Dear editor, This is an interesting study evaluating the harmful effects of the food additive tartrazine and the beneficial properties of blackthorn fruits (Prunus spinosa) on the blood and organs of albino Wistar rats. The study adds important data clarifying the mechanism of tartrazine toxicity development and elucidates the promising protective role of Blackthorn (Prunus spinosa) fine powder. But, some concerns need to be addressed to be fit for publication as follows in my evaluation reports.

Evaluation report The present study suggested that tartrazine was able to generate histopathological changes, as evidence of certain tissue injury and toxicity as liver, kidney, and spleen. The administration of this dye in the diet of rats can induce significant changes in all biochemical and hematological parameters of the blood. Also the study illustrated that oral administration of Blackthorn (Prunus spinosa) fine powder could alleviate the hematological and biochemical changes of blood of Wistar albino rats. While Prunus spinosa had no significant protective effects on tartrazine induced organ toxicity. The article may have a good application prospect. Some suggestions are given here.

Title: Good and relevant however, I recommend the addition of (potential or possible) before the word "protective". Abstract: good, summarized and comprehensive. 1-The aim is clear: the study aimed to evaluate the risky effects of the food additive tartrazine on the blood and organs of albino Wistar rats and the possible beneficial properties of (Prunus spinosa). 2-The methodology is quite clear with brief information. However author did not mention the period of the study. Also, author should be more concise regarding the dosing regimen and specify the mentioned doses per each rat or per group and whether the doses of treatment were daily based please, specify? 3-The results are clear 4-The conclusion should be rewritten to be more clear . Keywords are good however I recommend not to waste keywords and to use words other than that mentioned in the title. -References: are relevant to the topic, recent and including key study however, I recommend to revise and verify if you have arranged and referenced them correctly. Introduction: 1. The introduction is good and defining the problem. However, it should be abridged. 2. Paragraph no 1 and 2 are too long and i recommend splitting them to shorter ones or compressing them. 3. I recommend removing Paragraph no 3 and 4. I think these paragraphs will suite discussion section better as authors incorporated details about different doses, different toxic effects of Tartrazine and the probable mechanism underlying its toxicity of the previous literature. 4. The aims and purposes of the research are clear. Authors attempted to investigate the curative effects of blackthorn against the impact produced by tartrazine .as blackthorn fruits are a rich source of antioxidants, and may exert a potent protective effect in Wistar albino rats. The working hypothesis is clearly stated. Authors mentioned methods appropriate for the question.

Methods: 1-Is the rationale of the present study have been studied earlier also by any other group? How the authors arrived at the therapeutic dose of blackthorn? Any supporting literature? If yes please incorporate all the information in the manuscript. 2-I have some major queries concerning the experimental design and methods: - The arrangement of groups was misleading (i.e., I. group C, II. group I, III. group II, IV. group III). It is better to make the control group the last. -Why author didn’t assign an additional group that receives blackthorn only? Please, justify. - What were the 18 hematological parameters. They should be mentioned here. - The authors reported in the Statistical analysis section: [The normal distribution of the values for each biochemical parameter (AST, ALT, glucose, cholesterol, triglyceride, creatinine), hematological parameters (WBC, LYM, MON, NEU, LYM, MO, NE, RBC, HCB, HCT, MCV, MCH, MCHC, RDWc, PLT, PCT, MPV, PDWc) and morphological parameters (initial and final weight, liver, right and left kidney, spline and brain weight) were verified using the Shapiro–Wilk test.]. Each group of factors should be mentioned under its section but not to be detailed here (i.e., hematology, biochemistry). The authors should add also section for morphological parameters. - The statistical section should be the last part in the method section. Finally, methodology section should be reorganized and subtitles should be rearranged; subtitle "Materials" should be mentioned first before "Methods". -"Animals were randomly divided into 4 groups and each group has 5 animals" I wonder if author has used the sufficient number of animals as the statistical test methods calculated the minimum sample size suitable for the experiment and the studied parameters? -"Group II received 75 mg of tartrazine dissolved in drinking water and further, 200 mg of (Prunus spinosa) fine powder was added and mixed thoroughly in the tartrazine– water mixture (250 ml)" why author mix tartrazine and Prunus spinosa in the same solution ? Is there is any expected in-vitro interaction or inhibitory effect exerted by Prunus spinosa powders on tartrazine? -Author did not specify whether the used doses were calculated per kg BW or animal or group and if the doses calculated per group how you could confirm that each animal received an equal amount of the solution? -Author did not mention if the dry extract of (Prunus spinosa) has been reconstituted by HPLC or subjected to gas chromatography-mass spectrometry (GC-MS) analysis in the lab . -Author did not mention the dose or the route of administration of isoflurane (the atheistic drug used for animals narcosis). 3-What is the basis/importance for this selection of the doses level for Tartrazine. Whether authors have done any preliminary experiment on dose selection, kindly provide the data/evidence to prove this as effective doses to induce biochemical, hematological and pathological changes in the animals? How much the Tartrazine does of the LD50? Also, the authors should discuss the Tartrazine doses being used in the manuscript in light of the latest studies in the discussion section. 4-In toxicology, the dosage is very important. There were two doses of Tartrazine. How to view the correlation with actual human exposure? 5-Is the period of the study sufficient to induce the hematological and organ toxicity in albino rats? Justify, Please.

Results: - Each table should be understood without going back to text - Abbreviations should be put in the foot note of each table - Significant and non significant comparisons should be clear when reading the table - ANOVA test outcome should be presented followed by presentation of post-hoc test - The authors should not repeat results in tables & texts but only present them in tables with general comment in text then referring to tables. - As an example, the authors reported: [The biochemical analysis of blood showed significant variations of GLU (mg/dl) depending on the tartrazine dose applied. Significant values were obtained in the group III (200.24 ± 1.08) and the group I (191.76 ± 1.07) compared to the control group (181.96 ± 2.13). The values of total COL (mg/dl) did not show significant differences between groups. Regarding to TG (mg/dl), the significant values were observed in the group III (67.58 ± 1.33), which received the highest dose of food dye. TG values showed non-statistically significant differences among the control group, I and II (Table 1).]. All of this should be summarized into only: [the variables ...... showed significant difference between .... while the ...variables showed no significant difference (Table 1)] - The authors reported: [According to Arefin et al. significantly increased levels of CRE and TG are reported in Swiss mice, after applying tartrazine dose 400 mg/kg b.w. by oral gavage [52].]. This should be moved to discussion section. - Another example, the authors reported: [In the present study AST (U/l) showed statistically significant means in the group I (114.14 ± 1.44) and III (119.88 ± 1.43) compared to the control group (109.18 ± 0.92) and the group II with fruit pulp powder (108.42 ± 0.53). The highest dose of tartrazine was able to significantly increase the level of ALT (U/l) for the group III (63.54 ± 0.66) and for the group I (62.42 ± 0.67) compared to the control group (54.44 ± 0.60), respectively. In contrary, the application of dried fruit powder mixed in drinking water in the (group II) determined a greater effectiveness reducing ALT (U/l) (56.85 ± 0.57) values, modifying them similarly to control group.]. After reading this, I could not detect if there is a significant difference or not between C and Group II. The authors should simply report that there was a significant difference in level of AST between control group and ....groups but no significant difference between control group and ........ - Again, this should be moved to discussion: [Elevated levels of enzymes such as AST and ALT liberated into the bloodstream are directly correlated with damaged tissues of liver and heart [53]. - The authors reported: [Regarding CRE (mg/dl), the significantly increased values were registered for group III (0.53 ± 0.02) followed by the group I (0.49 ± 0.03), II (0.44 ± 0.01) compared to the control group (0.41 ± 0.01), respectively.]. Was there any significant difference between C and II in creatinine or not? - Again, all of this should be moved to discussion section: [Similar results with the present study were found in the study realized by Himri et al. [54], who measured AST (U/l) (165.16 ± 17.82) and ALT (62 ± 2.84) in groups of rats that received a tartrazine dose of 10 mg/(kg b.w.) and was significantly higher compared to the control group (120 ± 5.64), (54.16 ± 5.33), respectively. According to results of Alaa Ali et al. [32], the addition of tartrazine in water could greater increase parameters as Urea, AST and ALT. Khayyat et al. reported significantly increased biomarkers means of ALT, AST, urea, and CRE after administering tartrazine to rats [28]. Moreover, El-Desoky et al. attributed the increase of concentrations of TG, COL, AST, ALT and CRE in blood serum due to the application of tartrazine in rats compared to the control group [55]. Furthermore, Al-Seeni et al. reported that applied doses of 10 mg/kg b.w. of tartrazine in rat diet, showed a significant increase in biochemical markers as: ALT (77.16 ± 1.68U/l), AST (79.16 ± 1.57U/l), ALP (275.83 ± 3.56U/l), and TG (224.67 ± 3.25 mg/dl) compared with the control group [56]. In addition, concurrent treatment of rats with Nigella sativa oil demonstrated a curative effect on biochemical and histological parameters.] -So, I recommend that results should be organized as I can see authors had incorporated discussion for biochemical and hematological findings of the current study into the results section. However later on they separated histopathological results from discussion section. -The paragraphs of the results are too long. It is recommended to split them into shorter ones. Also, I suggest not repeating the mean values of the parameters and replications of data here as they have been already presented in the tables. -"Biochemical analysis" please, change to "Biochemical results. -"Hematological analysis" please, change to " Hematological results. -"Body weight and organ weight evaluations" please, change to "Body weight and organ weight results. -"Histological evaluation" please, change to Histopathological results. -Table 1: - The authors should put abbreviations in the footnote of each table - I could not understand how significance was reported: [?! Within columns, means followed by the same letter (a, b or c) are not significantly different between treatments according to the Tukey test (p = 0.05); While, within columns means followed by the different letter (a, b and c) are significantly different between treatments according to the Tuckey’s test (p = 0.05).] - The authors reported in the footnote some abbreviations but not used: [ns: not significant] - There should be a presentation of ANOVA outcome followed by presentation of post-hoc analysis -Tables 2-3 and the related text in results section: - The same comments on Table one and text regarding table 1 in the results section are also applied for Table 2-3 and any text related to them -I do not understand how personal communication is reported in the results section ?! [Kidney tissues of the group I, showed mild renal congestion and hydropic degeneration of renal tubular epithelium (personal communications Balta et al.).]. -In histopathological results; How could the authors give the term significant or no significant changes for pathological picture in slides? The authors should add any type of score for objective comparison. Also, authors displayed only figures of liver tissue however they reported some hisptopatholgical changes in kidney and other studied organs. Why? Justify, please.

Discussion 1-I have some major queries regarding the mechanism of action of Tartrazine and Blackthorn. -Data on the proper mechanism of action of Tartrazine and Blackthorn in the amelioration of Tartrazine toxicity based on the structure activity relationship is needed to explain the protective nature of Blackthorn. -Author should give their justification/ support for their co-treatment schedule for Blackthorn along with Tartrazine intoxication. Why they have not tried the pre/post treatment schedule of Blackthorn against Tartrazine toxicity. -Explain the fate of administered Tartrazine and its mechanism of distribution and its mechanism of inducing toxicity and its mechanism of excretion and the interference with Blackthorn should be given in a detailed manner in the discussion part for the better understanding of a lay man? 2- I suggest beginning with a statement related to the current research and focusing on your work followed by displaying the key findings of your work. Paragraphs needs rearrangement. 3-The paragraphs are too long. Please recast, and compress; no need for detailed repetition of results here. So, the discussion section should be abridged. Also, author should provide a statement about the importance of the operated research question 4-Authors displayed their results and discuss the results of the study in light of the previous study supporting study however they did not interpret the mechanisms underlying the results and the importance of the revealed findings especially regarding the results of Blackthorn - Tartrazine groups. - The discussion section should be rewritten and rearranged. Limitations of the study should be reported. -Authors did not mention other studies which their results against theirs with interpretations (In discussion). Conclusion: - I do not understand what mentioned in the conclusion section: [On the other hand, fruit powder demonstrated non-significant protective effects for analyzed organs.]. Authors should provide more clear and comprehensive statement. Meaningful recommendations and suggestions for future research should be mentioned. General comments -Please revise manuscript carefully and respond to my comments attached to each paragraph. I suggest you reply to each comment. -The manuscript should be thoroughly revised to avoid similarity with previously published papers. -Extensive Language editing is required. - The focus of the research is a good point. However, I recommend the author continue research in this point, using different parameters.