Title: Comparison of bee products based on assays of antioxidant capacities

Transcription

1 Author's response to reviews Title: Comparison of bee products based on assays of antioxidant capacities Authors: Yoshimi Nakajima Kazuhiro Tsuruma Masamitsu Shimazawa Satoshi Mishima Hideaki Hara Version: 2 Date: 23 January 2009 Author's response to reviews: see over

2 LETTER HEAD January 23, 2009 Dear Dr. Iratxe Puebla Senior Editor, BMC-series journals MS: Manuscript title: Comparison of bee products based on assays of antioxidant capacities Thank you for your letter concerning our manuscript entitled Comparison of bee products based on assays of antioxidant capacities. We describe the changes made in response to the reviewer s comments point-by-point as follows. We trust that the manuscript will now be acceptable for publication in BMC Complementary and Alternative Medicine. Reviewer(s) Comments to Author Referee 1 1. (Page 1/Lines 2-3): The title of a manuscript must be short and informative. Hence the title of manuscript should be corrected as Comparison of bee products based on assays of antioxidant capacities H 2 O 2 scavenging is not a radical-scavenging assay. As suggested by the reviewer, we have changed the title Comparison of bee products (including propolis, royal jelly, and pollen) based on assays of radical-scavenging capacities to Comparison of bee products based on assays of antioxidant capacities on page 1. As suggested by the reviewer, we have changed the word all of the manuscript, radical-scavenging assay to antioxidant assay, H 2 O 2 radical to H 2 O 2, O 2 - radical to O 2 -, HO radical to HO, and radical species to ROS. 2. (Page 12/Line 6): Caffeic acid has a strong antioxidant and radical scavenging properties. The authors should be mentioned it is here and give this reference Toxicology, 217 (2-3), in reference list. As suggested by the reviewer, we have added the sentence Caffeic acid which is a cinnamic acid 1

3 derivative has a strong antioxidant and radical scavenging properties. and reference Toxicology, 217 (2-3), on page 12, lines (Page 13/Line 7): After the sentence of Oxidative stress, which may be defined as an imbalance between the production and removal of ROS the following related references should be give Life Sciences, 78 (8), and Amino Acids, 32, We referred to Science, 262 (29), to discuss about imbalance between the production and removal of ROS. 4. (Figure 1): In this figure G, H, I combined as a new separate Figure. 5. (Figure 2, 3): At the same way, A, B, C combined in a new Figure, Also D, E, F and G, H, I combined in one Figure. Then, one figure must include these three figures. Same correction must do for Figure 3. As suggested by the reviewer, it is clear that G, H, I combined as a new separate Figure. However, fluorescence intensity of each ROS species was too different to combine axes range of ordinate. Therefore, we would like to keep the present style of figures. Referee 3 1. The introduction, considering the great number of articles in this area, can be more explored. As suggested by the reviewer, we have added the sentences, Furthermore, Turkey bee pollen has inhibitory effects against mycelia growth of microbes and several pharmacological activities (Ozcan et al., 2004). on page 5, lines 4-5 and Excess ROS generation has damage to various cell components and triggering of the activation of specific signaling pathways. Both of these effects can influence numerous cellular processes linked to ageing and the development of age-related disease (Finkel et al., 2000). on page 5, lines The Figure 1 (ABC) need be clarified. As suggested by the reviewer, we have changed the slight graph line to thick line and more clear in 2

4 figure 1 (A-C). 3. The legends need be summarized, and not need explanations about material and methods. As suggested by the reviewer, we have deleted and changed the sentences in figure legends about explanation about material and methods. In figure 1 legend, we have deleted the sentences ROS production was stimulated with H 2 O 2 at 100 µm, with KO 2 at 100 µm, or with H 2 O 2 at 1 mm plus ferrous perchlorate (II) at 100 µm, and fluorescence was measured at various time-points. on page 24, lines 5-7 and we have changed the sentences (A) Kinetics of the DCFH oxidation induced by H 2 O 2 in RGC-5. (B) Kinetics of the DCFH oxidation induced by O 2 - in RGC-5. (C) Kinetics of the APF oxidation induced by HO in RGC-5. to Kinetics of the DCFH oxidation induced by (A) H 2 O 2, (B) O 2 -, and kinetics of the APF oxidation induced by HO in RGC-5. on page 24, lines 7-8. In figure 2 legend, we have deleted the sentences One of the substances listed below was added to RGC-5 cultures for 1 h, followed by addition of CM-H 2 DCFDA (10 µm) or APF (10 µm) for 20 min. on page 24, lines In figure 3 legend, we have deleted One of the compounds listed below was added to RGC-5 cultures for 1 h, followed by addition of CM-H 2 DCFDA (10 µm) or APF (10 µm) for 20 min. on page 25, lines The discussion need have more comparisons with others studies. As suggested by the reviewer, we have added the sentences, In antioxidant-capacity assay using stable green radical cation of 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), caffeic acid and chlorogenic acid are stronger than ascorbic acid (Grace et al., 2000). Similarly, our results indicated that antioxidative effects of caffeic acid and chlorogenic acid against H 2 O 2 and O 2 - were 4-6 times stronger than ascorbic acid (Table 1). on page 15, line 9-page 16, lines 4 and In previous report, antioxidative effects of EEP are measured using chemiluminescence assay. Pretreatment with EEP scavenged the all ROS, although the IC50 values are different from our results. These assay may probably be due to the ph of the medium which permitted different redox potential of the propolis antioxidant compounds, and also due to the different kind of radicals formed (Marquele et al., 2005). Our results may be more reflected in biochemical reactions within the body because our study was measured using living cells. on page 13, lines16- page14, lines The author didn't explain because what they use a ethanolic extration for propolis and not for pollen. 3

5 We described methods both ethanol extract of propolis and pollen. In propolis, The Baccharis proplis was extracted with 95% ethanol at room temperature. on page 6, lines In pollen, It was extracted with 95% ethanol at room temperature. on page 7, lines The conclusion is very poor. As suggested by the reviewer, we have added the sentences in conclusion to Caffeoylquinic acid derivatives, main constituents of propolis, have strong antioxidative effects and equivalent efficacy of trolox and ascorbic acid. on page 16, lines Furthermore, we have changed the word propolis to propolis and its constituents on page 17, line 1. Referee 4 1. Hydrogen peroxide is not a free radical; it is reactive oxygen species. The authors should correct this concept along the manuscript including its title. As suggested by the reviewer, we have changed the title Comparison of bee products (including propolis, royal jelly, and pollen) based on assays of radical-scavenging capacities to Comparison of bee products based on assays of antioxidant capacities on page 1. As suggested by the reviewer, we have changed the word radical-scavenging assay to antioxidant assay, H 2 O 2 radical to H 2 O 2, O 2 - radical to O 2 -, HO radical to HO, and radical species to ROS. 2. The authors should give more details and references about the methods used to measure the scavenging ability of ROS. For this reviewer is clear that hydrogen peroxide penetrates to the cell diffusing through the membrane. Specifically, the generation of superoxide anion with KO2 and hydroxyl radical via Fenton reaction (using hydrogen peroxide and perchlorate hexahydrate) is occurring inside the cell? That is, the precursors of this ROS penetrate freely into the cells? In addition, the fluorescent marker APF is specific for hydroxyl radicals? APF is sensitive to hydrogen peroxide that is used to generate hydroxyl radicals? There are methods to measure ROS scavenging ability that are cell-free. 4

6 CM-H 2 DCFDA was converted with DCFH by being taken into the cell and being cut with the intracellular enzyme (esterase), therefore we think the precursors of this ROS penetrate freely into the cells. As CM-H 2 DCFDA was loading into cells, intracellular radicals have reacted with only DCFH, not CM-H 2 DCFDA. These assay was derived from the technique described by Sawada (Sawada GA, 1996, Cytometry, 25(3), ). APF is specific for hydroxyl radicals and little sensitive to hydrogen peroxide. APF was able to measure ROS scavenging ability both cell-free and cellular, but CM-H 2 DCFDA was unable to measure ROS scavenging ability that is cell-free. 3. Abbreviations should be used consistently along the text. For example in some cases, the abbreviations are used the first time when they are not defined. As suggested by the reviewer, we have founded the abbreviations are used the first time when they are not defined. We have changed the sentences ROS-sensitive probe CM-H 2 DCFDA or aminophenyl fluorescein (APF) to reactive oxygen species (ROS)-sensitive probe 5-(and-6)-chloromethyl-2, 7 -dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA) on page 2, lines There are also some spelling mistakes (for example oreder in Abstract) As suggested by the reviewer, we have changed the spelling mistakes word oreder to order on page 2, line The work may be improved significantly whether the authors could address the ability of the compounds studied here to scavenge another ROS such as peroxynitrite, singlet oxygen and hypochlorous acid (Discretionary revisions). As suggested by the reviewer, representative ROS are H 2 O 2, O 2 -, HO, peroxynitrite (ONOO - ), singlet oxygen ( 1 O 2 ), and hypochlorite anion ( - OCl). Further studies will be needed to clarify the antioxidative effects of bee products. Referee 5 1. General comments: 5

7 It is not clear for us the aim of this manuscript. We suggest that the author re-organize this manuscript, showing the chemical compostion of propolis extracts to compare the relationship between the quantity of chemical compounds present in the extract and its effects per se. The purpose of report was to investigate the antioxidative effects of three representative bee products (propolis, RJ, and pollen), and to identify any ROS (H 2 O 2, O 2 -, HO ) specifically scavenged by such bee products. Furthermore, we investigated the ROS scavenging effects of propolis components which were selected by reference to manuscript (Mishima et al., 2005, J. Ethnopharmacol., 99(1), 5-11). Their chemical structure was published by our previous report (Nakajima et al., 2007, Life Sci., 80(4), 370-7). The purpose of report was not to compare the relationship between the quantity of chemical compounds present in the extract and its effects, but to investigate the bee products scavenging ability against several ROS. We believed this antioxidative comparison of bee products is new report. 2. Background: Biological activities described in references 1-7 use European propolis and it is not related with Bracharis dracunculifolia propolis extract from Brazil. The correct is used the most and numerous actual references to this propolis sample (green propolis). As suggested by the reviewer, we described biological activities (antioxidant, anti-inflammatory, and antimicrobial activities) using a part of European and Canadian propolis. Therefore, we have changed the references using Brazilian propolis reference 2 (Drago et al., 2000) to (Souza et al., 2007), references 4 and 5 (Krol et al., 1990 and Scheller et al., 1990) to (Teixeira et al., 2008), and reference 3 (Mirzoeva et al., 1996) to (Paulino et al., 2006 and Barros et al., 2008) on page 4, line 9. References1, 6, and 7 are described biological activities using Brazilian propolis. 3. Methodology: Radical scavenging-capacity assay used by the authors is adequate to investigate the antioxidant effect of the bee products and very well described. Although, the results produced with this method alone is not conclusive to justify the conclusion suggested by the author. In this report, we elucidated antioxidative effects of bee products using antioxidant-capacity assay. Recently, we have reported that propolis and propolis components had antioxidant actions against 6

8 lipid peroxidation in mouse forebrain homogenates and against the diphenyl-p-picrylhydrazyl (DPPH) radical [Shimazawa et al., 2005, Evid. Based Complement. Alternat. Med., 2(2), and Nakajima et al., 2007, Life Sci., 80(4), 370-7]. Therefore, we think that propolis and its main constituents have various radical scavenging effects. 4. Results: The author present the radical scavenging-capacity to all bee products expressed in IC50 mean, although, royal jelly did not have effect and then royal jelly could not be compared with another bee product and neither show IC50 value (upper than 100µg/mL). We suggest that the author remove the royal jelly from rank order. In the same way, the author could not compare the main compounds with propolis extracts, because the author did not show how much of these compounds there are in each extract (water or ethanolic). The purpose of this study was to investigate the antioxidative effects of three representative bee products (propolis, RJ, and pollen), and to identify any ROS (H 2 O 2, O 2 -, HO ) specifically scavenged by such bee products. We want to compare the antioxidative effects of three bee products in the same condition, therefore the fact that RJ and constituent of RJ cannot scavenge ROS is very important in this report. In this study, we investigated the ROS scavenging effects of propolis using the same one and same extraction methods (water and ethanol) reported by Mishima et al., Therefore, this report was used as reference with propolis component fraction. 5. References: We suggest a update of the references and link as recent as possible to green propolis or Baccharis dracunculifolia propolis from Brazil. As suggested by the reviewer, we described biological activities (antioxidant, anti-inflammatory, and antimicrobial activities) using reference in Therefore, we have added the recent references as described by response of (2. Background). Referee 6 1. Figure legends are very long. It is recommended to make them shorter by transferring some explanations to the method section". 7

9 As suggested by the reviewer, we have deleted and changed the sentences in figure legends about explanation about material and methods. In figure 1 legend, we have deleted the sentences ROS production was stimulated with H 2 O 2 at 100 µm, with KO 2 at 100 µm, or with H 2 O 2 at 1 mm plus ferrous perchlorate (II) at 100 µm, and fluorescence was measured at various time-points. on page 24, lines 5-7 and we have changed the sentences (A) Kinetics of the DCFH oxidation induced by H 2 O 2 in RGC-5. (B) Kinetics of the DCFH oxidation induced by O 2 - in RGC-5. (C) Kinetics of the APF oxidation induced by HO in RGC-5. to Kinetics of the DCFH oxidation induced by (A) H 2 O 2, (B) O 2 -, and kinetics of the APF oxidation induced by HO in RGC-5. on page 24, lines 7-8. In figure 2 legend, we have deleted the sentences One of the substances listed below was added to RGC-5 cultures for 1 h, followed by addition of CM-H 2 DCFDA (10 µm) or APF (10 µm) for 20 min. on page 24, lines In figure 3 legend, we have deleted One of the compounds listed below was added to RGC-5 cultures for 1 h, followed by addition of CM-H 2 DCFDA (10 µm) or APF (10 µm) for 20 min. on page 25, lines In Figs, it is recommended to replace "total intensity" with "fluorescence Intensity". Total intensity was calculated by integrating the area under the DCF or reactive APF fluorescence intensity curve for 20 min after treatment with ROS-generating compounds. Therefore, we would like to leave total intensity. However, as suggested by the reviewer, we have changed the word fluorescence to fluorescence intensity on figure In the title of each Fig, I suggest to add the following: "in term of fluorescence Intensity". As suggested by the reviewer, we have changed the title of each fig, in figure 1 Time-kinetic and concentration-response data for antioxidant activities of Brazilian green propolis towards production of various radical species (H 2 O 2, O 2 -, HO ) in term of fluorescence intensity on page 24, lines 3-4. In figure 2 Antioxidant activities of bee products and trolox towards production of various radical species (H 2 O 2, O 2 -, HO ) in term of fluorescence intensity on page 24, line 15. In figure 3 Antioxidant activities of main constituents of WEP (caffeoylquinic acid derivatives) towards production of various radical species (H 2 O 2, O 2 -, HO ) in term of fluorescence intensity on page 25, lines Correct to: "one of the two." P. 9, line 3 (Results) 8

10 As suggested by the reviewer, we have changed the sentences, one of two to one of the two on page 9, line Correct: "through other mechanisms other than ROS scavenging P.14 As suggested by the reviewer, we have changed the sentences, through another mechanism (i.e., are not involving ROS scavenging) to through other mechanisms other than ROS scavenging on page 14, line Correct to "due to the hydroquinone moiety" P15. As suggested by the reviewer, we have changed the sentences, due to the by hydroquinone moiety to due to the hydroquinone moiety on page 15, line Provide full name of G6PD in the abbreviation section. I believe G6PD represent an enzyme not a gene. In that case please correct the related section of the text. As suggested by the reviewer, we have added unabbreviated word, glucose-6-phosphate dehydrogenase (G6PD) on page 15, line Correct to "EEP had a", P15. As suggested by the reviewer, we have corrected the sentences, EEP hada to EEP had a on page 16, line10. 9