Title: Physicochemical and antioxidant properties of Bangladeshi honeys stored for more than one year

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1 Author's response to reviews Title: Physicochemical and antioxidant properties of Bangladeshi honeys stored for more than one year Authors: Md. Asiful Islam Md. Nazmul Islam Mohammed Moniruzzaman Md. Abdul Mottalib Siti Amrah Sulaiman Siew Hua Gan Md. Ibrahim Khalil Version: 2 Date: 27 August 2012 Author's response to reviews: see over

2 Journal: BMC Complementary and Alternative Medicine. Manuscript title: Physicochemical and antioxidant properties of Bangladeshi honeys stored for more than one year. Manuscript tile ID: MS: Dear editor, For your kind information, we have modified the manuscript according to the reviewers comments, and the detailed corrections are listed below point by point: Reviewer 1: Maurizio Battino Point by point response: 1. Figures 3, 4, 5 and 6 should be eliminated since all their data can be better and useful collected in a Table. Thanks the reviewer's for his valuable suggestion. The figures (3, 4, 5 and 6) have now removed. The data for figures 3, 4 and 5 have been collected into a single table 3 while the data for fig 6 has been organized into table Table 4: is there any statistic significance? Please indicate which correlation values are significant (and at which level) and which not The statistical significant values have now been indicated with the corresponding statistical significance level. 3. Table 2 and 3: please indicate the level of significance. The levels of significance have been indicated in tables 2 and 3 accordingly.

3 4. What does it mean (N=8)? 8 samples per honey or 8 different honeys? If, as I understood, it is the latter case it is completely misunderstanding the use of "n=8" and it should be avoided throughout the text. Authors should only refer to the fact they used 8 diffferent honeys Thank you for the important comment. N=8 has now been avoided throughout the text to avoid misunderstanding as the study was conducted with eight different honey samples (and not with eight honey samples per honey) as the reviewer has suggested. 5. "Antioxidant activity" is wrong: Authors have never tested honey antioxidant activity but their "antioxidant capacity". Please, use the correct form throughout the text. The term antioxidant activity has been changed to antioxidant capacity according to the reviewer s comments. 6. As far as the previous point is regarded, Authors should also explain the readers why they assayed honey antioxidant capacity and why they chose AEAC, DPPH and FRAP assays and what they actually wanted to assay. In this sense Authors should cite a very recent paper (Niki E. Antioxidant capacity: Which capacity and how to assess it? Journal of Berry Research, 2011, 1(4): , doi /JBR ) which will help them in this aspect and in discussing their results. Thank you for this useful information. We have now cited Niki (2011) in the text as suggested as well as adding this reference in the reference section (ref no 13). 7. There is a large confusion in the reference list which appears especially careless. In fact, 7.1 Some references have issue number and some not

4 7.2 Some references have journal name in full, other the journal name is abbreviated. 7.3 Despite the prestigious (and expensive) certification there are some mistakes such as "Agricultrural" in ref 20. For all the above points, please check carefully throughout the ref list and correct the references according to the Instructions for Authors of THIS journal. The reference list has been carefully checked and corrected again for mistakes. Ref 26 (Previous ref 20) has already been corrected. 8. Finally, the refs list is quite updated but I consider that Authors should also cite PMID: , PMID: , PMID: , PMID: , PMID: , PMID: to give the readers a very, complete up-to-date information of the possible involvement of honey in human health. I have indicated PubMed ID number for brevity. More recent references relevant to our study have been cited as suggested by the reviewer. Five of them are cited in the relevant Background part (Ref No. 8, 9, 10, 11, 12) and one more in Result and discussion part (Ref No. 36).

5 Reviewer 2: Embong Zunaina Major compulsory Revisions: 1. Abstract (results) The mean color intensity was ± 17.5 mau,and the mean protein content was 8.6 ± 0.0mg/g. The values that were mentioned are not the mean but the highest level of color intensity and protein content. We apologise for this careless mistake. This has been corrected as highest accordingly. Thank you for this comment. 2. Result and discussion Total dissolved solids (TDS) and electrical conductivity (EC).of less than 0.8 ms/cm according to the [23]. There is missing word for this sentence. It was according to the EU & the missing word European Union has now been included. Thank you for pointing this out. Table 2 & Table 3 Significant values are represented by different letters. Should be defined clearly the superscript of the different letters. This has now been clearly defined. Figure 3, Figure 4 & Figure 5 also showed the presence of these different letters. Should mention which part the analysis of ANOVA was done. If ANOVA was done, then the exact p value should be stated. Figs 3, 4, 5 have been removed according to reviewer 1 s comments and have now been compiled in table 3. The specific p value has been stated in table 3.

6 Correlation between antioxidant properties and biochemical parameters It was mentioned in the text regarding the correlation of phenolics and flavonoids but there was no data of phenolics and flavonoids presented in Table 4. Suggest including the exact p value for each correlation in Table 4. We apologise for this error. The correlation value of phenolics and flavonoids has now been added to table 4 with specific p value. Minor Essential Revisions 1. Abstract Methods..proline, protein and antioxidantswere determined in Missing space for the word antioxidantswere Space given Results phenolic was ± 5.9 mg gallic acid/kg,and the ± 17.5 mau,and the mean protein Should be spacing after coma for acid/kg,and and also for mau,and Space added..aeac (34.1 ±1.4mg/100 g) and FRAP (772.4 ± 2.5 μm Fe (II)/100 g) were detected The value for FRAP (772.4 ± 2.5 μm Fe (II)/100 g) is not the same as mentioned in the text page 19 ( μm Fe [II]/100g). FRAP value was made (772.4 ± 2.5 μm Fe (II)/100 g) in both abstract and in page 19.

7 2. Background.presence of flavonoids, phenolic acids, ascorbic acid, catalase, peroxidase, carotenoids and products of Maillard reactions [2]. High levels of flavonoids, phenolic acids, vitamin C, ascorbic acid, catalase, peroxidase and.. Should be no spacing before coma for phenolic acids, and peroxidase, Should choose one, either vitamin C or ascorbic acid because both terms are the same. Space deleted before comma and ascorbic acid has been chosen. Thank you for this comment. 3. Material and methods The ph, moisture content and color analysis of honey samples Suggest mentioning the methods or procedures for each parameter (ph, moisture content and color analysis) in each paragraph and do not mixed. ph, moisture content and color analysis have now been separately stated. Thank you for this comment. Electrical conductivity (EC) and total dissolved solids (TDS) using a conductivity meter (HI 98311,Hanna. Should be spacing after coma for (HI 98311,Hanna Space given Proline content Should include a sentence The results were expressed as The sentence has been added. DPPH-free radical-scavenging activities..free radical-scavenging activity of the DPPH radical The determination was Missing full stop between the word radical and The determination Full stop given.

8 4. Statistical analysis difference (HSD) post hoc test (p < 0.05).Correlations were Should be spacing after full stop for (p < 0.05).Correlations Space given. 5. Results and discussion The ph, moisture content and color analysis of honey samples between 3.6 and 4.1 (Table 2).These values are. Should be spacing after full stop for (Table 2).These Space given. Color intensity Table 2 should be cited in the text. Table 2 cited. Total dissolved solids (TDS) and electrical conductivity (EC) Similar to honey samples from India [5], the EC values Should be no spacing before coma for India [5], the Space deleted. Protein The protein content of Bangladeshi honey samples was between 0.99 and 8.60 mg/g (Table 3). The values are not the same as presented in Table (in Table 3 was written 0.9±.02) 8.60(in Table 3 was written 8.6±.01) Values have been corrected. Thank you for this comment. Proline

9 .being the main residue[26]. Should be spacing between residue and [26]. Space given. FRAP..as did Beretta et al. [10](72.8 to μm Fe [II]/100g), Should be spacing between [10] and (72.8 to μm Fe [II]/100g) Space given. 6. Table 3 The data for flavonoids and proline should be standardized in term of decimal point. All the data of flavonoids and proline have been standardized in terms of decimal. Reviewer 3: Sandra Mendoza Page 2. In the Background section: There no available.. must be corrected for Background: There is no available.. In the methods section: antioxidantswere.. must be corrected for Methods: antioxidants were.. In the results section: ± 6.9mgcatechin/Kg.. must be corrected for ± 6.9 mg catechin/kg.. In all text give a space between the number and the units.

10 Page 4...between the months June 2009 and August 2009 and stored must be corrected for..between the months of June and August of 2009 and stored The stored conditions of the honey samples must be stated Page 7. The preparation of the honey extract for the total phenolics determination is missing The preparation of the honey extract for the total phenolics determination has been added. Page 8. The preparation of the honey extract for the total flavonoids determination is missing The preparation of the honey extract for the total flavonoids determination has also been added. Page 11...FRAP values were expressed as micromoles of ferrous equivalent (μm Fe [II]) per Kg honey. If it is micromoles such be written μmol Fe[II] instead of μm Fe [II]. Or it is micromolar? (Table 2).These must be corrected for (Table 2). These Basically the unit is Micromoles. So μm is corrected to μmol throughout the text. Thank you for this comment. Page 13. whereas BDH-4 is a multifloral honey.. must be corrected for whereas BDH-4 is a monofloral honey....according to the [23].. must be corrected for..according to the European Union [23].. BDH-4 is a monofloral honey. European Union added. Page 14 samples with their low ph must be corrected for samples with low ph

11 Page 15 Total phenolics section..using gallic acid standard was must be corrected for..using gallic acid as standard was In all text Galic acid/kg must be corrected for Gallic acid/kg Page 16 Total flavonoids section (155 ± 6.9 mg catechin/kg) must be corrected for (155 ± 6.9 mg Catechin/Kg) The results of ascorbic acid equivalent antioxidant content are not in agreement with the DPPH results. Since for both determinations the inhibition % of DPPH absorbance was used, it was expected a high correlation. The authors need to further explain.. AEAC is not expressed in % inhibition. Only DPPH is expressed in % inhibition. There was strong correlation of DPPH with AEAC (r= 0.74), however we have not mentioned in the text. Hopefully, there is no more confusion. Page 20 correlation between of color must be corrected for correlation between color Table 3..mg Galic acid must be corrected for..mg Gallic acid mg Catechin/Kg must be corrected for mg Catechin/Kg Figure 4..DPPH % inhibition at mg/ml must be corrected for..dpph % inhibition at 200 mg/ml Figure 4 already removed because of reviewer 1 s comment. Figure 5 Review the unit for the FRAP result μm or μmol Figure 5 already removed because of reviewer 1 s comment.

12 The manuscript has been resubmitted to your journal. We look forward to your positive response. Thank you, Dr. Md. Ibrahim Khalil Associate Professor Department of Biochemistry and Molecular Biology Jahangirnagar University Savar, Dhaka-1342, BANGLADESH Tel:

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