Sayaka Doi1, Mina Kawamura2, Keisuke Oyama3, Tetsuya Akamatsu2, Mizuki Mizobuchi4, Yasuo Oyama2, Toshiya Masuda1, Norio Kamemura4. 1. Faculty of Human Life Science, Osaka City University, Osaka City, Osaka, Japan. 2. Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima City, Tokushima Japan. 3. Surgery Division, Sakai City Medical Center, Sakai City, Osaka, Japan. 4. Department of Food-Nutritional Sciences, Faculty of Life Sciences, Tokushima Bunri University, Tokushima, Japan.
Abstract
Alginetin is the major product formed from pentoses and hexurionic acids. Alginetin is producted by cooking process of food including pection, a naturally-occurring polysacharride found in many plants. However, the biological interaction and toxicity of alginetin are not known at all. The aim of the present study was to investigate the cellular actions of alginetin on rat thymic lymphocytes. The effects of alginetin on the cell were examined using flow cytometry with fluorescent probes. Alginetin increased cellular content of non-protein thiols ([NPT]i) and elevated intracellular Zn2+ levels ([Zn2+]i). Chelation of intracellular Zn2+ reduced the effect of alginetin on [NPT]i, and chelation of external Zn2+ almost completely diminished alginetin-induced elevation of [Zn2+]i, indicating that alginetin treatment increased Zn2+ influx. Increased [NPT]i and [Zn2+]i levels in response to alginetin were positively correlated. Alginetin protected cells against oxidative stress induced by hydrogen peroxide and Ca2+ overload by calcium ionophore. It is considered that the increases in [NPT]i and [Zn2+]i are responsible for the cytoprotective activity of alginetin because NPT attenuates oxidative stress and Zn2+ competes with Ca2+. Alginetin may be produced during manufacturing of jam, which may provide additional health benefits of jam.
Alginetin is the major product formed from pentoses and hexurionic acids. Alginetin is producted by cooking process of food including pection, a naturally-occurring polysacharride found in many plants. However, the biological interaction and toxicity of alginetin are not known at all. The aim of the present study was to investigate the cellular actions of alginetin on rat thymic lymphocytes. The effects of alginetin on the cell were examined using flow cytometry with fluorescent probes. Alginetin increased cellular content of non-protein thiols ([NPT]i) and elevated intracellular Zn2+ levels ([Zn2+]i). Chelation of intracellular Zn2+ reduced the effect of alginetin on [NPT]i, and chelation of external Zn2+ almost completely diminished alginetin-induced elevation of [Zn2+]i, indicating that alginetin treatment increased Zn2+ influx. Increased [NPT]i and [Zn2+]i levels in response to alginetin were positively correlated. Alginetin protected cells against oxidative stress induced by hydrogen peroxide and Ca2+ overload by calcium ionophore. It is considered that the increases in [NPT]i and [Zn2+]i are responsible for the cytoprotective activity of alginetin because NPT attenuates oxidative stress and Zn2+ competes with Ca2+. Alginetin may be produced during manufacturing of jam, which may provide additional health benefits of jam.
Alginetin (3,8-dihydroxy-2-methylchromone) may be a forgotten compound, as nearly all papers concerning this molecule were published 65–84 years ago [1]. Moreover, there have been no reports of pharmacological and/or toxicological actions of alginetin. Therefore, our study is probably the first investigation of alginetin bioactivity.Pectin is a natural produced essential ingredient in preserves. Pectin is a type of starch, called a heteropolysaccharide, that occurs naturally in the cell walls of fruits and vegetables and gives them structure. Alginetin (3,8-dihydroxy-2-methylchromone) was first obtained by heating alginic acid with water in an autoclave and was also obtained from pectin and gummic acid in the 1930's [1]. Alginetin is a hydrolysis degradation product of pectin [2] Pectin is used as a food additive and occurs naturally in most fruits. Pectin is responsible for giving jellies their gel-like consistency, resulting in better spreadability. Use of a pressure cooker to make jam from fruits may result in alginetin production. Although bioactivity of alginetin has not been studied, the chemical structure suggests that the compound possesses antioxidant activity similar to other methylchromone derivatives with hydroxyl groups that have antioxidative properties [3]. In this study, we examined bioactivity of alginetin in rat thymic lymphocytes using flow cytometry with fluorescent probes. Beneficial cellular activity of alginetin would provide additional value for foods in which alginetin is produced during the manufacturing process.
Materials and methods
Preparation of alginetin
KH2PO4 and Na2HPO4 (9:1, 50 g) were added to a methanolic solution (50 mL) of D-glucuronolactone (1 g), then mixed well. After removal of methanol in vacuo, residual solids were heated at 130°C for 1 h under N2. The resulting brown solid was extracted twice with methanol (50 mL), and the combined extract was evaporated completely, resulting in a dark brown residue (210 mg). The residue was purified by chromatography using an octadecyl silane silica gel column with a stepwise gradient using 20%, 30%, and 40% aqueous methanol containing 1% acetic acid, and 100% methanol containing 1% acetic acid (each 120 mL). Eluate was collected in 60 mL fractions and the third fraction containing alginetin (17 mg) was evaporated in vacuo. These chemicals were purchased from Wako Pure Chemicals (Osaka, Japan). The structure of the isolated alginetin was characterized by nuclear magnetic resonance spectroscopy (NMR) as follows; 1H-NMR (400 MHz, CD3OD): δ 7.61 (1H, d, J = 7.6 Hz), 7.26 (1H, t, J = 7.6 Hz), 7.19 (1H, d, J = 7.6 Hz), 2.55 (3H, s). NMR spectra were obtained from a JNM-ECZ400S (400 MHz, JEOL, Tokyo, Japan).
Fluorescent probes and chemicals
Propidium iodide (PI) was used to assess cell death. Estimation of cellular content of nonprotein thiols was performed with 5-chloromethylfluorescein diacetate (5-CMF-DA) [4]. FluoZin-3-AMTM was used to study changes in intracellular Zn2+ levels ([Zn2+]i) [5]. These probes were commercially obtained from Invitrogen and Thermo Fisher Scientific Inc. (Eugene, Oregon, USA). Membrane-impermeable and -permeable Zn2+ chelators, diethylenetriamine-N,N,N',N",N"-pentaacetic acid and N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (DTPA and TPEN, respectively), were purchased from Dojin Chemicals (Kumamoto, Japan). Other chemicals were products of Wako Pure Chemicals (Osaka, Japan).
Cell suspension
The use of experimental animals in this study was approved by the committee of Tokushima University and Tokushima Bunri University (Tokushima, Japan) (Registration number in Tokushima university: T29-52, Registration number in Tokushima Bunri university: H30-3).Cell suspensions were prepared from thymus glands as follows. Briefly, thymus glands were dissected from 8- to 12-week-old male Wistar rats (Total number of rat; n = 8, 280–340 g / Charles River Laboratories Japan, Kanagawa, Japan) anesthetized with 50 mg/kg i.p. thiopental sodium (RavonalTM, Mitsubishi Tanabe Pharma, Osaka, Japan). The glands were sliced with a razor. The slices were triturated in chilled Tyrode’s solution (150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose) to dissociate individual lymphocytes. The pH of solution was adjusted at 7.3–7.4 with 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and appropriate amount of NaOH. The solution containing dissociated cells was passed through a mesh (diameter: 25 μm) to prepare the cell suspension (approximately 5 × 105 cells/mL). Cells were incubated at 36–37°C for 1 h before experimental use. The cell morphology images of the cells cultured in the present of alginetin were performed by a inverted microscope (Nikon Eclipse TS100, Nikon,Tokyo, Japan).
Fluorescence measurements of cellular parameters
Alginetin, 5-CMF-DA, FluoZin-3-AM, DTPA, and TPEN were initially dissolved in dimethyl sulfoxide (DMSO). DMSO was present at concentrations of 0.1–0.3% in final conditions, which did not induce cell death. Cellular and membrane parameters were measured using fluorescent probes and a flow cytometer equipped with a software package for data collection and analysis (CytoACE-150; JASCO, Tokyo, Japan). The excitation wavelength used for the fluorescent probes was 488 nm, and emission was detected at 530 ± 15 nm for 5-CMF and FluoZin-3 and at 600 ± 20 nm for PI. PI, which stains dead cells, was added to cell suspensions at a final concentration of 5 μM. 5-CMF and FluoZin-3 fluorescence were measured only in cells exhibiting no PI fluorescence (living cells with intact cell membranes). To estimate changes in [NPT]i, primarily glutathione, cells were incubated with 1 μM 5-CMF-DA for 30 min before fluorescence analysis. The correlation coefficient between flow cytometric and biochemical determination of glutathione was 0.965 [4]. To monitor changes in [Zn2+]i, cells were incubated with 500 nM FluoZin-3-AM for 60 min before fluorescence measurements were conducted.
Experimental protocol
Alginetin (3–100 mM in 2 μL of DMSO) was added to cell suspensions (1.998 mL per one test tube) and the mixtures were incubated at 36–37°C. Each cell suspension (100 μL) was analyzed using flow cytometry to assess alginetin-induced changes in cellular parameters. Fluorescence data acquisition from 3 × 103 cells required 10–15 s. In our previous study to examine the cytotoxicity of H2O2 as oxidative stress and A23187 (Calcimycin), a divalent cation ionophore (Sigma-Aldrich Co, St. Louis, MO, USA) as Ca2+ overload [6], the cells were incubated with H2O2 or A23187 for 3–4 h to induce cell death in 20–40% of cells. Change in [NPT]i by alginetin was examined 2 h after the application in order to suggest the [NPT]i before the occurrence of cell death induced by H2O2.
Statistical analysis
Statistical analysis was performed using Excel Toukei 2010 (Social Survey Research Information Co., Ltd. Tokyo, Japan). Statistical analysis was performed using ANOVA with post-hoc Tukey’s multivariate analysis. P-value < 0.05 was considered statistically significant. Data reported in this study were mean ± standard deviation of 4–8 experiments.
Results
Changes in 5-CMF fluorescence in response to alginetin
The histogram representing distribution of 5-CMF fluorescence in cells incubated with 100 μM alginetin for 2 h was shifted to a higher fluorescence intensity compared with the control histogram (Fig 1A) demonstrating that alginetin treatment increased [NPT]i. Alginetin augmented 5-CMF fluorescence at 10 μM and significant augmentation was observed across the range of 30–100 μM alginetin (Fig 1B). Moreover, alginetin was changed the cell morphology on rat thymic lymphocytes (Fig 1C). A previous study demonstrated that elevation of [Zn2+]i precedes increases in [NPT]i [7]. Therefore, to determine ifZn2+ contributed to increased [NPT]i, the effect of 100 μM alginetin was evaluated in the presence of 10 μM TPEN, an intracellular Zn2+ chelator. As shown in Fig 2, incubation of cells with TPEN for 2 h reduced steady state intensity of 5-CMF fluorescence. Furthermore, TPEN significantly reduced alginetin-induced 5-CMF, indicating involvement of intracellular Zn2+ in alginetin-induced elevation of [NPT]i.
Fig 1
Augmentation of 5-CMF fluorescence by alginetin.
(A) Shift of 5-CMF fluorescence histogram in response to 2 h alginetin treatment. The inset shows the chemical structure of alginetin. (B) Concentration-dependent augmentation of mean intensity of 5-CMF fluorescence induced by alginetin. Asterisks (**) indicate significant differences (P < 0.01) in 5-CMF fluorescence intensity between the control group (CONT) and the groups of cells treated with 30–100 μM alginetin. (C) The cell morphology images in response to 2 h alginetin treatment.
Fig 2
Augmentation of 5-CMF fluorescence by alginetin in the absence and presence of TPEN.
Asterisks (**) indicate significant differences (P < 0.01) in 5-CMF fluorescence intensity between the control group (CONT) and the groups of cells treated with 100 μM alginetin (ALG) in the absence (left pair / open column) and presence (right pair / filled column) of 10 μM TPEN. Pound signs (##) show significant difference (P < 0.01) between the groups of cells treated with alginetin.
Augmentation of 5-CMF fluorescence by alginetin.
(A) Shift of 5-CMF fluorescence histogram in response to 2 h alginetin treatment. The inset shows the chemical structure of alginetin. (B) Concentration-dependent augmentation of mean intensity of 5-CMF fluorescence induced by alginetin. Asterisks (**) indicate significant differences (P < 0.01) in 5-CMF fluorescence intensity between the control group (CONT) and the groups of cells treated with 30–100 μM alginetin. (C) The cell morphology images in response to 2 h alginetin treatment.
Augmentation of 5-CMF fluorescence by alginetin in the absence and presence of TPEN.
Asterisks (**) indicate significant differences (P < 0.01) in 5-CMF fluorescence intensity between the control group (CONT) and the groups of cells treated with 100 μM alginetin (ALG) in the absence (left pair / open column) and presence (right pair / filled column) of 10 μM TPEN. Pound signs (##) show significant difference (P < 0.01) between the groups of cells treated with alginetin.
Change in FluoZin-3 fluorescence in response to alginetin
To determine whether alginetin increases [Zn2+]i, alginetin-induced changes in [Zn2+]i were measured. As shown in Fig 3A, the histogram of FluoZin-3 fluorescence shifted to a higher intensity 1 h after treatment with 100 μm alginetin. Treatment with 30–100 μM alginetin for 1 h significantly increased the intensity of FluoZin-3 fluorescence (Fig 3B), indicating that alginetin induced elevation of [Zn2+]i. The observed changes in FluoZin-3 fluorescence induced by alginetin were positively correlated with those of alginetin-induced 5-CMF fluorescence (Fig 4).
Fig 3
Augmentation of FluoZin-3 fluorescence by alginetin.
(A) Shift of the FluoZin-3 fluorescence histogram in response to 1 h alginetin treatment. (B) Concentration-dependent augmentation of mean intensity of FluoZin-3 fluorescence by alginetin. Asterisks (**) indicate significant differences (P < 0.01) in FluoZin-3 fluorescence intensity between the control group (CONT) and the groups of cells treated with 30–100 μM alginetin.
Fig 4
Correlation between mean intensities of 5-CMF and FluoZin-3 fluorescence in cells treated with 10–100 μM alginetin for 1 h.
Augmentation of FluoZin-3 fluorescence by alginetin.
(A) Shift of the FluoZin-3 fluorescence histogram in response to 1 h alginetin treatment. (B) Concentration-dependent augmentation of mean intensity of FluoZin-3 fluorescence by alginetin. Asterisks (**) indicate significant differences (P < 0.01) in FluoZin-3 fluorescence intensity between the control group (CONT) and the groups of cells treated with 30–100 μM alginetin.Increases in [Zn2+]i can occur due to Zn2+ influx from the extracellular environment and/or intracellular Zn2+ release. To determine the source of alginetin-induced elevation of [Zn2+]i, changes in FluoZin-3 fluorescence induced by 100 μM alginetin were examined in the presence of 10 μM DTPA, an external Zn2+ chelator. Removal of external Zn2+ by DTPA significantly reduced steady state intensity of FluoZin-3 fluorescence and significantly attenuated alginetin-induced augmentation of FluoZin-3 fluorescence (Fig 5). Thus, alginetin-induced increases in [Zn2+]i were dependent on extracellular Zn2+.
Fig 5
Augmentation of FluoZin-3 fluorescence by alginetin in the absence and presence of DTPA.
Asterisks (**) indicate significant differences (P < 0.01) in FluoZin-3 fluorescence intensity between the control group (CONT) and groups of cells treated with 100 μM alginetin (ALG) for 1 h in the absence (left pair / open column) and presence (right pair / filled column) of 10 μM DTPA. Pound signs (##) indicate significant differences (P < 0.01) between the groups of cells treated with alginetin.
Augmentation of FluoZin-3 fluorescence by alginetin in the absence and presence of DTPA.
Asterisks (**) indicate significant differences (P < 0.01) in FluoZin-3 fluorescence intensity between the control group (CONT) and groups of cells treated with 100 μM alginetin (ALG) for 1 h in the absence (left pair / open column) and presence (right pair / filled column) of 10 μM DTPA. Pound signs (##) indicate significant differences (P < 0.01) between the groups of cells treated with alginetin.
Cytoprotective actions of alginetin
Glutathione protects cells against oxidative stress [8, 9]. Therefore, alginetin-induced increases in [NPT]i may result in resistance to H2O2toxicity. The effects of 10–100 μM alginetin on changes in cell lethality induced by 300 μM H2O2 were evaluated. As shown in Fig 6, incubation of cells with H2O2 for 4 h increased PI fluorescence, indicating increased cell death, while treatment with 100 μM alginetin for 4 h did not result in increased PI fluorescence. Cell death induced by H2O2 was significantly attenuated by simultaneous treatment with alginetin in a concentration-dependent manner (10–100 μM alginetin) (Fig 7A).
Fig 6
Changes in the population of PI-stained cells treated with alginetin, H2O2, or both.
The dotted line under the cytogram indicates the region of cells exhibiting PI fluorescence.
Fig 7
Protective effects of alginetin against oxidative stress induced by H2O2 (A) and Ca2+ overload by A23187 (B). Asterisks (**) show significant differences between the groups of cells under insult-induced stress in the absence and presence of 10–100 μM alginetin.
Changes in the population of PI-stained cells treated with alginetin, H2O2, or both.
The dotted line under the cytogram indicates the region of cells exhibiting PI fluorescence.Protective effects of alginetin against oxidative stress induced by H2O2 (A) and Ca2+ overload by A23187 (B). Asterisks (**) show significant differences between the groups of cells under insult-induced stress in the absence and presence of 10–100 μM alginetin.Treatment of cells with H2O2 increases intracellular Ca2+ concentration ([Ca2+]i) [10], and sustained elevation of [Ca2+]i is linked to cell death [11]. Furthermore, Zn2+ partially attenuates Ca2+-dependent cell death [12]. As alginetin treatment elevated [Zn2+]i, the possibility that alginetin treatment could protect against cell death induced by the calcium ionophore, A23187, was evaluated. As shown in Fig 7B, incubation of cells with 100 nM A23187 for 3 h significantly increased cell lethality. Simultaneous treatment with 30–100 μM alginetin significantly attenuated this effect.
Discussion
Our study demonstrated that alginetin protects cells against oxidative stress induced by H2O2 and Ca2+ overload induced by A23187. It is generally recognized that oxidative stress and Ca2+ overload trigger cell death [13, 11]. Therefore, alginetin may be cytoprotective against many damaging stimuli. Alginetin, a degradation product of pectin, may be produced during jam production [1, 2]. Development of a process for jam production which produces alginetin may be attractive to health-oriented individuals.
Cellular actions of alginetin
[NPT]i is required to maintain protein thiols in a reduced state and to support a variety of redox reactions for reducing ROS, detoxifying xenobiotics, and facilitating cell signaling. However, excessive oxidative stress results in indiscriminate and irreversible oxidation of protein thiols, depletion of [NPT]i and cell death [14]. Recently, Oyama et al. showed that the excessive of [Zn2+]i increased [NPT]i [15]. Our results showed that alginetin elevated intracellular Zn2+ levels and increased cellular content of non-protein thiols. Moreover, treatment with TPEN did not completely attenuate alginetin-induced increases in [NPT]i. Alginetin treatment also resulted in increased [NPT]i in the presence of DTPA, which almost completely suppressed alginetin-induced elevation of [Zn2+]i. These phenomena indicate that alginetin induced Zn2+-dependent and -independent increases in [NPT]i. Zn2+ increases glutathione synthesis through an ARE-Nrf2–dependent pathway [8]. Zn2+-independent mechanisms of action of alginetin will be evaluated in future studies. Increased [NPT]i in response to alginetin may protect against oxidative stress because [NPT]i is important in preventing pathological changes resulting from increased levels of reactive oxygen species [16]. A previous study demonstrated that removal of intracellular Zn2+ by TPEN increased cytotoxicity of A23187, a calcium ionophore that causes Ca2+ overload [12]. Zn2+ competes with Ca2+ at calcium binding proteins [17]. Therefore, increased [Zn2+]i in response to alginetin may reduce Ca2+ binding-related cell death, resulting in protection against Ca2+ overload. Elevation of [Zn2+]i in response to alginetin was extracellular Zn2+-dependent because augmentation of FluoZin-3 fluorescence by alginetin was almost completely attenuated by cotreatment with DPTA. Zn2+ influx is regulated by many zinc transporters such as ZIP4, 5, 6, 10, and 14 [18]. Alginetin may activate zinc transporters or increase membrane Zn2+ permeability via zinc transporter-independent mechanisms.
Conclusions
The biological interaction and toxicity of alginetin has not been studied in detail. Our study showed that alginetin increased cellular content of non-protein thiols and elevated intracellular Zn2+ levels on rat thymic lymphocyte. These results indicate that alginetin increases cell vulnerability to oxidative stress on rat thymocytes. This study provides that alginetin, possessing cytoprotective activity, would provide additional health benefits of jam if it is produced from fruit pectin during jam manufacturing.10 Aug 2020PONE-D-20-12715Bioactivity of alginetin, a caramelization product of pectin: Cytometric analysis of rat thymic lymphocytes using fluorescent probesPLOS ONEDear Dr. Kamemura,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.As you can see reviewer #2 suggested minor edits to your manuscript. While reviewer #1 recommended a rejection, I believe his/her comments can be addressed in a revised version.We look forward to receiving your revised manuscript.Kind regards,Patrick Lajoie, PhDAcademic EditorPLOS ONEJournal Requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2. Please add the sources and catalog numbers of all reagents as well as details regarding all equipment used in your study to the Methods section of your manuscript.In addition, please provide the total number of rats used in your study and clarify the method of euthanasia used.3. Thank you for including the following funding statement in your acknowledgements section; "The Japan Society for the Promotion of Science (Tokyo, Japan) supports this study with Grant-in-Aids for Scientific Research (C26340039, 18K14408). "We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)Reviewer #1: In this manuscript, the authors investigated the effects of alginetin on the cell behaviors. I think there are many issues should addressed before published.1. What is the “pection”?2. The chemical structure of the alginetin should be investigated. The author state that Alginetin was characterized by 1H-NMR, but no data were provided.3. The cell morphology images of the cells cultured in the present of alginetin should be provided.4. the conclusion is too short.Reviewer #2: Norio Kamemura’s manuscript, named as “Bioactivity of alginetin, a caramelization product of pectin: Cytometric analysis of rat thymic lymphocytes using fluorescent probes”, described the bioactivity algietin as an interesting story, which used fluorescent probes to analyze the zinc level and NPT level in the cytometric analysis. I thought it was good to accept after minor revision:1. Page 4, line 70 and line 71, the number of H should be identified in the 1HNMR.2. They didn’t declare clearly the relationship of NPT and oxidative stress.3. Page 4, line 90 and line 91, it was recommended to use “,” but not “;”.4. Page 5, line 117, A23187 wasn’t declare well when it was mentioned first time.5. In the figure 1B, 2, 3B, 5, 7, were these experiments performed once? The error bars weren’t showed in the appropriate way.**********6. 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PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.20 Sep 2020PROS ONEEditors-in-Chief:PhD, Patrick LajoieManuscript number: PONE-D-20-12715Title; Bioactivity of alginetin, a caramelization product of pectin: Cytometricanalysis of rat thymic lymphocytes using fluorescent probesAuthors: Sayaka Doi, Mina Kawamura, Keisuke Oyama, Tetsuya Akamatsu, Mizuki Mizobuchi, Yasuo Oyama, Toshiya Masuda, Norio KamemuraThank you for your e-mail of 11/Aug/2020. I was pleased to know of your positive evaluation of our manuscript. We have revised the manuscript PONE-D-20-12715 on the basis of the Referee's comments. I logged into the publisher website and submitted the following files related to the revised manuscript: (1) clean version of the manuscript (no mark-up, file name: PONE-D-20-12715-R), (2) marked-up copy showing changes made during the revision (file name: PONE-D-20-12715-R-M), (3) Reply to the comments raised by the reviewer (file name: PONE-D-20-12715-rev), (4) figure files (file names: PONE-D-20-12715-fig)Appended to this letter is our detailed point-by-point response to the comments raised by reviewer. I agreed with all the comments.Thank to your comments, I was able to rewrite my paper more wonderfully.We look forward to hearing from you regarding our submission. We would be glad to respond to any further questions and comments that you may haveSincerely,Prof. Norio KamemuraDepartment of Food-Nutritional Sciences, Faculty of Life Sciences, Tokushima Bunri University Tokushima, Nishihama, Yamashiro-cho, Tokushima, 770-8514, JapanTel.: +81 88 602 8095Fax: +81 88 656 9965E-mail: kamemura.norio@tks.bunri-u.ac.jpJournal Requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements,including those for file naming.Reply to the CommentWe tried to improve our manuscript.2. Please add the sources and catalog numbers of all reagents as well as detailsregarding all equipment used in your study to the Methods section of yourmanuscript.In addition, please provide the total number of rats used in your study andclarify the method of euthanasia used.Reply to the CommentWe tried to improve our manuscript.Briefly, thymus glands were dissected from 8- to 12-week-old male Wistar rats (Total number of rat; n=8, 280–340 g / Charles River Laboratories Japan, Kanagawa, Japan) anesthetized with 50 mg/kg i.p. thiopental sodium (RavonalTM, Mitsubishi Tanabe Pharma, Osaka, Japan).3. Thank you for including the following funding statement in youracknowledgements section; "The Japan Society for the Promotion of Science(Tokyo, Japan) supports this study with Grant-in-Aids for Scientific Research(C26340039, 18K14408). "We note that you have provided funding information that is not currentlydeclared in your Funding Statement. However, funding information should notappear in the Acknowledgments section or other areas of your manuscript. We willonly publish funding information present in the Funding Statement section of theonline submission form.Please remove any funding-related text from the manuscript and let us know howyou would like to update your Funding Statement. Currently, your FundingStatement reads as follows:"The author(s) received no specific funding for this work."Reply to the CommentWe tried to improve our manuscript.Reviewer 1#We thank referee for careful reading our manuscript and for giving useful comments1. What is the “pection”?Reply to the CommentWe added a new sentence to explain pection.In the revised manuscriptAbstract (Page 2, Line 24-25)a naturally-occurring polysacharride found in many plants.Introduction(Page 3, Line 49-51)Pectin is a natural produced essential ingredient in preserves. Pectin is a type of starch, called a heteropolysaccharide, that occurs naturally in the cell walls of fruits and vegetables and gives them structure.2. The chemical structure of the alginetin should be investigated. The author state that Alginetin was characterized by 1H-NMR, but no data were provided.Reply to the CommentWe added a new sentence to explain pection.In the revised manuscriptMaterial s and methods (Page 4, Line 75-78)The structure of the isolated alginetin was characterized by nuclear magnetic resonance spectroscopy (NMR) as follows; 1H-NMR (400 MHz, CD3OD): δ 7.61 (1H, d, J = 7.6 Hz), 7.26 (1H, t, J = 7.6 Hz), 7.19 (1H, d, J = 7.6 Hz), 2.55 (3H, s).3. The cell morphology images of the cells cultured in the present of alginetin should be provided.Reply to the CommentWe tried to improve our manuscript.In the revised manuscriptMaterial s and methods (Page 5, Line 102-104)The cell morphology images of the cells cultured in the present of alginetin were performed by a inverted microscope (Nikon Eclipse TS100, Nikon,Tokyo, Japan)Results (Page 6, Line 142-143)Moreover, alginetin was changed the cell morphology on rat thymic lymphocytes (Figure 1C).FigureFig 1 (C)Figure legends (Page 13, Line 317(C) The cell morphology images in response to 2 h alginetin treatment.4. the conclusion is too short.Reply to the CommentWe tried to improve our manuscript.In the revised manuscriptConclusion (Page 9, Line 216-222)The biological interaction and toxicity of alginetin has not been studied in detail. Our study showed that alginetin increased cellular content of non-protein thiols and elevated intracellular Zn2+ levels on rat thymic lymphocyte. These results indicate that alginetin increases cell vulnerability to oxidative stress on rat thymocytes. This study provides that alginetin, possessing cytoprotective activity, would provide additional health benefits of jam if it is produced from fruit pectin during jam manufacturing.Reviewer 2#We thank referee for careful reading our manuscript and for giving useful comments1. Page 4, line 70 and line 71, the number of H should be identified in the 1HNMR.Reply to the CommentWe tried to improve our manuscript. This NMR data shows that it is algnetin. However, this sentence is written in the paper that has already been submitted.In the revised manuscriptMaterial s and methods (Page 4, Line 75-78)The structure of the isolated alginetin was characterized by nuclear magnetic resonance spectroscopy (NMR) as follows; 1H-NMR (400 MHz, CD3OD): δ 7.61 (1H, d, J = 7.6 Hz), 7.26 (1H, t, J = 7.6 Hz), 7.19 (1H, d, J = 7.6 Hz), 2.55 (3H, s).2. They didn’t declare clearly the relationship of NPT and oxidative stress.Reply to the CommentWe tried to improve our manuscript.In the revised manuscriptDiscussion (Page 8, Line 192-197)[NTP]i is required to maintain protein thiols in a reduced state and to support a variety of redox reactions for reducing ROS, detoxifying xenobiotics, and facilitating cell signaling. However, excessive oxidative stress results in indiscriminate and irreversible oxidation of protein thiols, depletion of [NTP]i and cell death [14]. Recently, Oyama et al showed that the excessive of [Zn2+]i increase [NPT] i [15]. Our results showed that alginetin elevated intracellular Zn2+ levels increased cellular content of non-protein thiols.References (Page 12, Line 271-276)[14] Shahid P. Baba and Aruni Bhatnagar, Role of thiols in oxidative stress. Curr Opin Toxicol. 2018; 7: 133–139.[15] Akio Kinazaki, Hongqin Chen, Kazuki Koizumi, Takuya Kawanai, Tomohiro M. Oyama,Masaya Satoh, Shiro Ishida, Yoshiro Okano, Yasuo Oyama, Putative role of intracellular Zn2+ release during oxidative stress: a trigger to restore cellular thiol content that is decreased by oxidative stress. J Physiol Sci. 2011; 61:403–409.3. Page 4, line 90 and line 91, it was recommended to use “,” but not “;”.Reply to the CommentWe tried to improve our manuscript.In the revised manuscriptMaterial s and methods (Page 5, Line 97-98)(150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose)4. Page 5, line 117, A23187 wasn’t declare well when it was mentioned first time.Reply to the CommentWe tried to improve our manuscript.In the revised manuscriptMaterial s and methods (Page 6, Line 125-126)A23187 (Calcimycin), a divalent cation ionophore (Sigma-Aldrich Co, St. Louis, MO, USA)5. In the figure 1B, 2, 3B, 5, 7, were these experiments performed once? Theerror bars weren’t showed in the appropriate way.Reply to the CommentData reported in this study were mean ± standard deviation of 4–8 experiments.(127-129). However, we already submited the figure 1B, 2, 3B, 5, 7 which an error bar is written.In the revised manuscriptMaterial s and methods (131-134)Statistical analysis was performed using Excel Toukei 2010 (Social Survey Research Information Co., Ltd. Tokyo, Japan). Statistical analysis was performed using ANOVA with post-hoc Tukey’s multivariate analysis. P-value < 0.05 was considered statistically significant. Data reported in this study were mean ± standard deviation of 4–8 experiments.Submitted filename: PONE-D-20-12715-rev.docxClick here for additional data file.13 Oct 2020Bioactivity of alginetin, a caramelization product of pectin: Cytometric analysis of rat thymic lymphocytes using fluorescent probesPONE-D-20-12715R1Dear Dr. Kamemura,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.An invoice for payment will follow shortly after the formal acceptance. 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For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: No16 Oct 2020PONE-D-20-12715R1Bioactivity of alginetin, a caramelization product of pectin: Cytometric analysis of rat thymic lymphocytes using fluorescent probesDear Dr. Kamemura:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. 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Authors: Stefan W Ryter; Hong Pyo Kim; Alexander Hoetzel; Jeong W Park; Kiichi Nakahira; Xue Wang; Augustine M K Choi Journal: Antioxid Redox Signal Date: 2007-01 Impact factor: 8.401
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