Antioxidant Activity - Science topic

Might be its due to solubility, check the solubility of product.

Dear Prof Renjith Raveendran Pillai,

We also encountered this problem. I am wondering whether you finally tackle this Mo issue. Thank you.

Best wishes,

Hong

Yes

A sample solution was prepared by mixing 65 µL of linoleic acid with 5.0 mL sodium phosphate buffer (pH 7) and 2.5 mg of CCEs in 5.0 mL of 75% ethanol. Afterward, 2.5 mL of distilled water was added and the mixture was incubated at 40ºC for 15 minutes. 0.1 mL of sample solution of ferrous chloride (20 mM in 3.5% HCl), 0.1 mL of 30% ammonium thiocyanate and 5.0 mL of 75% ethanol were mixed. The absorbance of this solution at 500 nm was used to determine percentage inhibition using Equation.

I %= (Ac-As/Ac) x 100

Control (treatment without CCE) and sample absorbance values are represented as Ac and As at 3 weeks, respectively and I (%) percentage inhibition. Positive controls included butylated hydroxytoluene at 200 ppm.

In short, vitamin C plays a significant role in supporting the immune system. It helps stimulate the production and function of white blood cells, which are important for fighting infections. It also enhances the skin's barrier function and may improve the effectiveness of other immune responses...Vitamin C has anti-inflammatory properties that can help reduce inflammation in the body. This can be beneficial for conditions characterized by chronic inflammation, such as arthritis... Vitamin C enhances the absorption of non-heme iron (the type of iron found in plant-based foods). It converts iron into a form that is more easily absorbed by the body, which is particularly important for individuals who follow vegetarian or vegan diets... Vitamin C is involved in the synthesis of neurotransmitters such as dopamine. This process is important for brain function and mood regulation. Some studies suggest that adequate Vitamin C levels may support cognitive function and mental health.

For the hydrogen peroxide scavenging assay, use commercially available 3% or 6% hydrogen peroxide solutions instead of the 30% solution. Adjust the concentration to ensure the final concentration is 0.01 mM. Prepare the working solution by diluting it with phosphate buffer (pH 7.4) to reach the target concentration. Always prepare the working solution fresh before each experiment to avoid inconsistent results due to hydrogen peroxide decomposition.

Hello Avni Yildizbaş

You should first carry out a rough clarification of the extract by simply decanting, followed by a filtration step by using a six to seven layered muslin cloth. Then you may include a centrifugation step which may be required if the powder is too fine to be filtered.

I have attached a book below which may be helpful.

Best.

I used ethanol mixed with DPPH solution

I think you're calibrating a common colorimetric assay for ascorbate, using the radical dye 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH). This assay is known to produce non-linear results that are difficult to interpret. Your assay conditions will dictate your actual observations. I suggest you make certain your standard curve always includes values to provide a full sigmoidal range (0-100% inhibition). And run your standard curve in triplicate or more to get some statistical power for your result.

Alif Ismail

I use shaking during my test because when the sample precipitated it can not reacting with DPPH, therefor, I shaking my sample during test and in last five minutes of test I centrifuging microtube for 1 min for precipitated the large particles and I using the supernatant for determine absorbance

It is recommended to immediately put your samples in liquid nitrogen and store them at -80°C without hemogenization. Use specific buffer for each analysis. For example, for RNA or DNA extraction it is better to use TRIzol. For any analysis, you surly need its specific kit. Each kit has its own hemogenization buffer. You don't need to hemogenize all of your samples at first with a unique buffer for all analysis.

Dear Masoumeh

These quotes are effective, but in a variety of ways. SOD and DPPH techniques.

What methodology was followed?

Dear Wuraola,

Kindly read and digest the following articles.

Wish you all the best with your study

Check https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-015-1618-6

Dear Sahar

As Prof Shin wrote mostly we have more than one protocol , you should notice about the materials and all other points.

Deoxyribose assay is used to determine the hydroxyl radical scavenging activity in an aqueous medium. The reaction mixture containing FeCl3 (100 µM), EDTA (104 µM), H2O2 (1 mM) and 2-deoxy- D-ribose (2.8 mM) are mixed with or without the test sample (plant extract/fraction/compound) at various concentrations (10-250 µg) in 1 ml final reaction volume made with potassium phosphate buffer (20 mM, pH 7.4) and incubated for 1 hr at 37 0C. The mixture is then heated at 950C on water bath for 15 min followed by the addition of 1 ml each of Trichloroacetic acid (TCA) (2.8%) and Thiobarbituric acid (TBA) [0.5% TBA in 0.025 M NaOH containing 0.02% 2-Deoxy-D-ribose, butylated hydroxyanisole (BHA)]. Finally the reaction mixture is cooled on ice and centrifuged at 5000 rpm for 15 min. Absorbance of supernatant is measured at 532 nm. All readings are corrected for any interference from brown colour of the extract or antioxidant by including appropriate controls. The negative control without any antioxidant or sample is considered 100% deoxyribose oxidation. The % hydroxyl radical scavenging activity of test sample is determined accordingly in comparison with negative control. Ascorbic acid is taken as the positive control.

Hope it helps you.

Good Luck

Dear Dr Sahar, please kindly follow the attached article, hope it can be useful for you!

" according to reaction time (slow, fast kinetics), the linearity range of DPPH radical depending on the detection conditions as well as the kind of the investigated antioxidants (reference chemicals and the ground elder prepared from fresh and dry plants"

Md Jakaria Pimmada Junsathian hope you guys were using UV-compatible plates since the absorbance (230 nm) is quite low.

I think this is the role of a researcher, to find new techniques to evaluate biological activity.

What will we do if the IC50 values become negative after interepretation in excel?

Check https://journals.sagepub.com/doi/pdf/10.1177/1934578X1701200236

Hi, Dear Yunus Ahmed

checking the attached papers can be useful to calculate DPPH.

M regards

Dear Ayoub Najem

Depending on the inhibition test, it can reach 100%.

for example, in antibacterial tests it is easier to reach 100%.

In protein / enzyme inhibition tests it is more difficult, as there is the question of saturation and other parameters involved.

Dear Ayoub Najem thank you for your interesting technical question. I assume that citronellal can be viewed as a modest antioxidant because the aldehyde functional group –CHO can be oxidized to a carboxyl functional group –C(=O)OH. For a relevant reference supporting this please have a look at the following article entitled

(see attached pdf file)

An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols.

For more information check the file bellow will help you a lot

Official definitions by IUPAC

The terms “antioxidant activity” and “antioxidant capacity” have different meanings: antioxidantactivity deals with the kinetics of a reaction between an antioxidant and the prooxidant or radical itreduces or scavenges, whereas antioxidant capacity measures the thermodynamic conversion efficiencyof an oxidant probe upon reaction with an antioxidant.

"Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical Report)

The performance antioxidant activity of essential oil (EOs) is the result of a complex interplay between components and the oxidizable tested material. ABTS and DPPH are antioxidant assays to measure the activity of the compounds to scavenge free radicals. The essential oil especially terpenoid groups containing phenolic skeleton can react rapidly with peroxyl radical, therefore could protect oxidation by those radicals. Most of the phenolic compounds capable of scavenging free radicals by donating their electrons or H (from a hydroxyl (—OH) in the benzene ring) to the radicals. On the other hand, non-phenolic terpenoids can not protect lipid from oxidation due to (for example alpha-pinene or its similar compounds) undergo auto-oxidation. Furthermore, not all EOs have the ability to reduce reduces Fe³⁺ to Fe²⁺ (as measured in FRAP assay), even the phenolic structure with ortho-dihydroxy moiety (ex. hydroxytyrosol) could chelate Fe²⁺ whereas the tyrasol that has only one OH does not show that ability.

Harto Widodo The definition of antioxidant can't not be based on the mechanism. There area thousands of molecules which fit your definition, but they are not antioxidants. The concept of antioxidants belongs to life-sciences. In the field of chemistry dealing with oxidation by dioxygen the term "inhibitor" is commonly used. I think that the best definition of an antioxidant was given by Halliwell and Gutteridge: "an antioxidant is any substance that delays, prevents or removes oxidative damage to a target molecule”

Trolox is best

have you checked this?

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).

Also https://www.researchgate.net/publication/339575476_Characterization_and_application_of_red_pitaya_Hylocereus_polyrhizus_peel_powder_as_a_fat_replacer_in_ice_cream

You can find your answer through Ebers papyrus

Total Antioxidant Determination by FRAP Method

FRAP assay was carried out according to the previous study with some modification.[16] FRAP reagent was prepared from acetate buffer (1.6 g sodium acetate and 8 mL acetic acid makeup to 500 mL; (pH 3.6), 10 mM TPTZ solution in 40 mM HCL and 20 mM iron (III) chloride solution in proportion of 10:1:1(v/v), respectively. The FRAP reagent was prepared fresh daily and was warmed to 37°C in oven prior to use. A total of 200 μL of samples extract were added to 4 mL of the FRAP reagent and mixed well. The absorbance was measured at 593 nm using UV-VIS spectrophotometer (UV-VIS specord 205). Samples were measured in three replicates. Standard curve of ascorbic acid (125, 250, 500, 750, and 1000 μmol) and GA were prepared using a similar procedure.

Thank you Adam B Shapiro

So if i get 55% inhibition during ACE enzyme assay, what does it mean and correlate with substrate HHL and product hipuric acid

you may read this article

Dear Natasha,

It is based on reduction of the violet DPPH radical by the antioxidant via a hydrogen atom transfer mechanism to cause a change in the color to stable pale yellow DPPH molecules in the dark. This test provides useful information on the antioxidant capacity to donate hydrogen atoms. On the reactions reducing capacity and on mechanism between the free radical and the antioxidant.

The standard DPPH assay uses methanol or ethanol as solvents, or buffered alcoholic solutions to keep the hydrophobic hydrazyl radical soluble while offering sufficient buffering capacity.

The Beer-Lambert law states that there is a linear relationship between the concentration and the absorbance of the solution, which enables the concentration of a solution to be calculated by measuring its absorbance. This will be evaluated by % inhibition.

Regards,

Divya Jain

India

I suggest using pressurized fluid to extract the samples, then centrifuge for 10 min at 20000 ppm, separate by microfilter and bring HPLC-Mass to 10000 ppm

Dear Raul Diaz Molina

For ABTS radical scavenging assay, 0.1 M potassium phosphate buffer (pH 7.4) is the preferred diluent followed by methanol.

Regards

Thank you Jan Homolak

In my opinion, there are no simple methods to evaluate antioxidizing capacities of potential antioxidants. General tests like DPPH, ABTS... only (roughly) measure the ability of the antioxidants to scavenge free radicals. An antioxidant is much more than that; it can act as a scavenger of free radicals, repair of the oxidized species (reduction by one electron transfer) or by cascade (cumulative) effect . Furthermore, the antioxidant capacity can also depend on the agent responsible for the oxidative stress conditions (selective antioxidants).I recommend the evaluation of the mutual behavior of both antioxidant and the target to be protected in every specific oxidizing environment.For details see e.g.:

DPPH and TEAC (ABTS radical) are important methods belonging to single Electron transfer (sEt) based assays. In my opinion, you have to consider to apply both methods in order to test the antioxidant capacity with two different radicals. Your results can be expressed as value showing the concentration caused 50% scavenging of DPPH• radical (IC50 ) in  μg/ml

You can check also the following paper.

Deprnd on type of plant, and type of bioactive components an its concentraction in this plant.

I prefere wild and medicinal herbs, seasoning or spices

Have you checked whether the extract from the marine algae has potential antioxidant properties?

If No, You may go with spectrophotometric analysis (Antioxidant assays- DPPH,ABTS,ORAC,NO).

Further, you may proceed with cell lines or any normal cells to check the reduction in intracellular Reactive oxygen species of your extract.

Now, for the concentration you may start with the a higher say 1000uM and reduce it to lower concentrations based on the literature .

However, when you go for in Vivo model it is necessary to go for toxicity analysis to check the different parameters like mortality, malformations, heart rate etc. so that you can set a concentration. Also, the concentrations set for in vitro and Vivo antioxidant activity will vary.

You can go with DCFDA analysis. Further SOD,CAT,GPx for you in Vivo antioxidant studies.

https://www.researchgate.net/post/What_is_the_best_method_to_use_to_measure_antioxidant_activity_in_a_small_volume_of_isolated_exosomes_from_cancer_cells

To measure oxidative stress and the ability to oxidize in the plant is preferred to use

As for the animal, it is preferred to use clotathione

see this article phease

Best regards Dhifaf Zeki Susana Rey Alonso

I think garlic (Allium sativum) is the best plant.

yes is effect on antioxidant activity by many way......

these articles have a good informations about your question

http://www.jlr.org/content/22/5/763.full.pdf

https://www.ocl-journal.org/articles/ocl/pdf/2001/01/ocl200181p89.pdf

Dewi Asri Anggraini Namrata Dwivedi Priti Gupta Mohammed Ismael Rushdi

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.

you can use this research

in this research we used Querciten

the protocol is one

Hello Mellisa, for any blank solution we generally replace the sample with (say) DH2O. or sometimes the main reagent, here FRAP reagent with DH2O. But what I understand from your question is... you are somehow correlating DPPH assay with FRAP assay which is not correct. For DPPH, we calculate the % inhibition and therefore the concept of control absorbance. Here in case of FRAP assay, if you are following the Benzie and Strain method (1996) we have to draw a standard curve for FeSO4 just like others. FRAP assays estimates our sample's Ferric Reducing capacity and therefore we can't calculate % inhibition unlike DPPH or ABTS assays. Once you get the standard curve data, you can correlate to your sample data.

Hope this will help you.

What is names of the journals

Lactic acid bacteria don't produce in primary or secondary metabolic pathways phenolic compounds. The response provided by @Vineet Sharma is logical, but this phenomenon could be happen by different microorganisms including fermentative ones.

to my knowledge the product is unstable so it is difficult to measure thank you

Were your experiments done with ozone generated from air or from oxygen? If from air, you may find other molecules formed in the generator, are interfering with your results.

In answer to question 1, I would guess that it is the same as why ozone is not more used in Medicine i.e. there have been lo large scale trials. The reason for the lack is the same. There is no way of protecting your investment in a trial nor large money to be made.

It certainly is a cheap biocide. Even the equipment needed is cheap.

I have had saline ozone IVs and my condition did improve although it's back to where it was prior to treatment 2yrs ago.

Please take a look at the following RG link.

I would interprete these numbers as: 6 times extraction with 50 ml petroleumether, 3 times with 50 ml ethylacetate and 3 times with 5 ml n-butanol.

I doubt whether 6 extrations is essential. I learned that after 3 extractions (nearly) all has been removed.

Dear Dima Depp

Followed standard protocol for DPPH assay and additionally measure your sample colour absorbance for each dilution without DPPH (You need to do the same dilution as your DPPH addition). Then subtract the sample colour from reaction mixture colour(With DPPH).

Four cups of coffee a day

Best Regards Wesam Salah Mohamed Ibrahim

PLZ.look at the following link:

To be used for in vivo (e.g Wistar rats), What is the percentage of minimum DMSO that can be use to dissolve thymoquinone?

Las unidades de mg/g se refiere a los mg de antioxidante que se necesita para inhibir el 50% del radical ABTS o DPPH y los gramos (g) se refiere a gramos de hojas de ortiga, se reporta en mg/mL o mg/uL ya que se va a comprar con un estándar que esta disuelto en medio liquido por eso se utilizan estas unidades.

Hello Maryam Haqparast

Indeed, sperm cells are sensitive to freezing regarding to levels of DNA damages as measured by 8-OHdG immunostaining. Frozen samples have higher levels of DNA damages than fresh ones.

Considering this information, I must recommend to establish distinct norms for cryoconserved samples in one hand and fresh samples in the other hand. Furthermore, to ensure easier and reliable interpretation of your datas, it seems really important not to mix these two types of samples.

To answer entirely, I must add that the protocole of freezing, and particularly the cryoprotectant solution seems to be a key point and variation in its composition may have critical effect on DNA damages.

Regards,

Benoît

Dear Debi Prasad Sahoo,

The antioxidant power can be evaluated in vivo or in vitro by applying methods that measure the antioxidant capacity of a molecule or a natural extract. Given the complexity of the oxidation processes and the diverse nature of antioxidants, with both hydrophilic and hydrophobic components, there is not a universal method by which antioxidant activity can be quantitatively measured accurately. Most often, the responses of different and complementary tests must be combined to give an indication of the antioxidant capacity of the test sample.

Regards

Dr Benkhaled A

Hello, can't be evidence of ascorbate peroxidase in fungi. Peroxidase structures have significantly increased our understanding of the evolutionary and functional relationships within the plant peroxidase superfamily.

Three distantly related structural classes have emerged:

1. mitochondrial yeast cytochrome c peroxidase, chloroplast and cytosol ascorbate peroxidases, and gene duplicated bacterial peroxidase class I

2.secretory fungal peroxidases classII

3. classical, secretory plant peroxidases (class III).

So ascorbate peroxidase is class I peroxidase enzyme and catalyse the H2O2-dependent oxidation of ascorbate in plants, algae and certain cyanobacteria but not fungi. For this type of peroxidase your culture shuld be photosynthetic. My be we will see this activity in future in Radiotrophic fungus.

Good luck!

I've checked and in literature they freeze-dry the sample but I'have to use the extract in this way. I'm thinking to use FRAP method to overcame this problem or to dilute the extract before the DPPH analysis.

Thanks @royaha for you kindness.

But this method is even more complicated than spectrophotometry.

You can go for Autodock 4 version for docking studies with PDB suitable for antioxidant potentials..Good luck

A great number of companies offer polymer additives of various types for different properties.

Master batches can be like taking some liters of your intermediate liquid product and mixing in some additives before adding it all to the polymerization reactor. It makes mixing easier and can help with heat management.

Different master batch added after polymerization is usually a paint or other coating that is to be added to the surface of a product.

you can choose the journal according to your work from the below links

https://journalsuggester.springer.com/

I agree Parthiban Ezhumalai

Dr. Pagandai Vaithianathan Pannirselvam, noted.

Actually as you said I did tried with all different solvents. I used Methanol, 50% diluted methanol, ethyl acetate, hexane and distilled water to extract phenolic compounds separately and made comparison. And Yes as suggested methanol was very productive compared to other solvents. Thank you for the suggestions.

Dear Vikas,

Individual phenolic compounds may exhibit low to high antioxidant activity. This depends on aspects involving its structure and chemical function. In order for you to make sure that the antioxidant activity is low analyze several antioxidant methods FRAP, DPPH, CUPRAC and ABTS (for example). These methods have different principles and evaluate different forms of antioxidant activity.

Regards,

Alessandro