antioxidant activity of the native CA.

Comment No. 10

Table 2.  specify the conditions of the measurements, mainly the concentration (dose). Overally, all the tables and figures should be self-explanatory

Response

We have followed the suggestions of reviewer. More detail has already been added to Table 2 as following; “A: 10% CA, 64% Tween 85, 16% ethanol, 10% water; B: 10% CA, 53.3 Tween 85, 26.7% propylene glycol, 10% water; C: 10% CA, 46.7 Tween 85, 23.4% propylene glycol, 20% water; Asterisks denote significantly different between the results before and after stability test, P<0.05.”

Comment No. 11

which tested substances? only the whole oil was tested in the present paper.

Response

We have followed the suggestions of reviewer. The sentence “Additionally, CA possessed moisturizing efficacy of the tested substances evaluated on pig skin model” has already been changed to “Additionally, CA possessed moisturizing efficacy evaluated on pig skin model”.

Author Response File: Author Response.docx

Reviewer 2 Report

page 1, lines 37-38: China was the first country to use tea-this sentence supported with a book chapter (Paul, 1997) that deals with Indiand and kenyan tea but not chinese tea.

Line 43: The author states that 2.5 tons, but it is 2.5 million tons. This is a huge difference and the authors need to check the information very accurately.

Bibliography: Reference 3 & 4: enter the page numbers.

line 101: correct )DPPH assay( into '(DPPH assay)

Include the animal use protocol number.

Fig 4 and Fig 5a are same?

The research experiment is not described or detailed well.

The methods are not described well.

Why CA, CO1, and CO2 in 5% concentration was used?

what is the CA with Smix and water ratio?

Add details - heating-cooling stability test.

Overall, the methods were not described wel.. The references needs to be cited more appropriately.

Author Response

Author's Reply to the Review Report (Reviewer 1)

Comment No. 1

page 1, lines 37-38: China was the first country to use tea-this sentence supported with a book chapter (Paul, 1997) that deals with Indian and kenyan tea but not chinese tea.

Response

Although the publication of Paul et al. (1997) (https://doi.org/10.1007/s001220050408) was related to Indian and kenyan tea. But the introduction part mentioned that “South-East Asia is the original home of tea (Camellia sinensis (L.) O. Kuntze), and China was the first country to use tea as a beverage, which is now popular throughout the world.” These sentences could be found in line number 1-4 of the introduction part of the previous publication.

Comment No. 2

Line 43: The author states that 2.5 tons, but it is 2.5 million tons. This is a huge difference and the authors need to check the information very accurately.

Response

We are so sorry for the mistake. We have followed your suggestions. The number “2.5 tons” has been revised to “2.5 million tons” as shown in line number 43.

Comment No. 3

Bibliography: Reference 3 & 4: enter the page numbers.

Response

We have followed the suggestions of reviewer. Page numbers of reference number 3 & 4 have already been added as following;

3. Richards, A.V. The breeding, selection and propagation of tea. Tea Research Institute 1966, 154-160. Sri Lanka.

4. He, S.; Gu, Y. The comprehensive utilization of camellia fruits. American Camellia Yearbook 1982, 104-107. CAB Direct, United Kingdom.

Comment No. 4

line 101: correct )DPPH assay( into '(DPPH assay)

Response

We have followed the suggestions of reviewer. The “)DPPH assay(“ has already been changed into “(DPPH assay)” (line number 102).

Comment No. 5

Include the animal use protocol number.

Response

The piglet skin used in the present study was from stillborn piglets which were accidentally died before birth. Therefore, there was no animal use protocol number. Additionally, more detail about the piglet skin has already been described in line number 115-116 as following; “The tested skins were prepared from the frank area of stillborn piglets which were accidentally died before birth.”.

Comment No. 6

Fig 4 and Fig 5a are same?

Response

Since Fig 4 and Fig 5a are the same, we have already deleted Fig 4.

Comment No. 7

The research experiment is not described or detailed well.

The methods are not described well.

Response

More detail has already been described in the methods in line number 89-142.

Comment No. 8

Why CA, CO1, and CO2 in 5% concentration was used?

Response

We are sorry about the mistake. The pure oil and 100% tocopheryl acetate were used in the present study. The in vitro skin moisturizing effect determination has already been revised in line number 114-115 as following; “The tea seed oils including CA, CO1, and CO2 were examined in comparison with tocopheryl acetate (TA).”.

Comment No. 9

what is the CA with Smix and water ratio?

Response

More detail about CA with Smix and water ratio has already been described in line number 138-142 as following; “Three microemulsion formulations were developed, including microemulsion A, B, and C. Tween 85 and ethanal (4:1) were used as Smix in formulation A, where the CA:Smix:water ratio was 1:8:1. On the other hand, Tween 85 and propylene glycol (2:1) were used as Smix in formulation B and C, where the CA:Smix:water ratio were 1:8:1 and 1:7:2, respectively.”

Comment No. 10

Add details - heating-cooling stability test.

Response

We have followed the suggestions of reviewer. More detail about heating-cooling stability test has already been added in topic 2.3.4. Stability study, line number 57 as following; “6 heating-cooling cycles of 4°C (24 h) switching with 45°C (24 h)”.

Comment No. 11

Overall, the methods were not described well. The references needs to be cited more appropriately.

Response

More detail has already been described in the methods in line number 89-142 Additionally, all references has already rechecked.

Author Response File: Author Response.docx

Reviewer 3 Report

Revision of the manuscript  Cosmetics-318694

Title: Antioxidant and moisturizing effect of Camellia assamica seed oil and

its development into microemulsion

The manuscript under appreciation describes the antioxidant and moisturizing effect of Camellia assamica seed oil. The approach of the work is interesting however certain deficiencies in the presentation of the results have been manifested.

The manuscript may be acceptable if the comments below can be addressed.

Overall, the results should be argued better. There are two aspects to consider:

In the discussion of the      results it is necessary to comment on the different behavior of the      antioxidant capacity of the emulsions under conditions as a function of      stability. DPPH and inhibition of lipid peroxidation are two different      methodologies and also with a different behavior in the case of emulsions.      Although, despite being one of the central aspects of the work, its      behavior is not sufficiently discussed.

The      statistical analysis of the results should be improved, including the      results of the test of differences correctly in each case.

In short, the manuscript needs more discussion of the results to be accepted.

In addition, minor changes are necessary:

-          In the Abstract (line 20), “assumica” should be changed to “assamica”. In (line 28) “IC₅₀ against DPPH” it is not a correct expression, it would be better to express it as “the IC₅₀ value of DPPH assay”.

-          In the Introduction section (line 62), it would have to define “MEs.” In (lines 64 and 66) “assumica” should be changed to “assamica”.

-          In the Materials and Methods section:

(Line 88), “the moisturizing efficacy” should be changed  to “antioxidant activity”.  

(Line 91), “linoeic acid” should be changed to “linoleic acid”.

(Line 101 ) “scavening” should be changed to “scavenging”

(line 104) DPPH should be changed to DPPH·

-          In the Results and Discussion section

-          In figure 1 (a) letters should be revised, probably the order will be b, a, b, c. It’s not clear what means TA.

-          In figure 3, letter of statistical difference are not included.

-          In subsection (3.2.2. Microemulsion formulation) it will be important to include that formulation C, contains 20% tea seed oil.

-          In figure 10. It’s important express the meaning of the asterisk, from the statistical point of view. I understand that it is a comparison with oil without emulsifying.

-          In Table 1 and 2, It’s important express the meaning of the asterisk.

Strengths: The purpose of the work and the methodology developed are well developed.

Weaknesses: More concreteness of the results and better discussion of the data are necessary.

Author Response

Author's Reply to the Review Report (Reviewer 3)

Revision of the manuscript  Cosmetics-318694

Title: Antioxidant and moisturizing effect of Camellia assamica seed oil and

its development into microemulsion

The manuscript under appreciation describes the antioxidant and moisturizing effect of Camellia assamica seed oil. The approach of the work is interesting however certain deficiencies in the presentation of the results have been manifested.

The manuscript may be acceptable if the comments below can be addressed.

Overall, the results should be argued better. There are two aspects to consider:

Comment No. 1

In the discussion of the results it is necessary to comment on the different behavior of the antioxidant capacity of the emulsions under conditions as a function of stability. DPPH and inhibition of lipid peroxidation are two different methodologies and also with a different behavior in the case of emulsions. Although, despite being one of the central aspects of the work, its behavior is not sufficiently discussed.

Response

We have followed the suggestions of reviewer. More discussion has already been added in this part in line number 308-314 as following; “These two different methodologies revealed distinctly different results. Lipid peroxidation is a complex process involves various mechanisms, including lipid radical formation, oxygen uptake, and rearrangement of the double bonds in unsaturated lipids [28], whereas, DPPH assay was only related to the scavenging ability. This study revealed that the scavenging ability of microemulsions containing CA would not reduce over the time but the ability to inhibit the rearrangement of the double bonds in unsaturated lipids could be lower.”

Additionally, one more reference has been added as following;

28.   Buege, J.A.; Aust, S.D. Microsomal lipid peroxidation. Methods in enzymology 1978, 302-310. Academic press, United States.

Comment No. 2

The statistical analysis of the results should be improved, including the results of the test of differences correctly in each case.

Response

We have followed the suggestions of reviewer. The statistical analysis of the results has already been improved as shown in Figure 1, 3 and Table 1,2.

Comment No. 3

In the Abstract (line 20), “assumica” should be changed to “assamica”.

Response

We have followed the suggestions of reviewer. The word “assumica” has already been changed to “assamica”.

Comment No. 4

In (line 28) “IC₅₀ against DPPH” it is not a correct expression, it would be better to express it as “the IC₅₀ value of DPPH assay”.

Response

We have followed the suggestions of reviewer. The phrase “IC₅₀ against DPPH” has already been changed to “the IC₅₀ value of DPPH assay”.

Comment No. 5

In the Introduction section (line 62), it would have to define “MEs.”

Response

We have followed the suggestions of reviewer. MEs has already been defined in line number 58 As following; “Microemulsion (ME)”.

Comment No. 6

In (lines 64 and 66) “assumica” should be changed to “assamica”.

Response

We have followed the suggestions of reviewer. The word “assumica” has already been changed to “assamica”.

 Comment No. 7

In the Materials and Methods section:

(Line 88), “the moisturizing efficacy” should be changed to “antioxidant activity”.  

Response

We have followed the suggestions of reviewer. The phrase “the moisturizing efficacy” has already been changed to “antioxidant activity”.

Comment No. 8

(Line 91), “linoeic acid” should be changed to “linoleic acid”.

Response

We have followed the suggestions of reviewer. The word “linoeic acid” has already been changed to “linoleic acid”.

Comment No. 9

(Line 101 ) “scavening” should be changed to “scavenging”

Response

We have followed the suggestions of reviewer. The word “scavening” has already been changed to “scavenging”.

Comment No. 10

 (line 104) DPPH should be changed to DPPH·

 Response

We have followed the suggestions of reviewer. The word “DPPH” has already been changed to “DPPHŸ”.

Comment No. 11

In the Results and Discussion section

In figure 1 (a) letters should be revised, probably the order will be b, a, b, c. It’s not clear what means TA.

Response

We have followed the suggestions of reviewer. The order of letters has already been revised to b, a, b, c. Additionally, more detail of TA has been added in the figure caption as following; “tocopheryl acetate (TA)”.

Comment No. 12

In figure 3, letter of statistical difference are not included.

Response

We have followed the suggestions of reviewer. The statistical difference has already been included in Figure 3.

Comment No. 13

In subsection (3.2.2. Microemulsion formulation) it will be important to include that formulation C, contains 20% tea seed oil.

Response

All formulation contained 10% tea seed oil. The composition of formulation C were 10% tea seed oil, 70% Smix, and 20% water.

Comment No. 14

In figure 10. It’s important express the meaning of the asterisk, from the statistical point of view. I understand that it is a comparison with oil without emulsifying.

Response

We have followed the suggestions of reviewer. The meaning of the asterisk has already been expressed as following; “Asterisks denote significantly different form 10% CA, P<0.05”.

Comment No. 15

In Table 1 and 2, It’s important express the meaning of the asterisk.

Response

We have followed the suggestions of reviewer. The meaning of the asterisk has already been expressed as following; “Asterisks denote significantly different between the results before and after stability test, P<0.05.”

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript has been improved a lot. 

However, not all points have been clarified, especially the composition of CA. As the authors compare the effect of CA to commercial Camellia oleifera seed oils (CO1 and CO2), the composition of all three extract should be given and the differences should be discussed.

Moreover, the manuscript should be professionally proof read in order to further improve the English and the style.

Author Response

Author's Reply to the Review Report (Reviewer 2)

The manuscript has been improved a lot. 

Comment No. 1

However, not all points have been clarified, especially the composition of CA. As the authors compare the effect of CA to commercial Camellia oleifera seed oils (CO1 and CO2), the composition of all three extract should be given and the differences should be discussed.

Response

We have followed the suggestion of reviewer. The compositions of the tea seed oils have already been added in the manuscript in line number 174-178 as following;

3.1. Fatty acid composition of tea seed oils

              The fatty acid composition of CA and CO1 are shown in Table 1. The major components of CA were cis-9-oleic acid, cis-9,12-linoleic acid, and palmitic acid. CO1 had similar major components as CA but with higher amount.

Table 1. Fatty acid composition of tea seed oils (CA) and commercial tea seed oils (CO1)

The method of determination of fatty acid composition of tea seed oils has also been added in line number 86-92 and the related discussion has already been added in line number 272-279 as following;

2.2. Determination of fatty acid composition of tea seed oils

              Fatty acid composition of CA and commercial tea seed oils (CO1) were analyzed according to the method reported by Lucchetti et al. [11]. Briefly, fatty acids were converted to methyl esters before the analysis to reduce their polarity and increase their volatility. Then the methyl esters were quantified by gas chromatography (GC) equipped with a flame ionization detector (FID) (Perkin Elmer Autosystem, Waltham, MA, USA). The fatty acids were identified by their retention times as compared to those of standards purchased from Sigma–Aldrich (St. Louis, MO, USA).

4. Discussion

Tea seed oil is rich in vitamin A, B, and E with no cholesterol [2,17]. The major fatty acids of CA were cis-9-oleic acid (57.97%), cis-9,12-linoleic acid (18.57%), and palmitic acid (17.32%). The fatty acid contents of CO1 were slightly different from CA, as cis-9-oleic acid, the major component in both oils, was more pronounced in CO1 (79.96%) rather than that in CA (57.97%). Furthermore, the amount of cis-9,12-linoleic acid and palmitic acid in CO1 was about half of the amount that detected in CA. However, the results in this study were in an accordance with the literature which noted that tea seed oil contained high amount of oleic acid, medium amount of linoleic acid, and low amount of linolenic acid [5].

Comment No. 2

Moreover, the manuscript should be professionally proof read in order to further improve the English and the style.

We have followed the suggestion of reviewer. The article has already been proof-read by

Dr Karl Bailey, Scientific Officer, Department of Human Nutrition, University of Otago, New Zealand and Dr Pummy Krittaphol, assistant research fellow, School of Pharmacy University of Otago, New Zealand.

Author Response File: Author Response.docx

Reviewer 2 Report

The major problem with the manuscript is the English language. The article need to be critically proof-read by the native English speaker.

Did the authors measured the composition of the oils?; if not they need to add the piece of data.

The authors stated that 'CA was used as an oil phase' - it is not very clear; what is oil phase?

Correct ')DPPH assay('

Stability study - add more details.

Fig.1A - The first bar has the asterisk as 'b'

Statistical different needs to be explained in the legends.

Fig.2; why the 'X' axis were expressed as log concentration?

Fig 3 - statistics applied only to control, not to the rest of the four groups.

Pseudoternary phase diagram - explain in details.

Fig 8a - The statistics may be explained in detail.

The statistics were very poorly applied. Every figure has a different way of statistical display. The authors need to look into it.

Author Response

Author's Reply to the Review Report (Reviewer 1)

Comment No. 1

The major problem with the manuscript is the English language. The article need to be critically proof-read by the native English speaker.

Response

We have followed the suggestion of reviewer. The article has already been proof-read by

Dr Karl Bailey, Scientific Officer, Department of Human Nutrition, University of Otago, New Zealand and Dr Pummy Krittaphol, assistant research fellow, School of Pharmacy University of Otago, New Zealand.

Comment No. 2

Did the authors measured the composition of the oils?; if not they need to add the piece of data.

Response

We have followed the suggestion of reviewer. The compositions of the tea seed oils have already been added in the manuscript in line number 174-178 as following;

3.1. Fatty acid composition of tea seed oils

              The fatty acid composition of CA and CO1 are shown in Table 1. The major components of CA were cis-9-oleic acid, cis-9,12-linoleic acid, and palmitic acid. CO1 had similar major components as CA but with higher amount.

Table 1. Fatty acid composition of tea seed oils (CA) and commercial tea seed oils (CO1)

The method of determination of fatty acid composition of tea seed oils has also been added in line number 86-92 and the related discussion has already been added in line number 272-279 as following;

2.2. Determination of fatty acid composition of tea seed oils

              Fatty acid composition of CA and commercial tea seed oils (CO1) were analyzed according to the method reported by Lucchetti et al. [11]. Briefly, fatty acids were converted to methyl esters before the analysis to reduce their polarity and increase their volatility. Then the methyl esters were quantified by gas chromatography (GC) equipped with a flame ionization detector (FID) (Perkin Elmer Autosystem, Waltham, MA, USA). The fatty acids were identified by their retention times as compared to those of standards purchased from Sigma–Aldrich (St. Louis, MO, USA).

4. Discussion

Tea seed oil is rich in vitamin A, B, and E with no cholesterol [2,17]. The major fatty acids of CA were cis-9-oleic acid (57.97%), cis-9,12-linoleic acid (18.57%), and palmitic acid (17.32%). The fatty acid contents of CO1 were slightly different from CA, as cis-9-oleic acid, the major component in both oils, was more pronounced in CO1 (79.96%) rather than that in CA (57.97%). Furthermore, the amount of cis-9,12-linoleic acid and palmitic acid in CO1 was about half of the amount that detected in CA. However, the results in this study were in an accordance with the literature which noted that tea seed oil contained high amount of oleic acid, medium amount of linoleic acid, and low amount of linolenic acid [5].

Comment No. 3

The authors stated that 'CA was used as an oil phase' - it is not very clear; what is oil phase?

Response

We have followed the suggestion of reviewer. More detail about the components used for pseudoternary phase diagrams construction was added in line number 133-134 as following; “Three components used for the construction of pseudoternary phase diagrams were oil phase, water phase, and surfactant mixture (Smix).”

Comment No. 4

Correct ')DPPH assay('

Response

We have followed the suggestions of reviewer. The “)DPPH assay(“ has already been changed into “(DPPH assay)” (line number 109).

Comment No. 5

Stability study - add more details.

Response

We have followed the suggestions of reviewer. More detail about the stability study has already been added in line number 164-168 as following;

2.4.4. Stability study

MEs were kept in air-tight containers under the accelerated conditions, i.e. six heating-cooling cycles consisting of 4°C for 24 h switching to 45°C for 24 h. The ME were then characterized for the particle size by using photon correlation spectroscopy (Zetasizer® version 5.00, Malvern Instruments Ltd, Malvern, UK) and the viscosity by using a Brookfield DVIII rheometer (Brookfield Engineering Laboratories, Stroughton, MA) fitted with a bob spindle.

Comment No. 6

Fig.1A - The first bar has the asterisk as 'b'

Response

We have followed the suggestions of reviewer. The order of letters has already been revised to a, b, a, c.

Comment No. 7

Statistical different needs to be explained in the legends.

Response

We have followed the suggestions of reviewer. More detail about statistical different has already been explained in the legends as following;

Line 186-187:             (different letters (a, b, c, and d) denote significant statistically
differences between groups; P<0.05).

Line 198-199:             (Asterisks denote significant statistically differences from before application; P<0.05)

Line 242-244:             (different letters (a and b) denote significant statistically different
internal droplet size and viscosity between groups; P<0.05, whereas, different symbols (a and b) denote significant statistically different polydispersity index; P<0.05).

Line 251:                    (Asterisks denote significant statistically differences from 10% CA; P<0.05)

Comment No. 8

Fig.2; why the 'X' axis were expressed as log concentration?

Response

Figure 2 was plotted by using GraphPad Prism program which was used for the calculation of IC₅₀ value. The X axis need to be log concentration because the equation of sigmoidal curve was used for the IC₅₀ value calculation.

Comment No. 9

Fig 3 - statistics applied only to control, not to the rest of the four groups.

Response

The statistics have already been applied to all groups. However, there was no significant statistically differences. Therefore, no asterisk was denoted in the rest of the four groups.

Comment No. 10

Pseudoternary phase diagram - explain in details.

Response

We have followed the suggestions of reviewer. More detail about pseudoternary phase diagram has already been explained in line number 202-203 as following; “Pseudoternary phase diagram is an important tool for screening of self-dispersible formulation components and to assess the effect of different component of the microemulsion [15].”

Additionally, one more reference has already been added as following;

15.   Ahmad, J.; Amin, S.; Kohli, K.; Mir, S.R. Construction of pseudoternary phase diagram and its evaluation: development of self-dispersible oral formulation. Int j drug dev res 2013, 5(2), 84-90.

Comment No. 11

Fig 8a - The statistics may be explained in detail.

 Response

We have followed the suggestions of reviewer. The statistics have already been applied to Fig 8a and more detail has already been explained.

Comment No. 12

The statistics were very poorly applied. Every figure has a different way of statistical display. The authors need to look into it.

Response

We have added more detail about the statistical analysis in line number 170-172 as following; “Individual differences were evaluated by t-test and One-Way ANOVA with post-hoc testing. Statistical difference was set at P<0.05” Different way of statistical display was due to different method used. The t-test is an analysis of two groups, so asterisks were used for denoting the significantly difference, whereas, One-Way ANOVA is an analysis of at least three groups, so different letters were used for denoting the significantly difference.

Author Response File: Author Response.docx

Reviewer 3 Report

I agree with the changes made. I believe that the modifications have been positive and have made it possible to improve the quality of work.

Therefore I believe that the work can be accepted for publication.

Author Response

Thank you for your valuable suggestions and comments that will permit to improve the paper quality.

Round 3

Reviewer 2 Report

Fig.3: Did the authors compared the statistical difference between control vs TA; Cont vs CA; Cont vs CO1; Cont vs CO2?

Reference 22; Mehta, S.K.; Kaur, G. Microemulsions - add page numbers.

Reference 29; Clint, J.H. Microemulsions. Surfactant - add page numbers.