I am following this research paper
https://www.nature.com/articles/nprot.2007.102
In the very first step I took 0.1g of fresh sample and homogenized it in 2ml of 95% methanol, centrifuged and then for phenolic estimation 0.5ml supernatant was added in 2.5 ml F-C reagent( 10%) and 2 ml Na2CO3 and then OD was taken at 750nm. For flavonoid estimation 0.5ml supernatant was added with 0.3 ml sodium nitrite, 0.3ml aluminum chloride and 2 ml sodium hydroxide and the OD was taken at 510 nm through spectrophotometer. I have also develeoped gallic acid standard curve for total phenolic and quercetin standard curve for Flavonoid. So am I using the right method? and whats the further formulae for total phenolic and flavoonoid in terms of mg/g of fresh weight?
Dear Maria, as an inorganic chemist I'm absolutely not an expert in this field of research. However, I made the experience that is often pays off to use RG directly as a good source of information. Check particularly the "Publications" and "Questions" sections of RG for helpful literature references and closely related questions. For example, it might be worth having a look at the answers given to the following question which has been asked earlier on RG:
Calculation of TOtal phenolic content and total flavonoid content?
https://www.researchgate.net/post/Calculation-of-TOtal-phenolic-content-and-total-flavonoid-content
(6 answers)
Also please check the following helpful articles about this topic:
Estimation of Total Phenols and Flavonoids in Extracts of Actaea spicata Roots and Antioxidant Activity Studies
Article Estimation of Total Phenols and Flavonoids in Extracts of Ac...
and
Total phenolic, flavonoid content, and antioxidant activity of bulbs, leaves, and flowers made from Eleutherine bulbosa (Mill.) Urb
Article Total phenolic, flavonoid content, and antioxidant activity ...
Both papers have been posted by the authors as public full texts right here on RG. Thus you can freely download them as pdf files.
I hope this helps answering your question. Good luck with your work and please stay safe and healthy! With best wishes, Frank Edelmann
Dear Dr Maria Babar,
I have prepared for you two protocols (in attached), that may help you calculating total phenolic and flavonoid contents from fresh sample.
Best wishes,
Sabri
Check https://www.hindawi.com/journals/ecam/2014/253875/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341144/
Also check https://www.researchgate.net/publication/357206129_Effect_of_Extraction_Methods_on_Polyphenols_Flavonoids_Mineral_Elements_and_Biological_Activities_of_Essential_Oil_and_Extracts_of_Mentha_pulegium_L
Check on this paper
https://iopscience.iop.org/article/10.1088/1755-1315/299/1/012021/pdf
Dear Maria
First, you have to determine the slope of the standard curve (using different concentrations of Gallic Acid for Total Phenolic Content and Quercetin for Total Flavonoids as function of their absorbances). Then you divide the absorbance of your extract by the slope in order to obtain the concentration in mg/ml.
In a second time, and in order to obtain the concentration in mg GAE/g DM or mg Quercetin/g DM (dry matter), you multiply the concentration obtained above (in mg/ml) by "a", and you divide the result by "b" and by "c", where "a" is the volume of solvent (water or else), "b" is the mass of solid matrix extracted and and "c" is the percentage of dry matter of the matrix.
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