Phenolic Compounds - Science topic

The question is so wide it's hard to answer. Here are a few options that come to mind:

1. You are doing the tests wrong. Too much bacteria, wrong media, wrong time of growth,...

2. Your extraction is wrong - wrong extraction solvent, temperature, etc.

3. You misidentified the plant you extracted (if you did extract a plant), or you are seeing the effect of ecotypes - geographical region, growth conditions, season, sun/shade, et.

4. The paper you looked at got one of the above wrong

5. Something else ...

I you want help try to define your question and conditions

Dear Trieu Phu Hau , wouldn't both products come from the intra-cyclization or a partially epoxidized pyrogallol unit? depending on the position of the OH and of the O-CH2-Epox groups (both ortho to each other) one would get one of the 2 structures you propose. Thanks for your time and consideration.

Infrared spectroscopy is another method that would give you clues about the chemical structure.

Dear Wahiba,

Regarding the antimicrobial activity of plant extract which contains phenolic extract, firstly you do...

Disk diffusion method (Agar Cup - plate method ) which giving the antimicrobial potential of your phenolic extract by observing the zone of inhibition (potential of your drug to fight microbial organism).

For carrying this kind of activity you select any two gram positive, two gram negative and one fungal organism.

After observing the zone of inhibition, you carry the MIC (minimum inhibitory concentration) value of your phenolic extract for confirming the antimicrobial potential of your isolated phenolic extract from plant

If I understood you correctly, then to obtain an extract for the DPPH test, is it possible without using thermal techniques? and still obtain reproducible results with the subsequent reading in the spectrophotometer?

For liquid-liquid extraction of phenolic compounds from aqueous solutions for subsequent GC-MS analysis, you can use organic solvents that are immiscible with water. Commonly used solvents for this purpose include: Ethyl Acetate, Diethyl Ether, Methyl tert-butyl ether (MTBE), Hexane.

I use the RNeasy kit but for my disruption/homogenization (I am working with zebrafish brains that have been stored in RNALater previously) I just use a pestle and so far has been working well. 1% agarose gel (not denatured) has also sufficed. when preparing my samples for the gel, I make a known amount (ie 2 ug) of the sample and then add DEPC treated water to it. if that volume is 10 uL, my loading dye would also be 10 uL and load 20 uL in total.

The following is a useful paper for you: 10.1016/j.lwt.2010.12.009

I have a recipe for a flow cytometry buffer that uses 4% PVP-10. What does this mean? Is it PVP with a Mw of 10,000?

Estimation of total polyphenolics

The reaction mixture contain 50 μl of sample, 3 ml water, 0.25 ml FC reagent and 0.75 ml 20 % Na2CO3. The total volume make up to 5 ml using water. Mixed well and incubated the mixture at 50°C for 2 hours. Read the absorbance at 765 nm using spectrophotometer [23]. Here gallic acid was used as the standard. The concentration of total polyphenolic content was obtained from gallic acid standard curve by using the following formula.

T = C × V/M

T = Total polyphenolic content expressed as mg/g of the sample extract in GAE

C = Concentration of Gallic acid from caliberation curve (mg/ml)

V = Volume of extract (ml)

M = Weight of sample extract (g)

Marah Al Taher Aicha Akouz Nadjet Debbache Benaida

I motivate all to read the following article:

Noel W Davies thank you very much

You are right. Positive control is necessary. The results can be expressed with reference to the Positive control Vitamin C. While doing the analysis it is better to take Methanolic extract from the oil. That is the accurately weighed oil may be mixed with excess Methanol and boiled over water bath for few minutes. Then the mixture can be kept inside the freezer for over night. By that time the whole oil (Triglyceride) gets solidified and the

Methanol stands apart. This Methanol fraction bearing all phenolic and other polar compounds can be decanted and evaporated to dryness to get rid of Methanol. This can be taken for DPPH free radical scavenging activity study. @

Some extraction methods, such as percolation, Macerassion and Soxhlet, can be useful. However, according to the type of secondary metabolite, The best method of them should be tested and selected using experiment (trail and error).

Regards

The problem is in the order of treatment. Is LPS treatment prior to inhibitor and phenolic compound or vice versa?

Dear Pablo Reguera ,

In short it is basically as said here by the Sigma-Aldrich site (https://www.sigmaaldrich.com/NL/en/product/sial/f9252) “Addition of the phenol reagent generates chromogens that give increasing absorbance between 550-750nm. Normally, absorbance at the peak (750nm) or shoulder (660nm) are used to quantitate protein concentrations between 1-100 mg/ml while absorbance at 550nm is used to quantitate higher protein concentrations.”.

Or as said in one of the most cited papers when it comes to Folin–Ciocâlteu reagent (FCR) “Because of the breadth of these peaks and the fact that other components in biological samples do not absorb in this region, analysis can be carried out at a wide range of wavelengths, 760 nm generally being chosen for FC.” (Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent).

See for what they mean by breadth for example the absorbance spectra in the following paper (Figure 1): https://www.researchgate.net/publication/237096981_Application_and_Analysis_of_the_Folin_Ciocalteu_Method_for_the_Determination_of_the_Total_Phenolic_Content_from_Limonium_Brasiliense_L

Although the overall message is that every wavelength between 750-765 nm is fine, personally, I would go for 765 nm. This is based on the official JAOAC method: https://academic.oup.com/jaoac/article/102/1/320/5658204 but as said every wavelength is fine as long as you clearly indicate in your method which wavelength you choose (and on which paper you based that choice).

Best regards.

All right, thank you

you can do the maceration extraction method by using ethanol 70% or 96% for 24 hour and then it can be evaporated by using rotary evaporator instrument. so you can collect the extract.

by using the reference chemical compound (eg. quersetin) and setting the HPLC condition you can check the consentration the specific chemical compound (eg. quercetin) by AUC result.

if you do fraction part (non polar; semi polar; polar) you know the compound part of solvent type.

for the next part, you can do purification by isolation the specific compound of herb.

thank's

Are they natural product extracts? Do they contain water or ethanol?

The polymerization process can occur leading to a loss in activity, it is desirable to store very diluted extracts to avoid this. Filtration through 0,45 Nylon prior to storage is helpful also.

If your extract is a mixture of alcohol and water, polymerization is frequent and you should consider drying your extract and preparing new solutions when needed.

Hydroalcoholic solvents are the best to use...phenolic acids diffuse to water, flavonlids prefer the organic such as ethanol...80_90% ethanol is worth to try...for ph issue, if you choose alkaline rougly saying 9 , 10 most of the phenolics come up as anion thus water solubility is increased... more acidic ph needs more organic percentage and higher alcohol compositio os needed due to the increased hydrophobicity of the target phenolics...

My pleasure dear colleague, I wish you good luck in your experiments.

Dear Hermine Boukeng Jatsa , the best way to achieve what you want is to do hydroalcoholic extraction from the plant material, you can use an extraction system such as Methanol-water (80:20), then prepare your extract for MPLC then prep-HPLC in order to fractionnate then purify compounds.

If you don't have prep-HPLC you can use the classical methods, following these steps:

1/ extract plant material with cyclohexane or heptane --> terpenoids

2/ then extract the residue with Methanol --> alkaloids + phenolic compounds

3/ recover the alkaloids using the acid extraction method (liquid/liquid extraction where the acidification of the aqueous phase make the alkaloids soluble on the organic one)

4/ you will obtain the alkaloids alone, the other phase will have the polyphenols and other polar to medium polar compounds.

best regards

The answer depends on whether the concentration of NH3 or NH4Cl is specified. Once the ingredients are mixed the pH will be adjusted using HCl or NH3I suggest that solution.

Hello!!! This is an article that contains a standard protocol, explains step by step.

and also see this paper here:

3 years later, I am having the same problem (Waters Xevo G2-XS Q-TOF), please Daniel, could you tell me how figured up that problem?

Visit kindly also the following useful links: https://www.semanticscholar.org/paper/Significance-and-applications-of-phenolic-compounds-O%E2%80%99Connell-Fox/d50a6791db01ebaf646b930fd97fed3cfa329e46

In 2.5 mL Folin-Cioleauteu’s reagent (10 %) 500 μL of sample was dissolved and kept it for 2 min. Then added 2 mL of Na2CO3 (5 %) and volume was adjusted up to 10 mL with distilled water. After vigorous mixing, the solution was heated at

45 C for 15 min and was kept for cooling. After cooling, the absorbance was measured against the blank at 765 nm.

I am also interested to know about this. How to preserve the color and quality of chlorophyll in wheat juice and preserve it for many days.

The simple reason is that the phytochemicals responsible for the anti-inflammatory activity are more abundant in the stem, or the stem may contain secondary metabolites different from those contained in the Leaves. Different plant parts are known to contain different kinds or different proportion of phytochemicals. I will be necessary to isolate the compounds from both parts and study them

You may benefit from toxchem model

Check https://www.mdpi.com/2071-1050/12/14/5804/pdf

Also check https://www.researchgate.net/publication/224890906_Application_of_in_vitro_gastrointestinal_model_to_evaluate_nitrogen_and_phosphorus_bioaccessibility_and_bioavailability_in_fish_fed_ingredients

The reason for the application of Choline chloride in DES as a replacement for ionic liquids is based on advantages such as ease of preparation, low cost of DES, environmental friendliness and ability to work at low temperatures to achieve similar results.

Also check https://applbiolchem.springeropen.com/articles/10.1186/s13765-021-00608-3

Also check https://www.researchgate.net/publication/248511209_Phenolic_compounds_in_olive_oil_and_olive_pomace_from_Cilento_Campania_Italy_and_their_antioxidant_activity

First, you should investigate what polarity the metabolites of your interest have and according to that, you will look for solvents that have a similar polarity. You can also base on the extract you already have and perform liquid-liquid extractions with immiscible solvents to eliminate the compounds you are not interested in.

Which detector was coupled to your HPLC? Accordingly, you can rely on previous studies where they used a system similar to yours, and even if possible of the same plant species.

Dear AAntonio, this is a very interesting technical question which has been frequently discussed on RG. I also suggest that you directly search the "Publications" section of RG to find and access relevant publications on this topic. Fore example, please have a look at the following useful articles which have all been posted as public full texts on RG:

Isolation and Characterization of Novel Flavonoid from Methanolic Extract of Pongamia pinnata Pods

Article Isolation and Characterization of Novel Flavonoid from Metha...

Flavonoid from methanolic extract of Limoniastrum Feei (Girard) batt (Plumbaginaceae)

Article Flavonoid from methanolic extract of Limoniastrum Feei (Gira...

FLAVONOID FROM METHANOLIC EXTRACT OF LIMONIASTRUM FEEI (GIRARD) BATT (PLUMBAGINACEAE)

Article FLAVONOID FROM METHANOLIC EXTRACT OF LIMONIASTRUM FEEI (GIRA...

Isolation and Characterization of Flavonoid from Methanolic Extract of Mirabilis jalapa Linn. Tuber and Evaluation of its Cardioprotective Property

Article Isolation and Characterization of Flavonoid from Methanolic ...

Preliminary Phytochemical Screening and HPLC Analysis of Flavonoid From Methanolic Extract of Leaves of Annona squamosa

Article Preliminary Phytochemical Screening and HPLC Analysis of Fla...

and various others. Good luck with your research and best wishes!

hi

the below links may be helpful

wishing all the best for you

Thank you @Kobra Ghobad for your suggestion. But the sample looks to be insoluble in cold MeOH unless they are warmed to about 50 degrees, when left for a while they precipitate. This means there is a risk of killing the HPLC column.

Hi Unays!

Not sure if I understood what you need. Are you studying metal-protein interactions? And if so, what are you interested in? Interaction? Function?

There is a good database (Brenda), where you can explore several enzymatic parameters, such as metal interactions. When I want to confirm my metal sites, for instance, I use CMM (Check my metal), which analyses coordination, occupancy, etc.

Not sure if that's related to what you need, but I hope this can help.

Best,

Lorenzo

Maybe your target compound can be recrystallized.

Shantanu Das Can you write all the protocol steps? Then we can discuss.

You can precipitate the mucilage with ethanol at a low temperature, similar to the methodology of article

Specifically in this part:

The supernatant was again precipitated with the addition of cold absolute ethanol to a final concentration of 80% (v/v) and kept at 4 °C overnight. The resulting precipitation was vacuum concentrated and freeze dried, yielding Grifola frondosa heteropolysaccharide (GFP).

Dear Jinadasa

Thank you for your information. I will check and update you.

Fruit Juices may be extracted exhaustively by maceration with acidic methanol over 24 h at room temperature. The extract is filtered and concentrated in vacuo at 37C. The extract is partitioned sequentially with hexane and ethyl acetate, and the aqueous fraction containing phenolics may be lyophilized. Removal of other water-soluble constituents, such as sugars and ascorbic acid, is accomplished by column chromatography by applying the dried extract to a Diaion HP-20SS resin column and allowed to adsorb for 20 min. The column is sequentially eluted with 500 ml water to remove sugars etc. and then with 300 ml acidic methanol to elute all of the phenolic compounds (flavonoids/anthocyanins).

Folin-Ciocalteu colorimetric method can be a useful and easy technique foe analizing TPC

Dear Walaa A Negm

Although it is a traditional method, I depend on the utilization of column chromatography. This is usually performed after establishing the solvent mixture by applying TLC.

All the best

PVP is basically a polymer, which will only mention as %. So its better to go with dilution. In my experience, CTAB-buffer with 2% PVP works well for the Isolation of DNA. Incubate your CTAB with PVP for 15 mins at 65 C and then vertex.

Dear Natasha,

The major plant compounds characterized by antioxidant activity are polyphenols. These are present in most plants and are considered to prevent free radicals associated damages in numerous ways including direct scavenging of free radicals and inhibition of enzymes involved in free radical production.

Whereas, TPC activity is the process to figure out the amount of phenolic content present in the samples. Phenolic compounds that contained in the plants have redox properties, and the properties allow them to act as antioxidants.

After checking antioxidant activity in the samples, We perform quantitative assay through total phenolic content, total flavonoid content, and total saponin content, etc. to check the total quantity of secondary metabolites present in the sample.

Regards,

Divya Jain

India

This link maybe useful for you :

ESTIMATION of TOTAL PHENOLIC CONTENT

Please see the following RG links and PDF attachment.

Dear Dr. Pierre Villeneuve,

Thank you so much. Your answer made me very pleased. So, what do you think about chemoenzymatic methods?

How can I understand if esterification has occurred?

What should I use as standard in chromatographic methods?

Please excuse me if I ask very basic questions. I'm a baby researcher yet.

you can use 0.5 g and more

this enough

It is certainly possible and XIX century chemists proved it by extracting and purifying many natural compounds from plants!

Yes Polarity decreases down the group I.e methanol> ethanol>propanol>butanol etc...

@mansi i did that but it didn't worked...

I'm attaching the picture.. Navy Blue is protein only.. Yellow and light blue are added compounds... The baseline for these is same concentration of compounds

Dear Duried Alwazeer ,

If I dissolved (10) g of dried pomegranate peel with (25) ml of boiled distilled water to prepare aqueous extract of pomegranate peel, my question is how I can calculate the concentration of extract solution.

Thanks in advance.

are you mean phenolic compounds or phenol compounds.

Condensation of 2x1,3-diphenol with 1,2 di-benozic anhydride (Flourescein dye)and condensation of 2x 3-aminophenol with 1,2 di-benozic anhydride (Rhodamine dye)

If you have LC-MS data, I would suggest you try XCMS Online (https://xcmsonline.scripps.edu/) for relative quantification.

Here you can find an example of an application:

Hope it helps!

Both of them are not phenolic compounds they are heterocyclic compounds. Pyrone is heterocyclic chemical compounds, contains an unsaturated six-membered ring containing one oxygen atom and a ketone functional group while Kojic acid or5-Hydroxy-2-(hydroxymethyl)-4-pyrone or 2-Hydroxymethyl-5-hydroxy-γ-pyrone .

attached a good article on pcr inhibitors. Not so good on modern dna purification methods but hopefully plant geneticists will give you the best preparation protocols

Jean Rene Grezes

yes I am using Camag TLC scanner 4 ,working on natural phenolic compounds and how degradation can affect readings if I am preparing fresh stock solutions every time and measuring readings instantly after developing plates and air drying the developing system (I also tried oven drying of eluent with the same problem).

I compared it with Agilent Cary eclipse spectroflorimeter, unfortunately it couldn't even detect concentrations in low nanogram range( TLC scanner is far more sensitive)

Chathura Abeywickrama

yes absorption mode gave the same behavior as florescence going up and down in the same way. how to avoid this problem???

Please look at the following below links which may help you in your analysis:

Thanks

Hello J.D. Franco-Navarro

Your question can’t be answered as @Fahad Shafiq has correctly pointed out at the end of the comments because there is almost complete lack of information about the methods. The study seems to have been done with rather small plants, and in such a way that there would be a strong interaction between nutrition and water supply, making interpretation very difficult. Let us assume you grew the plants in soil, applied a liquid fertilizer and allowing some plants in each nutrient treatment to dry by stopping watering, while the control was given ample water. That is a standard molecular biology/biochemistry approach to such studies.

Which was the treatment supplying the most nutrients - A I suspect, with C supplying least. So plants in A would grow better than in C, and have more, larger leaves. When you started the drought treatment plants in A would dry the soil much faster than those in C – simply a matter of greater surface area in A. So plants in A would become severely drought stressed before those in C, which would appear to grow and be much more `drought resistant’. They would also have very different contents of metabolites. The lesson from this – if my supposition is true - is that it IS ESSENTIAL TO COMPARE PLANTS AT THE SAME RELATIVE WATER CONTENT. If this is not done then the study is invalid. I suggest my paper explains the problem and ways of doing such studies correctly – it focuses on GM plants but the problem is absolutely the same for all studies involving water supply.

Lawlor DW. Genetic engineering to improve plant performance under drought: physiological evaluation of achievements, limitations, and possibilities. J Exp Bot. 2013 Jan;64(1):83-108. doi: 10.1093/jxb/ers326. Epub 2012 Nov 16.

By ignoring the complexities involved with water supply in solid matrices and the effects of different rates of water loss, studies of the metabolism, genetic modifications etc are invalid. Hope this rather strong critic does not apply to your study. Please provide the necessary information about methods.

Best wishes

David

Flavrnoids are subgroup of phenolic compounds. You must focus in the lecture to see if their any metabolic bathway consumed phenol content. Also make this test again to be sure about your results

Please take a look at this useful RG link.

Change in bioactive compounds pattern, thus the bioactivities could be different compared to the original substrate.

@ Wasif Nouman Can you please provide full reference to Gull 2014? Thanks!

@ S. Wenkart .. hey, Dear, I worked most about aromatic and phenolic compounds and biodegradations, but in new my research I was looking an effective method for prevention of occurrence of browning and reduce the effects of phenolics during micropropagations for obtaining higher efficiency...

Kamilla Linggam The method you mention here is only applicable for plant extract or extraction is required to utilize this method. Is there any other method apart from F-C reagent and chromatographic method (attached) which could be used to quantify phenolic compounds in wastewater where there is no extraction required?

Thank you and kind regards

In addition, the amounts of phenolics usually increase after pathogen attack as a defence mechanism. So when we decide to use biological agents against pathogens, we prefer to use isolates which has also more effective on plant defence.