Hi Sandeep, indeed, like others also indicated, your question is too general. Start with the material you have. Did others do identification studies using the same or related herbal species? Can you make a list of expected phenolic compounds? That will facilitate the analsysis with DAD and MS as well as MS/MS. For complete ID you will need additional structural information such as from NMR, but for initial annotation LC-DAD-MS(-MS) will do. Quantification is another issue: it involves having the standards available, or being able to purify and quantify the metabolites from the extract. Good luck!

What do you mean by whole polyphenolic components ?

- is it the quantification of total polyphenolic content ? and therefore you can estimate by UV absorption and have a more accurate value using Folin & Ciocalteu's phenol reagent

- or do you want to quantify all individual polyphenol from your extract ? This might be more complicated and will take much more. HPLC analysis should be the best approach.

Dear Dr. Bertrand, I would like to quantify all individual polyphenol present in that extract if i don't know how many polyphenol are present there.

therefore, the only solution is to identify each of them. This is not always complicated it depends of your extract (if it is a studied plant or not).

The HPLC quantification with standards should do the job.

Then your first step is bibliographic search to look for known polyphenols in your plant extract.

For total phenolics use Folin-Ciocalteu's, for profile the goal standard is HPLC-DAD-MS/MS (QqQ)

In our lab we quantify a number of phenolic compounds by HPLC-UV. To choose a certain method, you need to know first which compounds you are interested in. After that, there are a couple of options for separation and quantitation depending on the instrumentation you have available (DAD or MS), the number of compounds and which ones they are, etc.

to quantifie total phenolic compound you have the folin method; for separating tou have to use HPLC

Check our recent papers on analysis of sage (we analyzed for FC-index, using LC/MS, and NMR)

Thank to all for your valuable suggestions.

Dr. Lachenmeier, Can you please post here link of your recent paper, which you mentioned above

what do you mean with "herbal extract"? I presume you actually have an extract from the aerials of a certain plant. If you want to perform a complete phytochemical study, you should proceed with a cascade extraction with different solvents. Not to mention the volatiles, of course. On the other hand, if you have already indications about the type of extract (water infusion, decoption, lipidic) your work is much more simple. You usually get this info from the use in local folk medicine. Regarding the identification and quantification of the components, I agree with those saying HPLC/DAD/MS is the best choice.

https://www.researchgate.net/publication/51515759_Rapid_UHPLC_determination_of_polyphenols_in_aqueous_infusions_of_Salvia_officinalis_L._(sage_tea)

https://www.researchgate.net/publication/223974920_Holistic_Control_of_Herbal_Teas_and_Tinctures_Based_on_Sage_(Salvia_officinalis_L.)_for_Compounds_with_Beneficial_and_Adverse_Effects_using_NMR_Spectroscopy

https://www.researchgate.net/publication/51920837_Antioxidant_Capacity_and_Polyphenolic_Composition_as_Quality_Indicators_for_Aqueous_Infusions_of_Salvia_officinalis_L._(sage_tea)

https://www.researchgate.net/publication/51509923_Determination_of_the_biologically_active_flavour_substances_thujone_and_camphor_in_foods_and_medicines_containing_sage_(Salvia_officinalis_L.)

Article Rapid UHPLC determination of polyphenols in aqueous infusion...

Article Holistic Control of Herbal Teas and Tinctures Based on Sage ...

Article Antioxidant Capacity and Polyphenolic Composition as Quality...

Article Determination of the biologically active flavour substances ...

Hi Sandeep, indeed, like others also indicated, your question is too general. Start with the material you have. Did others do identification studies using the same or related herbal species? Can you make a list of expected phenolic compounds? That will facilitate the analsysis with DAD and MS as well as MS/MS. For complete ID you will need additional structural information such as from NMR, but for initial annotation LC-DAD-MS(-MS) will do. Quantification is another issue: it involves having the standards available, or being able to purify and quantify the metabolites from the extract. Good luck!

For total polyphenols you can use Folin-Ciocalteu method, but for profile you need HPLC, the best with HPLC-MS

TLC can work for some identification of polyphenols too, but you must have standards..

Hi, I am doing this for my PhD work. You have to use LC-MS coupled with HPLC or UPLC. I have attached two papers, hope they help you.

Here is another paper

The question is indeed quite general. You have various classes of phenols and each one can be specifically assessed and the individual compounds identified. Please go through the following papers which might provide ypu with a comprehensive approach.

D. Ramful, E. Tarnus, O. I. Aruoma, E. Bourdon and T. Bahorun Polyphenolics, Vitamin C composition and antioxidant propensities of Mauritian citrus fruit pulps. Food Research International (2011) 44, 2088–2099 (FOR FLAVONOIDS)

D. Ramful, B. Aumjaud , V.S. Neergheen , M.A. Soobrattee , O.I. Aruoma and T. Bahorun, Eugenia pollicina leaf extract: A rich source of polyphenolic antioxidants for potential use as prophylactics and food preservatives. Food Research International (2011) 14, 1190-1196 ( FOR FLAVAN-3-OlS and derivatives)

M. A. Soobrattee, V. S. Neergheen, A. Luximon-Ramma, O. I. Aruoma and T. Bahorun. Phenolics content and antioxidant actions of the Rubiaceae, Ebenaceae, Celastraceae, Erythroxylaceae and Sterculaceae families of Mauritian endemic plants. Toxicology in vitro (2008) 22, 45-56 ( MAJOR PHENOLIC CLASSES IN HERBAL EXTRACTS)

] A. Luximon-Ramma, T.Bahorun, A. Crozier, V. Zbarsky, K. K Datla, D. T Dexter and O. I Aruoma Assessment of the polyphenolic composition of the organic extracts of Mauritian black teas: A potential contributor to their antioxidant functions. Biofactors, 2006, 27, (1-4), 79-91 ( TEA PHENOLICS)

V.S. Neergheen, T. Bahorun, M.A Soobrattee, and O.I. Aruoma. Characterisation of phenolic contents of selected Mauritian endemic plants from the Rubiaceae and Myrtaceae families and assessment of their antioxidant activities. Journal of Plant Physiology, 2006, 163, 787-799 ( MAJOR PHENOLIC CLASSES IN HERBAL EXTRACTS)

M.A. Soobrattee, V.S. Neergheen, A.Luximon-Ramma, O.I. Aruoma and T. Bahorun, Phenolics as potential antioxidant therapeutic agents : Mechanisms and actions. Mutation Research/fundamental and Molecular Mechanisms of Mutagenesis, 2005, 579, 200-213

Do you want to isolate the pure compounds or just want to confirm the style and quantity of the total polyphenolic component?

You can also use Folin -ciocalteu. This a classical method here is paper for that:Antioxidant, Total Phenolic Content and Cytotoxicity Evaluation

of Selected Malaysian Plants

Suhailah Wasman Qader 1,*, Mahmood Ameen Abdulla 2, Lee Suan Chua 3, Nigar Najim 4,

Mazatulikhma Mat Zain 4 and Salehhuddin Hamdan

www.mdpi.com/1420-3049/16/4/3433/pdf

Nothing is new in research.kindly search the previous data from all sources especially,from chemical abstracts.Then start with the known using T.L.C ,and SPECTROMETRIC STUDIES IN U.V AND VISIBLE, Confirm with pure samples available from sigma .When you finish these,you would have gained expertise to complete the remaining compounds. Kindly click this link

http://curing-fungal.blogspot.in and view my thesis in references.

regards.prof.dr.s.d.

Dear Dr. Su, infact I want to know total polyphenolic content first, then identify them too separately....

Dear dr. Su, i too want to know the same as to isolate the pure compounds and to know the quantity of the total polyphenolic component?

try the following books for your phenolic content determination in your herbal extract

1. British pharmacopoeia

2. US pharmacopoeia

3. Chinese pharmacopoeia

4. Japanese pharmacopoeia

5.Different volumes of A.O.A.C related to herbal medicine

i guess..we can go for initial qualitative analysis..trial n error method..for detection of the same...Phenolics...by TLC method...n once the systems are finalised we can compare the same wid the standars...to finalise the compounds!!!

USE LC MS technique to solve this problem

I agree with Nagendra...

Hi

First u identify the extract containing the polyphenols using Folin Ciocalteu and TLC and then proceed with any analysis, that will be the best. This will make your further analysis easy and fruitful.

Regards

Thanks to all for yours valuable suggestions.

For identification purpose LC-MS and HPLC: and for quantify need markers compunds

For identification, I believe you could use FeCl3 solution test, you may get change of colour due to iron complexation with polyphenol, mean while you could run TLC and stain it with FeCl3 staining solution, I think this solution is selective for phenols. For quantification, you need to use HPLC or NMR

Simply; You can determin the total phenolic contents of any herbal extract spectophotometricaly using Folin Ciocalteu reagent, and gallic acid as standerd

Can you please suggest me an appropriate protocol for folin ciocalteu assay and what is requirements for the same??

Best to use the Folin Ciocalteu method for quantification of polyphenols. Method depends on your extract really.

GC-MS analysis post-silylation will give you m/z and fragmentation, which by NIST database will give you hits.. literature of these hits will help you get the exact answers..

You do not require markers/standards for the use of the technique

Hi,

POLYPHENOLS SEPARATION-

1)0.5 mL of methanolic extract and 0.2 mL oftetrachloromethane were added to give a clear solution.

2)Adding a further 0.3 mL of distilled water to the solution resulted in the formation of a two-phase layer.

3) shake thoroughly ,centrifuged at 10000 rpm for 10 min

4)u can see sharp interface.

5)The aqueous methanolic phase was separated, dried, and dissolved in methanol. use it for further analysis

IDENTIFICATION

To determine the TOTAL PHENOLIC CONTENT, use Folin–Ciocalteu reagent test.

after this use selected polyphenols to identify in TLC and HPTLC, and HPLC

Hi.

I used folin ciocalteu to determine de total phenolic content in my extracts (aqueous ethanolic extract obtained from E. globulus bark).

It is a very simple method but besides phenolic compounds other compounds can alsi react like proantocyanidins and tannins (it depends in what you are expecting to obtain). But it is a standard method generally used for comparing with other extract sources and to make an assessment of the potential of extracts regarding total phenolic compounds.

This method is very simple I used the following method: 0.5 mL of extract (liquid extract or solution prepared with dried extract) + 2.5mL of folin ciocalteu reagent diluted 10 times V/V with water+ 2 mL of NaCO3 75g/L. The mixture is kept for 5min @ 50ºC. After 10 min of cooling, the absorbance is measured at 760 nm. A blank is prepared at the same time as samples using 0.5 mL of extraction solvent. Gallic acid can be used as standard for the calibration curve. (for more detailed information you can look at my paper: Mota, I., Pinto, P., Novo, C., Guerreiro, O., Guerra, A., Duarte, F., Rodrigues, A., Extraction of polyphenolic compounds from Eucalyptus globulus bark: process optimization and screening for biological activity. Ind. Eng. Chem. Res., 2012, 51(20), pp 6991-7000, DOI: 10.1021/ie300103z.

As for individual separation of phenolics using HPLC-MS seems a good choice. However you probably need to simplify your sample and remove other interferences. See this paper it has an example of HPLC-MS identification and quantification of some polyphenolics: Santos, S. O.; Freire, C.; Domingues, M. R. R.; Silvestre, A. J.; Neto, C. P. Characterization of Phenolic Components in Polar Extracts of Eucalyptus globulus Labill. Bark by High-Performance Liquid Chromatography−Mass Spectrometry. J. Agric. Food Chem. 2011, 59, 9386−9393.

Yes, even i have used folin ciocalteau method, and it does work very well.

Yo can follow our article

http://jnatpharm.org/article.asp?issn=2229-5119;year=2010;volume=1;issue=1;spage=40;epage=45;aulast=Goyal

Its freely accessible.

Dear Prof Inês Mota

Can you upload papers mentioned in your answer

Thank You

Sushil

Total phenolics: use Folin-Ciocalteu method (Singleton and Rossi, 1965) For identification in first HPLC-DAD is good with standards, in second LC-MS/MS for unknow molecules

The best is using Folin Coicalteu method. is the best. though questions arise wether to use ethanol or methanol because of tractability with the extraction solvent of polyphenols

To assay "total phenolic contetnt" there are two main methods:

"Folin-Ciocalteu" and "Prussian Blue assay".

You can find a comparison between these methods. To sum up, "Prussian Blue assay" is better method. Please find the paper for more details.