Title:Inhibition of hematopoietic prostaglandin D2 Synthase (H-PGDS) by alkaloids from Combretum molle

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1 Author's response to reviews Title:Inhibition of hematopoietic prostaglandin D2 Synthase (H-PGDS) by Authors: Rejoice Moyo Theresa Chimponda Stanley Mukanganyama Version:4Date:4 June 2014 Author's response to reviews: see over

2 Reviewer's report Title: Inhibition of hematopoietic prostaglandin D2 Synthase (H-PGDS) by Version:1Date:15 May 2014 Reviewer: Fu-Ming Tsai Reviewer's report: The manuscript by Moyo et al. report that the alkaloid from Combretum molle has potent to inhibit the H-PGDS activity. These results based on an enzyme's kinetics assay. Alkaloids are a group of naturally chemical compounds. Since the crude extract is the source for studying the kinetics of enzyme activity, it is difficult to reproduce the same results all over the world due to the different place of the product for plant or the various components within alkaloids. Therefore, similar result will be more convinced when used the pure components in this study. Major Compulsory Revisions 1. The authors should state the components in crude extract. Is the alkaloid from Combretum molle a pure compound? What is the structure or the composition? If the extract is a mixture, the use of major compound in purified alkaloid to validate the inhibition effect on H-PTGDS will be very helpful. The alkaloids isolated from C. molle are not pure. These were actually a mixture. The method we used was selective for isolation of any nitrogen-containing cyclic phytochemicals. Although working with a pure compound may be more useful, however, the pure compound may not possess activity as compared to the crude alkaloid extract. We isolated alkaloids as we wanted to determine if the alkaloid extract possessed anti- H-PGDS activity. 2. In the result section, the H-PGDS expression should be presented (Coomassie blue staining and Western blot for poly-his tag). Data not shown is not appropriated to mention the expression of H-PGDS. The expression has been changed to Coomassie blue staining and Western blot for poly-his tag as stated and Data not shown has been deleted. 3. The discussion is limited and too descriptive of the data. A more in depth discussion of the possible results of the demonstrated changes to H-PTGDS function on cells or immune system, or comparison of each compound (drug) on inflammatory response would be beneficial. We have tried to give a possible response to the immune system. However, as indicated there are not as many studies that have been done. Since a crude alkaloid extract was used, we cannot discuss the effects of each drug on inflammatory response. 4. In table I legend, does alkaloids have a significant effect in blocking the kinetic of GST? Please perform the statistical analysis. One-way analysis of variance test

3 (ANOVA) with Dunnett s Multiple Comparison Test was used to analyse the results. Values represent the mean + SD for N= 2. The values with a p-value < 0.05 or less were considered statistically significant. * P < 0.05, ** P < 0.01, *** P< Graphical and Statistical analyses were carried out using Graphpad Prism 5 Software (Version 5.0, Graph pad Software Inc, San Diego, USA). The results indicate that increasing the concentration of the extracts especially at 10 and 20 µg/ml significtanly reduced the Km for GSH and CDNB as well as the K cat for both CDNB and GSH at 20 µg/ml. 5. The figure legends should be rewritten. Where appropriate, these legends have been re written. 6. Whether alkaloids may affect the PGD2 production? The authors evaluated the H-PGDS activity using the substrates CDNB and GSH. Can these results represent inhibiting effects of alkaloid in PGD2 production in physiology? The authors should mention the possibility. The results may represent the results in vivo. The studies with CDNB allows for selection of potential inhibitors even when PGH2 is used. However, further experiments with PGH 2 need to be carried out. Minor essential revisions 1. The abbreviation should only be used when a word appears at least three times in abstract or main article (from introduction to discuss). The full name of a word only appears the first time, followed by typing its abbreviated name. The GSH in abstract need to type full name, whereas the full name glutathione in methods and discuss need to be changed to abbreviation. Also, authors need to check the CDNB typing throughout all manuscript. The abbreviations have been used as advised. 2. There are many errors in describing the city of the manufactures of chemicals and equipment. (page 7, line 2, 14, 15; page 8, line 2, 19, city need to be stated), page 7 line 16, German? CA or California? USA or U.S.A? New York or NY? The cities have been written in full. 3. The H-PGDS plasmid is obtained from someone else. What is the tag carried when H-PGDS is expressed. In other hand, when the protein is expressed, what is the predict M.W. of the tagged protein. The construct detail needs to be stated in detail in the method section. The H-PGDS is 6-Histidine tagged. The pjexpress 401 expression vector was used. The estimated M.W of the protein is 23.4 kda. This has now been stated in the method section.

4 4. In method section, the authors mentioned that cells were then incubated at 160 rpm at 37 oc overnight in a SI 300 Lab companion, (Jeio Tech, Korea). Is this sentence exactly? The H-PGDS protein expression needs overnight induction by IPTG? We followed the procedure for the purification of other GSTs as outlined in the papers below. Kolm RK, Danielson UH, Zhang Y, Talalay P. and Mannervik B. Isothiocyanates as substrates for human glutathione transferases: structureactivity studies. Biochem. J. (1995) 311, Y.S. Zhang, R.H. Kolm, B. Mannervik, P. Talalay. Reversible Conjugation of Isothiocyanates with Glutathione Catalyzed by Human Glutathione Transferases. Biochemical and Biophysical Research Communications (1995) 206: Mukanganyama S, Bezabih M, Robert M, Ngadjui BT, Kapche GFW, Ngandeu F and Abegaz B. The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1. Journal of Enzyme Inhibition and Medicinal Chemistry DOI: / After addition of IPTG, E. coli cells were grown for a further hours before harvesting them by centrifugation. This has been done for other GSTS such as GST A1-1, P1-1, M2-2, M4-4. Level of interest: An article of importance in its field Quality of written English: Needs some language corrections before being published Statistical review: No, the manuscript does not need to be seen by a statistician. Declaration of competing interests: I declare that I have no competing interest. Reviewer's report Title: Inhibition of hematopoietic prostaglandin D2 Synthase (H-PGDS) by Version: 1Date:31 March 2014 Reviewer: Sabariah Ismail Reviewer's report: Major Compulsory Revisions Since no specific alkaloid compounds of the plant extract were isolated and identified, it is recommended that the authors refer to their plant extract as the alkaloid extract of Combretum molle throughout the manuscript. The plant extract has been referred to the alkaloid extract of Combretum molle. Under the Discussion section, second paragraph, last line, "The investigation therefore, narrowed down to specific compounds.' That sentence can be taken out. This sentence has been deleted.

5 Minor Essential Revisions 1. Methods (i) Screening for inhibition of H-PGDS by alkaloids from C. molle The second sentence may be rewritten as "enzyme activity was determined through the measurement...and was done in quadruplicate". The sentence has been corrected and has been rewritten as advised. (ii) Determination of time-dependent effects The incubation mixtures contained H-PGDS (final concentration ? unit missing) "was investigated" at the end of the first sentence is to be deleted. The units have been inserted and the statement "was investigated" has been deleted. Results (i) Effect of the alkaloids on H-PGDS kinetics Spelling of "uncompetitive" and some minor grammatical errors to be corrected. The spelling has been corrected. Level of interest: An article of importance in its field Quality of written English: Needs some language corrections before being published Statistical review: No, the manuscript does not need to be seen by a statistician. Declaration of competing interests: I declare that I have no competing interests.