Title: Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L.

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1 Author's response to reviews Title: Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. Authors: Naima Saeed Muhammad R Khan Maria Shabbir Version: 2 Date: 9 August 2012 Author's response to reviews: Dear Editor The manuscript has been revised according to the suggestions of the reviewers. Mnuscript is also checked for the English language. Yours Sincerely Dr Muhammad Rashid Khan Reviewer's report Title: Findings on antioxidant activity and total phenolic and flavonoid contents of Torilis leptophylla L. Version: 1 Date: 2 July 2012 Reviewer: Ilhami Gulcin Reviewer's report: In the present study, the authors evaluated effect of solvent extracts of whole plant of Torilis leptophylla with respect to its antioxidant activity and total phenolic and flavonoid content in order to find new potential source of natural antioxidant. The manuscript was well documented. It can be accepted after some corrections. These corrections were listed below: 1. After the sentence of Antioxidant activity is an important and fundamental function in life systems. Many of the biological functions such as the anti-mutagenic, anti-carcinogenic, and anti-aging responses originate from this property the following references should be given: Antioxidant activity of food constituents-an overview. Archives of Toxicology, (2012). 86(3), and Caffeic acid phenethyl ester (CAPE): Correlation of structure and antioxidant properties. International Journal of Food Sciences and Nutrition, (2011). 62(8): incorporated

2 2. After the section of DPPH radical scavenging activity assay the following actual reference should be given: Polyphenol contents and in vitro antioxidant activities of lyophilized aqueous extract of kiwifruit (Actinidia deliciosa). Food Research International, (2011), 44 (5), incorporated 3. At the same manner, after the information of The antioxidant capacity of test samples was expressed as EC50, the concentration necessary for 50% reduction of ABTS. the following actual reference should be given: (2011). Pomological features, nutritional quality, polyphenol content analysis and antioxidant properties of domesticated and three wild ecotype forms of raspberries (Rubus idaeus L.). Journal of Food Science, 76(4), C585 C593. incorporated 4. The reference of Park et al (2008) [27] should be corrected as Park et al. [27]. incorporated 5. After the sentence of Hydrogen peroxide occurs naturally at low concentration levels in the air, water, human body, plants, microorganisms and food the following reference should be given:.(2005). Antiradical and antioxidant activity of total anthocyanins from Perilla pankinensis decne. Journal of Ethnopharmacology, 101, incorporated Level of interest: An article of importance in its field Quality of written English: Acceptable Statistical review: No, the manuscript does not need to be seen by a statistician. Review of the manuscript Findings on antioxidant activity and total phenolic and flavonoid contents of Torilis leptophylla by Saeed, Khan and Shabbir. I reviewed the above manuscript and made the following general observation: The paper has some good science and warrants publication but also has some deficiencies that need to be addressed. I was by the way the authors used the literature to support the relevant sections of their manuscript. The major and pertinent reference material in the field are cited (a total of 47 references are cited). The results and discussion sections are reasonably well done and supported with relevant references. The reference section is fairly done but there are a few corrections to make. The English language is reasonably good

3 and of acceptable standard Mandatory changes: (a) The material and methods section is however wanting and needs to be re-written putting necessary experimental details to enable a scientist familiar with the work to repeat their experiments and be able to get comparable results. This section is revised according to the suggestion. (b) Error treatment and uncertainties in measurements: Table 1: needs to be redone with the right error treatments with both measured values and uncertainties given to the correct number of decimal places. In this regard Table 1 needs to be redone. I have reason to believe authors have the raw data given to the correct number of decimal places as evidenced by the data they gave in the write up on page 2, page 12, page Correct decimals are provided in the required sections 1. Section: preparation and Extraction-Page 5: There are two problems in this section. The authors did a sequential liquid- liquid partition using nhexane, ethyl acetate, chloroform and n-butanol in that order. Problem: pure chloroform is less polar than ethyl acetate! Thus extracting with ethyl acetate before chloroform disrupts the polarity order which is, in increasing order,: n-hexane << chloroform < ethyl acetate << n-butanol. Thus the polarity order of chloroform and ethyl acetate is reversed. The second problem is that chloroform is heavier than water (aqueous layer). Thus when one extracts with chloroform, all the heavy (e.g. emulsions) soluble material which is not otherwise soluble in chloroform ends up in the bottom layer by virtue of density. This really messes up the fractionation as one ends up with some amounts of water soluble in the chloroform layer. In short extracting into the bottom layer presents problems mentioned above and gives an organic layer that is more polar than it should be. I believe since chloroform and ethyl acetate are of comparable polarity (with ethyl acetate more polar) the authors could do one of the two things: (a) Totally disregard all results from chloroform extract then all problems will be solved. Or (b) extract with n-hexane followed by chloroform (make sure now heavy material ends up in the bottom layer- the layer may also need to be dried), then ethyl acetate and then n-butanol. The plant section Extract preparation should read something like The..sample, which was later it was coursely extraction. Line 5: The filtrate (residue).n-hexane, chloroform, ethyl acetate (if you decide to reverse order and keep chloroform data) and n-butanol (a total of 200 ml each, give details of whether it was one aliquot of 200 ml of several aliquots of say 50 or 100 ml)..added, shaken..layers were..soluble residual fraction. Each of the fractions were concentrated and dried up passing through using a rotary evaporator [18], to give (give the dry masses of n-hexane, [chloroform], ethyl

4 acetate, n-butanol and the residual water fraction). The experiment is repeated as suggested 2. Correct Table 1: give measured values and errors to the correct and corresponding number of decimal places. Correct the corresponding write up on pages 2, 12, Corrections are made Minor Corrections: (page 1): Title The title would read better if changed to Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. title is changed Abstract (page2): Background:.phenolic and total flavonoid. In order to find potentially new source of natural antioxidants. corrected (page 2: Material and methods) Shade dried and powdered whole plant of. Filtrate The residue was.fractionated successively with.used as residual aqueous fraction (TLA). All the fractions were subjected for The total yield, total phenolic and total flavonoid contents of all the fractions were determined. corrected (page 2): Results: The exhibited the most distinguished potent DPPH (give values and errors in this section to correct decimal places). Note: The positive correlation coefficient values given in Table 3 though significant (p <0.05) are just marginal (R2 > to R2 > 7458; for good correlations R2 > ). It is important to reflect this in your findings. Thus for sentence beginning with For total phenolic content hydroxyl radicals I suggest A significant but marginal/weak positive correlation was found between TPC and EC50 values for DPPH, hydroxyl, phosphomolybdate and ABTS, while also another marginal positive correlation was found between TFC and EC50 values for superoxide anion and hydroxyl radicals.

5 Qualitative analysis Phytochemical screening of TLM. (page 4): Background Last paragraph: The 95% methanol extract was also subjected to phytochemical screening to determine Page 5: Extract preparation: see earlier comment. Page5: Chemicals: 2, 2 -azino-bis( ); Folin-Ciocalteu; cancel second gallic acid, change deoxyribose to 2- deoxyribose. Change this in other places in text. You need to mention also the chemical purity of your extracting solvents, n-hexane, chloroform, ethyl acetate and n-butanol in this section. Page 6: Antioxidant Assays: Each methanol at to make a concentration. Page 6: DPPH radical scavenging assay: to be consistent with what you wrote on page 5 change 1, 1, diphenyl-2-picrylhydrazyl to 2,2,diphenyl-1-picrylhydrazyl. For value (±0.02) the decimal places in the value and error to not correspond. Correct this elsewhere in the text. Page 7: Hydroxyl radical scavenging assay: line 2 Change Fe3+ to Fe3+. Page 9: Estimation of total flavonoid content(s): Total flavonoid content was determined by following a method by Park.. Page 10: Test for alkaloids: first sentence change MCL to TLM and add and the between HCL, and mixture. Page 12: Qualitative analysis of TLM: the sub heading is vague, change it to Phytochemical screening oftlm Also change analysis to screening in the sentence below. Page 12: DPPH radical scavenging activity: Change TLN to TLH (two places in this section). Watch out for decimal places in your reported values. The last sentence could read better if phrased like the presence of phenolic

6 compounds (containing phenolic hydroxyls) are mainly found in these two fractions and could be attributable to the observed high antiradical properties of these fractions. Page 13: Phosphomolybdate Assay: last sentence. Strong indicates strong antioxidants in this fraction and these could be attributable to the presence of phenolic compounds. Page 12: Correlation of EC50 values of antioxidant activities with Phytochemical contents: heading is vague: change to Correlation of EC50 values of antioxidant activities with TPC and TFC. Use earlier comments (page 2 results) to polish up this section. Page 17: Second paragraph last sentence: All the fractions possessed highly strong ABTS scavenging activity, an observation that is supported by other researchers [39]. Last paragraph change Fe3+ and Fe2+ to Fe3+ and Fe2+. Make these changes elsewhere in the text. Page 18: line 3-4: change the to their and delete the word ability. Page 20: References: Ref 2: Period (full stop) after Wagenitz, delete one after Rec. Ref3: delete word J before Pharm Biol. Ref4: delete issue number i.e. (3) to be consistent with other references, same for refs15, 16, 21, 22, 27, 38. Ref8: capital A for Algerian; Ref11: add initial A for last author. Ref 15: all initials for authors are wrong: correct to Baranski R, Baranska M, Schulz H, Simon PW, Nothnaged T. Ref 16: same as 15: correct to Bigdeli M, Rustaiyan A,. Also capital O for Oil. Ref27: Some of the authors first names are hyphenated and this should be reflected correct the following Park Y.-S, Jung S.-T, Kang S.-G and the rest are okay. Ref 33: no comma (,) after Nithyanandam.

7 Ref41. J. Agric Food Chem Ref42: J. Chromatography A Ref 46: no comma (,) after Bravo. Tables: correct table 1 (see earlier comments: Change aqueous fraction (table 1and 2 and also in text) to residual aqueous fraction to show that it is what remains of the water suspended 95% methanol extract after sequential extraction with organic solvents used. Other comments (discretional): It would be nice to have at least one antioxidant assay that involve in vivo or a close mimic thereof. That result would add more value to the paper though, in my opinion, absence of such measurements should not prejudice publication of this manuscript. Studies are included Conclusion: I believe this manuscript is publishable after minor revisions that involve two mandatory changes to the write up (stated above)

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