I left academia in 1997 after having worked in the field of "oxidative stress" for several years. It was already known back then that in vitro antioxidant activity could not be directly correlated with in vivo antioxidant activity. One paper I eventually had published on this subject in 1997 [1] was initially rejected by the editor of another journal on the basis that it "provided only negative results".

Much of what is written on antioxidants is just "hype", whether in the field of cosmetics, nutrition, or medicine. It tells me something about how much background literature research is not done when designing experiments in this field!

Just about anything and everything can be shown to have some kind of antioxidant activity. Water can be very effective at putting out a small fire; so can a plank of wood. These are both antioxidants.  Context matters.

Living systems are essentially reducing environments in which oxidative processes are allowed to occur under carefully controlled conditions. That control is exerted by various antioxidant molecules and systems. These oxidative processes release energy from fuel molecules. Without antioxidant defences, we would ultimately self-combust. Water plays a significant part in our antioxidant defences. So do free-radical scavenging antioxidants; so do other classes of antioxidant molecules. But we have to understand that antioxidant chemistry is just chemistry. It was demonstrated many years ago that when a water soluble radical-initiator is added to whole blood, endogenous antioxidants are consumed sequentially: this is driven by the oxidative / reductive electrochemical properties of the reactants. Ascorbic acid is consumed most rapidly, followed by bilirubin then uric acid then plasma vitamin E [2].

Ascorbic acid acts as a sacrificial antioxidant. In taking a hit from a potentially-damaging oxidant free radical species, it traps the unpaired electron to form a particularly benign free radical species, namely semidehydroascorbic acid. This can take a second hit to become dehydro(ascorbic acid), the original ascorbic acid molecule having quenched two unpaired electrons in the process. But perhaps most importantly, the dehydro(ascorbic acid) can then be metabolically reactivated back to ascorbic acid at the expense of glutathione. The glutathione, in turn can be metabolically reactivated at the expense of NADPH. And NADPH is produced in mitochondria, our intracellular "batteries" [3].

Innumerable examples of substances with ascorbic-acid-like antioxidant activity in vitro can be found in the literature. But ascorbic acid is probably unique in the fact that when it quenches a potentially damaging free-radical, it does not itself become a new potentially-damaging species. Ultimately, this is why results from in vitro antioxidant studies are so difficult to interpret. In silico predictions are only as good as the data used to develop the in silico model.  "Garbage in; garbage out".

So, we humans [probably] just need to ensure we have an adequate supply of ascorbic acid / vitamin C in our diet. And, more importantly, that our diets do not add to the background oxidative stress we all inevitably have to endure as the price we pay for extracting energy from the food we eat. Adding random new antioxidant wonder-molecules to our diet is probably a bad thing to do unless these molecules are first shown to produce chemically and physiologically benign products on trapping reactive oxygen and reactive nitrogen species.

1. http://dx.doi.org/10.1016/S0378-8741(97)01510-9

2. Niki E. Antioxidant compounds. In: Oxidative Damage and Repair (Ed. KJA Davies). Oxford: Pergamon Press, 1991, pp 57-64.

3. Schmidt RJ. Redox homeostasis and microbial colonisation of wounds: new insights into the energy economy of chronic wounds, Journal für Anästhesie und Intensivbehandlung 3(3): 26-31 (1996) [http://www.pabst-publishers.de/Medizin/med%20Zeitschriften/jai/1996-3/art-6.html ; downloadable from http://bit.ly/VQBqm4]

Actually, in vitro antioxidant assays are not waste of time. Maybe, in vivo is. However, these experiment (in vitro and in vivo) are useless. I agree with that. There is some in silico methods such as QSAR and you can be researching the toxic effects of materials or drugs. antioxidant assays do it already, Don't they?. It is also, there are different results between in vivo and in vitro assays, even if doses, experimental conditions and materials are same!! In my opinion, we must determine the toxic effects of a material using in silico programs. These are cheap and so fast. 

Best regards

 Dear Mahesha,

In vitro Antioxidant activity of crude extract is just a preliminary study, many journals even not accepting papers on the same.

If some one is working on ageing, inflammation and liver diseases these preliminary data is useful, otherwise it is just waste of time.

 In vitro Antioxidant activity of isolated compounds and work on related disease might give impact to the work. otherwise really it is useless.

I might be wrong, if i am wrong suggest me too.

Dear Mahesha,

This actually depends on the objective and design. If I have a plant extract rich in antioxidants, apart from determining its in-vitro antioxidant properties, I should test its effects on certain disease markers. Stopping at the level of antioxidant potentials will be of little benefit if not applied. You may wish tocheck out the profile and publications of Ganiyu Oboh in research gate to have a proper perspective on how to make antioxidant studies useful.

Regards

Dear Manesha,

You do not assess antioxidant activity for each and every plant you find on your way, because there are hundreds of thousands and more plants and this would really make no sense. One usually investigates antioxidant activity of these plants which have been on the lists of traditional medicines (Chinese, Ayurvedic, Tibetan, etc.) for thousands of years now, to better understand their curative potential. In the other words, antioxidant activity of medicinal plants is a valued information which modern science can add to traditional medicine pharmacopoeias (owing to an excellent analytical potential of modern times).

Now then, when you have many different species of one and the same genus, you can compare antioxidant activity among these species to see which one has the highest antioxidant potential, in vitro at least. Then you can introduce certain amendments to the traditional medicine pharmacopoeias, suggest introduction of commercial plantations of novel species, etc.

I disagree with a statement that modern science does not respect the results of the in vitro antioxidant measurements. Last time, I was invited as guest editor to organize a special section on this particular issue by the J AOAC Int, a serious and influential analytical chemistry journal (see my special section in issue 4, 2015, of J AOAC Int.

From our special section, it comes out that the most serious drawback of antioxidant assessmenya is that they are based on different models of antioxidant (gallic acid, ascorbic acid, trolox etc.) and on different models of the free radical scavenger (DPPH, ABTS, etc.), so that the results obtained with use of different models have a somewhat different chemical meaning and cannot be directly compared. The worst drawback of antioxidant potential measurements - both in vivo and in vitro - is that the scientists have not yet agreed for one standard assessment method, by which an antioxidant potential of plants might be compared between different laboratories. In this sense there is a chaos and the results obtained by different groups are incomparable and makind little sense. But I personally believe that the phytochemists and those engaged in pharmacognosy will find a consensus, stick to one pharmacopoeial method and start better perform interlaboratory comparisons.

Best wishes to All,

Teresa

I left academia in 1997 after having worked in the field of "oxidative stress" for several years. It was already known back then that in vitro antioxidant activity could not be directly correlated with in vivo antioxidant activity. One paper I eventually had published on this subject in 1997 [1] was initially rejected by the editor of another journal on the basis that it "provided only negative results".

Much of what is written on antioxidants is just "hype", whether in the field of cosmetics, nutrition, or medicine. It tells me something about how much background literature research is not done when designing experiments in this field!

Just about anything and everything can be shown to have some kind of antioxidant activity. Water can be very effective at putting out a small fire; so can a plank of wood. These are both antioxidants.  Context matters.

Living systems are essentially reducing environments in which oxidative processes are allowed to occur under carefully controlled conditions. That control is exerted by various antioxidant molecules and systems. These oxidative processes release energy from fuel molecules. Without antioxidant defences, we would ultimately self-combust. Water plays a significant part in our antioxidant defences. So do free-radical scavenging antioxidants; so do other classes of antioxidant molecules. But we have to understand that antioxidant chemistry is just chemistry. It was demonstrated many years ago that when a water soluble radical-initiator is added to whole blood, endogenous antioxidants are consumed sequentially: this is driven by the oxidative / reductive electrochemical properties of the reactants. Ascorbic acid is consumed most rapidly, followed by bilirubin then uric acid then plasma vitamin E [2].

Ascorbic acid acts as a sacrificial antioxidant. In taking a hit from a potentially-damaging oxidant free radical species, it traps the unpaired electron to form a particularly benign free radical species, namely semidehydroascorbic acid. This can take a second hit to become dehydro(ascorbic acid), the original ascorbic acid molecule having quenched two unpaired electrons in the process. But perhaps most importantly, the dehydro(ascorbic acid) can then be metabolically reactivated back to ascorbic acid at the expense of glutathione. The glutathione, in turn can be metabolically reactivated at the expense of NADPH. And NADPH is produced in mitochondria, our intracellular "batteries" [3].

Innumerable examples of substances with ascorbic-acid-like antioxidant activity in vitro can be found in the literature. But ascorbic acid is probably unique in the fact that when it quenches a potentially damaging free-radical, it does not itself become a new potentially-damaging species. Ultimately, this is why results from in vitro antioxidant studies are so difficult to interpret. In silico predictions are only as good as the data used to develop the in silico model.  "Garbage in; garbage out".

So, we humans [probably] just need to ensure we have an adequate supply of ascorbic acid / vitamin C in our diet. And, more importantly, that our diets do not add to the background oxidative stress we all inevitably have to endure as the price we pay for extracting energy from the food we eat. Adding random new antioxidant wonder-molecules to our diet is probably a bad thing to do unless these molecules are first shown to produce chemically and physiologically benign products on trapping reactive oxygen and reactive nitrogen species.

1. http://dx.doi.org/10.1016/S0378-8741(97)01510-9

2. Niki E. Antioxidant compounds. In: Oxidative Damage and Repair (Ed. KJA Davies). Oxford: Pergamon Press, 1991, pp 57-64.

3. Schmidt RJ. Redox homeostasis and microbial colonisation of wounds: new insights into the energy economy of chronic wounds, Journal für Anästhesie und Intensivbehandlung 3(3): 26-31 (1996) [http://www.pabst-publishers.de/Medizin/med%20Zeitschriften/jai/1996-3/art-6.html ; downloadable from http://bit.ly/VQBqm4]

Dear Teresa,

That's a really wonderful answer. I also believe that in vitro antioxidant assay gives a preliminary idea on bioactivity of the extracts and also about the possible compounds present in the extract (like polyphenols show high antioxidant activity due to -OH group). 

You are right that we must have a standard protocol to measure in vitro antioxidant assay, taking standard amount of radicals and solvents and also maintaining a standard conditions of analysis. Then we can a comaprative data about different plant extracts/phytochemicals. 

Professor Kowalska is totally wright. Now we have so many methods and protocols for comparing antioxidant properties of plant extracts, molecules, food etc. that eventually nobody knows which is the most active because the methods are not comparable. Very often the protocols have also very low recurrence. Therefore we need some gold standard to be able to compare the results for different species, substances etc.

I also think that the antioxidant research have bright future and many things can still be done on that subject.

Best regards for all

I totally agree with Richard.

In my opinion yes, in vitro antioxidant assays are mostly useless and without significance. Unless you are looking for something to replace synthetic antioxidants like BHA or BHT, and even then.

Why?

- because many antioxidants (and certainly those from plant origin like polyphenols) are very poorly bioavailable, they are present in circulating blood , and in addition at levels of magnitude lower than the body's own antioxodant enzymes etcas metabolites with different structures and usually much lower antioxidant assays (btw, I also think that assessing bioactivity of secondary metabolites on cells has little significance, as what will reach the cells will be the metabolites, not the native molecules);

- the one part of the body where antioxidants may have an interest is in the stomach as preventing the formation of toxic compounds from the oxidation of polyunsaturated fatty ac ids.

- because the "many antioxidant assays" are ac tually for a large part redundant, because they have the same underlying chemical mechanisms (Folin Ciocalteu and DPPH for example...). I do not know how many articles I have read that marvel at the close correelation between those two, which is more or less mechanical.

- effects should be linked to specific activities, interactions with various proteins and receptors, classical mechanisms; polyphenols share similar actions probably because they share colonic metabolites, which are bioavailable.

- there are clear indications tha tantioxidant supplementation is not beneficial and even detrimental for health, what is proven is the interest of eating (varied) fruit and vegetables.

Cordially

It can be implied from the discussion above that contributors believe that we will one day find a new wonder antioxidant that we can take as a food supplement or even as a medicine for the purpose of extending life / avoiding or even treating illness / preventing wrinkles / gaining eternal youth / etc. On the basis of what I know as a pharmacist and toxicologist (and therefore as somone who knows what medicines can and can't do), and as someone who has also dabbled in electrochemical studies of antioxidants, and who for many years pre-2000 contributed to the oxidative stress literature, I do not expect ever to be able to extend my life or improve my health with a new, yet-to-be-discovered wonder antioxidant.

The point I make in my earlier contribution is that whatever exogenous antioxidant molecule we might introduce into our bodies, it will simply add to and integrate with the significant bulk of antioxidant substances and processes that already exist in our bodies. And where ascorbic acid levels are adequate, the new wonder antioxidant will [probably] serve no purpose whatsoever because ascorbic acid is perfectly "tuned" for its role as the ultimate aqueous phase sacrificial antioxidant. Only when ascorbic acid acid levels are depleted and metabolic pathways to regenerate ascorbic acid are similarly depleted of glutathione and NADPH, will the new wonder antioxidant begin to take over the antioxidant role of ascorbic acid. This begs an obvious question: why not just take a vitamin C supplement? And make sure that you are taking in enough vitamin E to maintain lipid phase antioxidant capacity. And perhaps enough N-acetylcysteine to maintain glutathione levels?

In vitro assays, standardised or not, gold standard or not, tell us essentially nothing about how a molecule with antioxidant activity will behave in a living system. You need a living system in which to do the assays. Even whole blood evidently lacks the antioxidant regenerative capacity of the whole animal, as appears to have been demonstrated by Niki (1991) - see reference 2 in my earlier contribution.

Eternal youth is not going to be delivered by a new wonder antioxidant. We just need to take in much, much less sugar so as to minimise the formation of advanced glycation end-products (AGEs). AGEs pick up decompartmentalised iron and become "Fenton-reactive". These complexes generate oxidative stress. We just have to eat plenty of fresh fruit and vegetables containing vitamin C (and potassium, but that's another story) and vegetable oils containing vitamin E. Such a diet will also supply [antioxidant] flavonoids, which were at one time known as vitamin P before their antioxidant activity was recognised.

Dear All,

It is nice that the question posed by Mahesha instigated such a turbulent discussion. This means that he managed to pose a very good question, congrats to Mahesha! This also means that the issue itself is important.

Due to this "turbulence" of the discussion, let me get engaged in it for the second (and hopefully and presumably for the last!) time.

First of all, may I refer to some statements made by Catherine and Richard. Ladies go first, so I will first comment on the comments given by Catherine.

It is absolutely not true that all methods of evaluating an antioxidant potential are chemically equivalent in this sense that they measure one and the same thing, one and the same magnitude. This is obvious even to my BSc undergraduate students who hold their BSc PowerPoint seminars at which they specially emphasize differences among what's measured with use of F-C, DPPH, ABTS, and the other spectrophotometic methods. They also comment on the results obtained with such sophisticated instrumental measuring technique, as electron spin resonance (EPR) spectroscopy. They simply show stoichiometric reactions standing behind each methodology and then even for a "lay person" it becomes evident without any extra explanation that these reactions substantially differ in chemical terms, hence an interpretation of the results thereof must be different as well. When standards (a free radical and a free radical scavenger) are different, then an outcome must be different as well. At this pont, we even forget to mention that sensitivities of all these - mostly colorimetric - methods are also different, which additionally adds to possible discrepancies.

Now let me answer to Richard. This hopefully scientific discussion certainly is not about an eternal youth, Cleopatra bathing in donkey milk, or Dr. Faustus and a recipe for immortality, as suggested by Richard. But in certain sense, it is on precisely  these topics! Let me remind in this place the so-called "French paradox" and French addiction to red wine, in combination with surprisingly low levels of cardiac situations, in despite of a highly caloric French diet. Nowadays, the 'responsible' molecule seems to be trans-resveratrol, but who knows.

At the end, let me repeat once again:

(i) Analytical equpment becomes increasingly more sophisticated, so now we can measure such magnitudes which one or two decades ago were impossible to measure.

(ii) Due to relatively late access to these analytical techniques, no standardized measuring protocols have been elaborated so far, but one day (rather sooner than later) they certainly will.

(iii) Please,don't forget the synergy of the discussed effects, which can be due to a mixture of compoents provided by this, but not another plant.

(iv) Please, don't forget that ca. 70% global population of this planet rely on exclusively herbal medicines; firstly, out of the poverty, but secondly, because these medicines have a real curative potential (which to a large extent is an antioxidant potential of the so-called secondary metabolites, which are more abundant with one plant and less abundant with another). 

It has to firmly be stated that skepticism toward herbal medicines (and an antioxidant potential thereof) mainly serves to promote synthetic drugs and in the first instance, to promote pharmaceutical companies with their gains even surpassing those of the warfare industry.

Good night everybody!

Teresa

I agree with them , there is no near future for medicinal plants , pharmaceutical societies cares about monoclonal antibodies more than bioactive compounds  ( my opinion)

but don't forget that there are some plante that we don't know there antioxydante activity so what should we do ?

Why should we care about this knowing that we have a good synthetic antioxydants with out toxicity !

For sure, results provided by the in vitro antioxidant assays have no correlation with those from in vivo studies. The in vivo is more complex and may depend on many factors's interactions, not only on the compound that you are interested in. Nevertheless, the antioxidant assays could give information about your matrice/plant as preliminary study to further analytical studies, economically more expensive.

The science of Nutrigenomics in human health has moved well away from putative benefits of direct-acting antioxidants - not entirely but certainly the clinical value of phytochemicals is now more targeted to activating transcription factors which activate gene expression of the endogenoussly-generated antioxidant enzymes.  Polyphenols have low bioavailability and are now considered to exhibit direct antioxidant activity on contents of the intestinal lumen or as their metabolites following contact with colonic microflora.  The FDA in 2010 removed reference to ORAC Tables from its website becasue these in vitro comparative measures of antioxidant value of plant bioactives can not be supported in vivo.  Supplement manufacturers were using these values to claim that one supplement by virtue of its ORAC value had higher antioxidant thn another.  Examples includes Goji berries, Acai berries, blueberries.  As a result, such data have limited clinical value.

In vitro antioxidant results sometimes may look bogus but they serve as preliminary steps for in vivo studies. Using in vitro antioxidant assays, we will be able to identify the plant that is best suited for in vivo studies. Carrying out in vivo without in vitro would amount to guessing the antioxidant activity of such a plant. We can not solely rely on synthetic antioxidant compounds because they have side effects which can be detrimental. 

I can see wonderful answers from researchers across the globe. If consider a standard method for antioxidant assays (in vitro) which method do you recommend? What are the critical points to be considered? which standard reference we shall take? At the end, how can we conclude our results? Can we say our 'extract' or 'single molecule' is highly antioxidant with the proposed standard method? 

I agree with Ms Kiwalska ,Houghton and Mr Azeez. We need in  vitro results to proceed with in vivo studies. The problem is the correct interpretation of these results and not the assays. There are many research papers relative to in vivo studies. Another problem is the bioavailability  of the antioxidans and their  interaction with other substances present in foodstuffs. This is also a very interesting point for research.

As I recall Linus Pauling (two time Nobel Prize winner) advised adults to take up to 15 g ascorbic acid supplement per day, then titrate down a bit if one encountered diarrhea.

Personally I would prefer food-derived or GRAS antioxidants in my gut rather than synthetic molecules and any impurities they may contain.

Somehow against my own will, I feel forced to come back to this dicsussion. All voices participating so far, can be divided into two sub-classes: those who disregard experimental results and those who think otherwise. I must say that I am gravely  stupefed by these voices which try to disregard (or at least to marginalize) experimental results, no matter how modest they might be. Once natural / life scientists start talking about superiority of in silico simulations over the results originating from the bench top, for me this is a real end of the world (literally!!!), a shock and a sacrilege in pure form. Please, keep in mind that each - even the most modest - numerical estimate of an antioxidant potential of a weed growing next to a dirty railway track has a superiority over an in silico simulation by the very nature of being real, REAL! Dear Natural Scientists, please wake up and re-consider your sacred convictions!

Again I have to congratulate Mahesha for being sober and for recently asking a very well posed question. He asks how the standardized method of an in vitro assessment of the herbal antioxidant potential measurement should look like. My answer is: a consensus should be made over the measuring protocol from start to end. The analysis has to start from plant material (dried herbs) - how many grams? - and many / most papers start preaching their story from a liquid extract only (with the quantity of dried plant not even mentioned, neither mentioning the details of the extraction procedure). The amount of dried herbs has to be agreed on, the extractant, the extraction technique, and at the end on the method of assessment itself.

Hydrodistillation or accelerated solvent extraction (ASE)? This is the next serious question which has to be answered and the technique should be assumed by a standardized protocol.

Colorimetric techniques are not very precise and therefore my own preference in the electron spin resonance (EPR) spectrometry, otherwise expensive and unavailable in most laboratories. But we are talking about precise instruments and not about pleasing the Third World.

Finally one should standardize the free radical model and the free radical scavengers model. A consensus about these issues should be obtained, but DPPH and trolox seem a nice choice. Once there is serious will in the community to standardize the measuring protocol, sharp measuring tools already are there.

Matthew, you refer to Linus Pauling but please remember that his research is 40-50 years old and his consumer-level books 40 years old.  At the time, no-one knew about Nrf2 as a transcription factor that governs the expression of around 2000 cytoptotective genes.  There have also been subsequent studies on the pharmacokinetcs of Vitamin C (Mark Levine has published 2 papers on this) and he shows that in all cell types, asocrbate absorption plateaus at aroudn 400 mg daily.  Robert Cathcart, one of Pauling's contemporaries coined the term 'bowel tolerance' to claim the level being absorbed.  His theories (no-one else has published on this) state that one continues to ingest ascorbate until diarrhoea and that this determines the correct dose, assuming that  teh quantity ingested prior to the onset of diarrhoea is absorbed.  I doubt that this can be verified.  I woudl suggest that Pauling's theories (Nobel prize winner in 2 other fields) do not hold up under scientific scrutiny.

http://advances.nutrition.org/content/2/2/78.full

In vivo antioxidant studies are no doubt the true values of antioxidants. These does not mean that the others who are doing in-vitro studies are fools or senseless and their data are of no sense. This sort of views expressed by those who only advocate only  in-vivo studies are doing research with a biased mind set, which it self is against the principles of research methodology.

The relative value of in vitro vs in vivo studies depends on what you want to know.  My point is that direct-acting antioxidants are performing quite a different function from indirect-acting phytochemicals that induce expression of genes which code for endogenous antioxidants.  Joseph Kanner an Israeli biochemist has published extensively on the function of polyphenols which are active in the gut but are too poorly-bioavailable to act systemically as antioxidants.  This fact is being missed especially by supplement manufacturers who promote green tea, grape seed, goji berries and so on based on in vitro data that is clinically irrelevant.

As Christine Houghton points out, the relative value of in vivo vs in vitro studies depends on what you are trying to find out.

If you just want to rank substances according to activity in a particular in vitro test, then go ahead. Compare their activity to that of ascorbic acid, or glutathione, or NADPH, or whatever else you want to use as a comparator substance. But then ask yourself why do you want to do this?

Even a tissue culture system is not a good model for a living organism when it comes to antioxidant studies. It was discovered by the earliest pioneers of tissue culture that vitamin C added to tissue culture medium would kill cells. It was later worked out that this happened because cell culture medium contained traces of decompartmentalised iron sufficient to initiate autoxidation of vitamin C and thus generation of reactive oxygen species. This does not happen in a healthy functional living organism (e.g. man), except under inflammatory disease conditions, because free iron is kept at vanishingly low levels. This same process will occur when any substance with ascorbic-acid-like antioxidant activity is added to a tissue culture. But researchers who have no understanding of this phenomenon fail to control for this process by conducting their studies in the presence of, for example, catalase to remove autoxidatively-generated hydrogen peroxide.

The implication of what I have written above is that a good strategy for developing or discovering a new wonder antioxidant would be to find a substance that binds iron without rendering it "Fenton-reactive". No radical-scavenging type of in vitro assay will detect this kind of activity. And if you do not even understand what "Fenton-reactive" means, you need to look into this area of chemistry as a matter of some urgency!

And what would an in vitro or even an in silico system tell you about alk(en)yl catechols and alk(en)yl resorcinols? These are really active naturally-derived ascorbic-acid-like antioxidants. They have the added advantage that they have a vitamin E-like gross structure, so might be regarded as potentially useful lipid-phase antioxidants. But would you ever contemplate taking these substances orally as an antioxidant supplement or even for medicinal purposes? I think not ... unless you know nothing about poison ivy!

Like others have mentioned, very important question, Mahesha. Thanks for bringing it up. I also agree that in vivo and in vitro antioxidant tests do not really correlate. 

I'm more on the food chemistry side, in particular, fats and oil, and in increasing their oxidative stability using antioxidants, mostly from natural sources. For my use, I find that in vitro antioxidant tests  are a convenient screening option, they are fast, inexpensive, highly reproductible and ... relevant, in that their results correlate somewhat well with validation in food products. 

I am agree with Teresa Kowalska. As it is part of research and it gives immense idea about the antioxidant effect, how can we ignore that.

Dear Mahesha, Though by now, many scientists have already addressed your question. I also agree with the fact that the in vitro antioxidant studies and in vivo antioxidant studies will not correlate completely. If they do, then ofcourse in vitro antioxidants assays would have completely replaced all in vivo antioxidant studies.

But as per my opinion, in vitro antioxidant studies are not waste of money and time. These protocols atleast help researchers to filter out the best antioxidant molecules which can be sequentially assayed by in vivo means. Just think how many animals you can save out of it.

And if we talk about wastage of money and time, then I think we should do all studies directly on humans. This is because more than 90% of the animal (rat/mice/dog/monkey horse) based results doesn't correlate with the results obtained after administration in humans. That is why out of 10,000 molecules only 1 or 2 molecule successfully passed phase 4 of clinical trial. So in a word, I believe that these methodologies are important for us to atleast filter out the odd molecules.

In vitro antioxidant assay is a very expensive demonstration of not being pro-oxidant / toxic ?!

Dear Dr. Rajeev, 

Thanks for your answer. I completely agree with your answer. The fact which most of the researcher argue is the 'screening out' phase using in vitro method itself is inefficient and insignificant. Of course, almost all the molecules (organic) in the nature exhibit atlest a small level of antioxidant behavior in the given environment due to the nature of their chemical structure. 

But these in vitro assays does not take care of bioavailaiity and bioaccessibility issues. What if you loose a molecule which is TRULY antioxidant but excluded due to its low level antioxidant behaviour in vitro?

whats your suggestion?

Dear Mahesha

True, bioavailability and bioaccessability are 2 important aspects of say an antioxidant. Again the low-level has to be viewed from these 2 aspects also. If the molecule is lost during in-vitro tests, we need not worry about it, because there is very little chance that it will be available in-vivo when you consider the pharmacokinetic and pharmacodynamic aspects of it. It will remain obsecure in the animal body too. 

Dear Dr. Mahesha and Dr. Ghosh,

I agree with Dr. Mahesha and in disagreement with Dr. Ghosh. Dr. Ghosh, I guess Dr. Mahesha wants to point out the concept of prodrug here, where inactive drug becomes active after metabolism, so in those particular cases, in vitro shows it to be an inactive drug.

But Dr. Mahesha, this concept lies with all in vitro screening protocols, and not restricted only for antioxidant. But before selecting any procedure, we need to rationalize the risk/benefit ratio. I believe that judicial use of animals in drug discovery is much important, we can sacrifice some tentative best prodrugs, but not life of any organisms on the cost of inactive drug/prodrugs.

In vitro antioxidant is  really time consuming, but not time wasting.. there are no direct co-relation between in vitro antioxidant & in vivo antioxidant. without in vitro-antioxidant u cant find of  antioxidant  activity of any plant..There are 4 test  named 1. Reducing power 2.Total phenol 3. Flavenoids 4.DPPH in in-vitro antioxidant test.. on these test I got good antioxidant assay on some plant.. so I think that its not totally time & money wasting.

Dear Dr. Rajeev,

That's the point! You are absolutely right. That is what I was thinking! active-inactive mechanisms. We must design a confident method to screen these molecules/antioxidants. 

There was a time back in the 1970s when plants were being screened for phytosterols. It is possible to find many papers from that time describing the isolation and characterisation of phytosterols from one plant after another. It soon became evident that most if not all plants contain phytosterols and that it would actually be more interesting to discover a plant that does not contain these compounds. The journal Phytochemistry then stopped publishing detailed reports describing the isolation and characterisation of phytosterols even as Short Communications, being content with simple declarations (which were not subject to peer review) of the fact that a particular plant was found to contain a particular phytosterol.

A similar situation pertains with antioxidant compounds in plants. All plants contain antioxidant substances. It's not a big deal anymore. So, I return to an earlier point I made: why do you want to screen plant extracts for antioxidant substances?

If the answer is that you just want to find antioxidant substances in plants then you are, I fear, wasting research time and money.

If you are wanting to find a new wonder antioxidant for use in cosmetic products on the skin, you are again, I fear, wasting research time and money (see http://dx.doi.org/10.1111/j.1600-0536.2007.01146.x).

If you are wanting to find a new wonder antioxidant to be developed for use as a medicine, you will need to carry out in vivo studies (i.e. clinical trials) to demonstrate safety and efficacy, unless of course these are not yet required in the country where you would anticipate selling such a product. You are unlikely to be able to convince anyone with any business sense that this would be a good investment.

If you are wanting to find a new wonder antioxidant to be developed as a food supplement for the "worried well" (or do I mean "the gullible and dim-witted" ?) then you can use any in vitro assay you like.  But just make sure that the plant material you are screening is recognised as edible.

So, Mahesha, to what end purpose are you wanting to screen plant extracts for antioxidant substances? If you can tell us this, I think I and others following this interesting discussion will be able to better help you.

Dear Christine,

I was reading your interesting answer regarding Puling’s ideas and contentions on vitamin C. Dr. S.J. Padayatti (NIDDK, Clinical Center, NIH) is the first author of the papers on vitamin C bioavailability in humans that you are alluding to (Levine). Let us put their articles in perspective. Vitamin C saturation issue that you have mentioned applies to oral administration (see below) whereas the cancer treatment studies of Dr Cameron and Dr Pauling were primarily based on parenteral routes of administration of vitamin C.

In vivo animal studies have reported that hydrogen peroxide is selectively generated by pharmacologic concentrations of vitamin C in extracellular fluid around normal and tumor tissues, and that these concentrations of vitamin C retard tumor growth. Padayatti et al reported that such pharmacologic concentrations of vitamin C are achieved by parenteral administration in patients in a phase I clinical trial. 

Plasma vitamin C concentration rises sharply at oral doses between 30 and 100 mg/day and reaches a steady-state concentration at doses of 200 to 400 mg/day in healthy young adults.  Ingestion of vitamin C at doses up to 200 mg at a time results in one hundred percent absorption efficiency. When plasma ascorbic acid levels reach saturation excess vitamin C predominantly undergoes elimination in the urine. Parenteral administration of vitamin C, however, results in much greater plasma concentrations in comparison with that of oral dosing (Padayatti et al 2004). At the highest dose of 1.25 g, mean peak plasma vitamin C concentrations from intravenous administration were 6.6-fold greater than that obtained with the same dose by oral administration. Peak plasma vitamin C concentrations increased concomitantly with increasing intravenous doses. Padayatti et al (2004) documented that intravenous administration  of vitamin C to healthy young volunteers produced plasma concentrations 30- to 70-fold higher than that obtained with the maximum tolerated oral doses. The authors concluded that the efficacy of vitamin C treatment cannot be reliably assessed from clinical trials that use only oral dosing and opined that the role of vitamin C in cancer treatment needs reevaluation.

A few decades ago, Dr. Ewan Cameron, a surgeon, reported the effectiveness of oral and intravenous administrations of vitamin C, at doses of up to 10 g/day, in the treatment of cancer. In 1979, Cameron, Pauling and Leibowitz reported that they increased the time of survival of cancer patients and improved their quality of life by parenteral administration of high doses of vitamin C.  However, the outcome of such increase in survival time was not observed in double-blind placebo-controlled clinical trials when similar doses of vitamin C were orally administered.

It has now been shown that the route of administration causes major differences in the bioavailability of vitamin C. Intravenous administration of pharmacological concentrations were reported to be effective in cancer therapy while oral administrations were not.  Padayatti et al have advanced a plausible scientific rationale for the observed ineffectiveness of orally administered vitamin C in clinical trials, and have furnished a scientific basis for employing parenteral vitamin C in cancer treatment.  To wit, Linus Pauling would stand the test of time!

Padayatty SJ, Sun H, Wang Y, Riordan HD, Hewitt SM, Katz A, et al. (2004) Vitamin C pharmacokinetics: implications for oral and intravenous use. Ann Intern Med 140: 533–37.

Padayatty SJ, Sun AY, Chen Q, Espey MG, Drisko J, Levine M (2010) Vitamin C: Intravenous Use by Complementary and Alternative Medicine Practitioners and Adverse Effects. PLoS ONE 5(7): e11414. doi:10.1371/journal.pone.0011414

 Hoffer LJ, Levine M, Assouline S, Melnychuk D, Padayatty SJ, Rosadiuk K, et al. (2008) Phase I clinical trial of i.v. ascorbic acid in advanced malignancy. Ann Oncol 19: 1969–74.

 Padayatty SJ, Levine M (2000) Reevaluation of ascorbate in cancer treatment: emerging evidence, open minds and serendipity. J Am Coll Nutr 19: 423–25.

http://cancerres.aacrjournals.org/content/39/3/663.long  Ascorbic Acid and Cancer: A Review. Ewan Cameron, Linus Pauling, and Brian Leibovitz. Confer with #  56, 57, 60, 253.

The RDI of vitamin C for healthy adult males (above 19 years of age) is 90mg/day. In 1974, Dr Linus Pauling wrote that the (the then) RDA of vitamin C, 45 mg/d, was just sufficient to prevent scurvy, and therefore this RDA be named “Minimum Dietary Allowance”.  http://www.pnas.org/content/71/11/4442.full.pdf

As an aside, Dr. Linus Pauling (Nobel prizes for chemistry and for peace) is held as  one of the 20 greatest scientists of all time (New Scientist magazine). Professor Pauling used the term, “molecular medicine” in 1949, in his epochal paper in "Science" on the mechanism of production of sickle cell anemia. This article formulated that the origin of disease was influenced by specific mutations of genes, which could manifest an altered "molecular environment". Consequently, modified physiological functions associate with specific diseases. Pauling L, Itano HA. et al. Sickle cell anemia: a molecular disease. Science 1949; 110:543-548:

"Molecular Architecture and the Processes of Life." May 28, 1948

http://scarc.library.oregonstate.edu/coll/pauling/dna/papers/1948p.13-01.html

Chithan: You assert that "In vivo animal studies reported that hydrogen peroxide was selectively generated by pharmacologic concentrations of vitamin C in extracellular fluid around normal and tumor tissues; such concentrations of vitamin C retarded tumor growth."  Could you please identify the studies (or do you mean study?) to which you are referring?

Dear Richard,

What I meant was that "animal studies indicated that ..." I can modify that sentence.

Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR, et al. (2005) Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl Acad Sci U S A 102: 13604–9.

 Chen Q, Espey MG, Sun AY, Lee JH, Krishna MC, Shacter E, et al. (2007) Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo. Proc Natl Acad Sci U S A 104: 8749–54.

Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, Krishna MC, et al. (2008) Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc Natl Acad Sci U S A 105: 11105–9.

Thank you!

I published a paper many years ago showing that ascorbic acid added to a tissue culture would autoxidise to produce reactive oxygen species including hydrogen peroxide [JBiomedMaterialsResearch 27(4): 521-530 (1993); http://dx.doi.org/10.1002/jbm.820270413].  And I have also published a paper describing the pro- and anti-proliferant effects of hydrogen peroxide in tissue culture [Schmidt R J, Chung L Y, Andrews A M and Turner T D — Hydrogen peroxide is a murine (L929) fibroblast cell proliferant at micro- to nanomolar concentrations, Proceedings of the 1st European Conference on Advances in Wound Management (Editors Harding K G, Leaper D L and Turner T D). Macmillan Magazines, London : 117-120 (1992) ; ISBN 0-333-58645-X ].  These weren't entirely new observations even then. And this hydrogen-peroxide-generating effect is mediated by catalytic quantities of iron, which are to be found in tissue culture medium, but not in normal healthy tissue in vivo.  I haven't yet looked at the paper by Chen et al. 2007 that you cite, but I wonder exactly how the detection of hydrogen peroxide "in extracellular fluid in vivo" was carried out.  Perhaps large doses of ascorbic acid do cause release of (catalytically active) iron from (catalytically inactive) iron stores (perhaps transferrin) ... see Richardson DR (1999) http://dx.doi.org/10.1016/S0022-2143(99)90166-X.  Much will depend on exactly how much hydrogen peroxide is generated in this way.  Low concentrations are pro-proliferant; higher doses are anti-proliferant ("hydrogen peroxide induced senescence"); higher doses still will cause apoptosis.

Dear Mahesha,

Antioxidant activity is a chemical/molecular property of an agent. A compound can be said to possess antioxidant activity. In the midst of the almost exponential growth of substances that have such activity, it may turn out that many compounds that are indiscriminately termed as antioxidants today were studied as natural products in the early to mid 20th century.

The story of naturally occurring molecules with antioxidant capacity goes back to the 1930s’ – back to the pioneering work of Albert Szent-Györgyi. In 1936, Rusznyák and Szent-Györgyi showed that two flavonoids derived from citrus fruits decreased capillary fragility and permeability in humans, which is related to free radical biology. Rusznyák S, Szent-Györgyi, A. Vitamin nature of flavones. Nature 1936; 138:798

Oxidation, Energy Transfer, and Vitamins. The Nobel Prize in Physiology or Medicine 1937. Albert Szent-Györgyi, MD, PhD

http://www.nobelprize.org/nobel_prizes/medicine/laureates/1937/szent-gyorgyi-lecture.pdf

In addition to hydrogen peroxide mediated effects, other mechanisms have also been considered that might increase the sensitivity of cancer cells to vitamin C- induced toxicity; these include the hypoxia-inducible factor and the modification of oxidized GADPH. 

Dear Mahesha,

Thank you for your interesting and intriguing question. You have recently participated in two conferences in Europe, and the highlights projected by researchers out there are tantamount to the (may be sheer) lack of any use of in vitro antioxidant studies, and that these studies do not delineate any “real significance; “they have argued that the results will never have any significant correlation with in vivo studies.” Such a quandary is well known (the “antioxidant paradox").  One of your conferences is related to foods and the other is related to lipids, fats and oils, we presume. Were the arguments articulated therein largely confined to 1) food and 2) lipid and fat oxidation, or did they extend to other realms?

In animal studies, the biological effects of natural antioxidants, for instance, dietary flavonoids, are exerted at supra physiological (pharmacologic) doses, which are realistically not achievable in humans by ingestion. At such extreme concentrations, the observed effects may be highly nonspecific and may be owing to, in part, genomic effects. An association between antioxidant intake and the possible prevention of cancer has been inferred based on population studies (retrospective, prospective, cross sectional, case-control, cohort etc.). However, no human studies have unequivocally and convincingly established that consuming antioxidant supplements can help mitigate the risk of cancer. In fact, certain studies have even documented an accentuated risk of some cancers, depending upon the time of administration of the test substance and patient history (prior smoking for instance). Accrued evidence centered on the use of vitamin and mineral supplements for the prevention of chronic diseases, including cancer, conducted for the United States Preventive Services Task Force (USPSTF) likewise found no clear evidence of benefit in preventing cancer.

It is worth remembering that in clinical studies, wherein there was a lack of benefit, the tested antioxidants were largely consumed as pure chemical moieties in contrast to when they are ingested in food, which contains complex mixtures of antioxidants, vitamins, phytochemicals and minerals. Several scientists and physicians have criticized these studies; they opined that these trials have had major flaws relating to poor study design, insufficient comprehension of science behind oxidants and antioxidants and inadequacy of appropriate patient cohorts for testing.  A serious and critical issue with such trials appears to be the latency period; the duration of these trials was generally 8-10 years. Latency period for cancer development in humans may be about 25-30 years or even longer. 

The positive results obtained from a series of in vitro studies such as the inhibition of LDL oxidation in macrophages and other systems) by nutritional antioxidants were not seen in clinical trials involving coronary artery disease.

Several human intervention trials have failed to corroborate the ascribed roles of the traditional dietary antioxidants such as ascorbic acid, tocopherols, and carotenoids, etc. While thousands of bioactive constituents with presumptive antioxidant activity are elaborated by vascular plants (antioxidants) the compounds (recognized as natural/dietary antioxidants) tested in these clinical trials probably number half a dozen.  During the course of evolution, plants have attained the possibility to elaborate a bewildering array of molecules with antioxidant potential, which can possibly confer protection upon plants (a well-known example is photoreception/protection of plant DNA from ultraviolet rays from the sun). For instance, as of the year 2000, more than 8,500 distinct individual flavonoid structures have been identified in vascular plants; the total number of flavonoid molecule that could occur in nature might be even greater. This sounds like a legion! Hundreds of flavonoids can be present in a single plant food item, whereas a test study focuses on an individual moiety.   

Let us discuss the in vivo significance of in vitro findings gleaned from studies on the potential role of antioxidants in cancer prevention. The prevention of cancer caused by chemicals (chemical carcinogenesis) by the intervention of chemical entities is known as chemoprevention. The issue regarding antioxidants and cancer has two aspects: one is prevention while the other is therapy (a therapeutic benefit; antineoplastic/antitumor effects).  Intervention consisting of consumption of substances exhibiting antioxidant function is purported to prevent oxidative damage. Oxidative stress, chronic inflammation and cancer appear to be interrelated. The exposure of antioxidants to individuals who already possess prolonged or chronic inflammation (example cigarette smokers) can be counterproductive. In this case, antioxidant consumption in trials can increase the incidence of cancer.

The cancer preventive potential of dietary fruits and vegetables has emerged from association studies. More than 175 population studies have amply highlighted the cancer preventative effects of vegetables and fruit consumption; the benefit may be owing to varied antioxidants in a network, non- antioxidant nutrients and minerals, functioning in a judicious, interrelated way, in a natural milieu. Thus, we already have a potential and prudent (and physiological) solution to the prevention of cancer. However, this has not been replicated in antioxidant intervention trials with single antioxidants (or two in certain instances), which is most disappointing.

I would suggest that the chemopreventive effect of plant foods is more likely to be related to the subtle signalling by weakly pro-oxidant compounds that activate  transcription factors such as Nrf2 as a means up upregulating the endogenous defences.  Antioxidant vitamins in supraphysiological doses typically mask such signals.  As a result, increased expression of cytoprotective genes does not occur.

Dear All,

Let us be more sincere and frankly admit that for the time being, our up-to-the-date and seemingly sharp modern tools of analytical chemistry (e.g., chromatographies, MS, 1H and 13C NMR, etc.), no matter how sophisticated they seem to us, are still by far insufficient to reliably trace subtle physiological effects. They are still neither sensitive enough to directly operate on physiological levels, nor fast enough (to provide the results with a kinetic or at least semi-kinetic importance), nor both. Moreover, the chemistries of amino acids, peptides and proteins as well as the chemistry of sugars (both classes belonging to the so-called primary metabolites) are full of still unanswered question marks. Consequently, our ideas about physiological roles of secondary metabolites in many cases are nothing more than wishful thinking.

To this effect, interpretation of the results of the in vivo experiments acts more like (often unfounded, sorry about that!) speculations rather than a hundred percent documented and reliable truth. Just due to that, an occasional superiority air in our present public discussion, when referring to the in vivo results (in comparison with those obtained in vitro) seems to me largely unfounded.

My understanding of the discussion subject matter is such: Sharpness of our analytical tools just allows to obtain more-less reliable in vitro results and for the time being, let us be happy with this. It is rather impossible that no relationships exist between antioxidant effects observed in vivo and in vitro, but for obvious reasons and in the first instance due to the aforementioned instrumental shortcomings, strict algorithms of these relationships still remain obscure.

So that instead (openly or less openly) disregarding the in vitro results and discouraging young scientists like Mahesha from collecting them, let us encourage building huge databases on antioxidant properties of as many as possible representatives of the kingdom of plants and peaceful wait for REAL elucidation of the in vitro / in vivo dependences.

One is not entirely sure if Mahesha's intent is to undertake an in vitro assay? In the third  segment of his question he is seeking opinions as to the need to stop doing such studies. The first part of his well formulated question is on his impressions gathered from his conference attendance (perspectives), and the the second concerns broad observations and his impression. 

The difference between an in vitro assay and an in vivo situation is that linear dynamics are observed in the former (i.e. predictability) whereas non-linear dynamics are observed in the latter (i.e. "chaos theory" applies).

In any system where there are more than two co-existing processes and where what happens in one process affects the other, chaotic behaviour will occur. Living organisms, like populations, like ecosystems, like the weather, like the economy, are "fundamentally complex" chaotic systems. Therefore, outcomes cannot be predicted with certainty unless one has an infinite dataset. Even rounding errors at the 10th (or 1000th or 10000th) decimal place in a mathematical calculation will affect outcomes downstream in a chaotic system. This is why we will never be able to predict the weather in anything more than general terms more than about 5 days in advance. And whilst politicians tell us that global warming will cause the world to warm up, chaos theory tells us that as global temperature rises, it will reach a bifurcation point where the temperature will either start to increase further and the whole world will turn tropical or start to decrease and we will enter a new ice age. Because of the unpredicatability of chaotic systems, we cannot know which way the weather will turn, or indeed when this will happen if at all.

So Teresa's suggestion that we should encourage the creation of a huge database on antioxidant properties clearly disregards what is already known about chaotic systems. A huge database would still not be huge enough. It would have to be infinitely huge!

We cannot model in vitro the chaotic system that is our pro-oxidant / anti-oxidant balance in vivo. Even when testing the effect of one isolated substance in an in vivo system, e.g. in a clinical trial, outcomes are unreliable because we are always constrained by the fact that we cannot test the substance in an infinite number of subjects. That is why we never rely on the results from one clinical trial but endeavour instead to carry out a meta analysis of the results from many similar trials.

The bottom line is that if we observe in a clinical trial that apples are good for you, then we should simply advise people to eat apples. The belief that we have to take apples apart to see if we can find an "active ingredient" has evolved from the notion that "there is a pill for every ill". But the more you know about "pills", the more you realise that they don't actually fix very much at all. There is now more illness in western societies than there ever has been. Yes, we are living longer. But all the "pills" are doing is to help us rattle on for longer with an increasing burden of [often self-inflicted] illness. Prevention of illness is what we should be striving to do. Eat more apples!

There are lots of books and articles on chaos theory. See for example, https://en.wikipedia.org/wiki/Chaos_theory and http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2465602/

Dear Dr. Chithan and Ruchard, 

Thank you for your excellent feedback. 

I have spent a lot of time and money on in vitro antioxidant assays during the last few years. Meantime, I can still find that 1000 articles online regarding in vitro antioxidant assays of plant extracts and food stuffs. I really do have my plans to conduct in vitro antioxidant assays of my food samples during the next few months.

Once I heard the statements as 'in vitro antioxidant assays are really a waste of time and money', I just wanted to have opinions of researchers across the globe. The statement was given by a well known researcher from the University of California, Davis. Surprisingly, the same statements were repeated by two researchers from Italy in a different conference. Of course, these two conferences were held on food and lipid aspects. 

My concern is, if it is really NOT useful, why we have to waste our time and money? Indeed, in vitro studies are not that inexpensive. 

I am really happy to see excellent comments from researchers. But you may see that still people are in a dilemma! 

Scientists develop hypotheses then design experiments to test those hypotheses.  That is how scientific understanding progresses.  What you try to do when designing an experiment is to disprove your hypothesis.  Only when you and others fail to disprove the hypothesis will it become established.  So, what hypothesis are you testing?

If your hypothesis is that foods contain antioxidants, that is established.

If your hypothesis is that there is more antioxidant activity in one plant extract than in another, you just need to carry out an in vitro assay to demonstrate that.  But why do you want to know this?  Are you looking for a source of a natural antioxidant for a particular application?  What are you trying to discover?  What hypothesis are you trying to test?

What the discussion above is telling you is that if you are hoping to find a new wonder antioxidant that will prevent or cure cancer, you are wasting your time and money.  These sorts of discoveries are usually made serendipitously, not by rational scientists designing rational experiments.  Books on the history of scientific discoveries make interesting reading.

Dear Richard ,

The last paragraph your answer actually gave the answer to my question and doubts :) 

I am looking to extract and isolate some novel antioxidants! 

Being an antioxidant is a relative thing.  It just means that one compound in a particular chemical environment will oxidise preferentially and thereby protect another from the same oxidising event.  For hydrogen atom abstraction reactions (such as those driven by DPPH), the order of play is determined by the "bond dissociation energy". Phenolic, allylic, and benzylic hydrogen atoms, for example, have quite low bond dissociation energies.  So compounds containing these functional groups will behave as antioxidants in the DPPH assay. The CRC Handbook of Chemistry and Physics (http://www.hbcpnetbase.com/) contains tables of bond dissociation energies.

But what matters in vivo is whether the free radical species produced by the antioxidant  does or does not go on to do other potentially harmful things as it integrates itself into the chaotic processes of the pro-oxidant / anti-oxidant balance.

Being an antioxidant is a relative thing.  It just means that one compound in a particular chemical environment will oxidise preferentially and thereby protect another from the same oxidising event.  For hydrogen atom abstraction reactions (such as those driven by DPPH), the order of play is determined by the "bond dissociation energy". Phenolic, allylic, and benzylic hydrogen atoms, for example, have quite low bond dissociation energies.  So compounds containing these functional groups will behave as antioxidants in the DPPH assay. The CRC Handbook of Chemistry and Physics (http://www.hbcpnetbase.com/) contains tables of bond dissociation energies.

But what matters in vivo is whether the free radical species produced by the antioxidant  does or does not go on to do other potentially harmful things as it integrates itself into the chaotic processes of the pro-oxidant / anti-oxidant balance.

Yes, now I am clear. So we can go for in vitro studies to generate a comparative data. But in vivo gives the true relevance of the antioxidant. 

It has been an interesting discussion.

Dear Mahesha,

Thank you for your lucid answer!

The issue of the plausible significance of in vitro study results in connection with the outcomes of clinical trials of cancer and coronary artery disease was earlier brought up here just to indicate the aspect of correlation. Such is the capricious nature of this issue. This is the aftermath of your question (the first paragraph on "significance of in vitro results"). The answers touching upon the outcomes of these clinical trials are in no way intended to discourage or dissuade you.

In vitro studies generally can be primarily useful in exploratory work. Such studies may yield certain pointers, provide some general framework, on which further work may be judiciously predicated, and may yield certain important benchmarks. The results could also be useful in studying drug design. In vitro studies on potential pharmacologic activities of single plant-derived molecules and plant extracts have been numerous, and are continuing at a faster pace.

Earlier, Dr. Rajeev Singla has provided an excellent answer to your question. In particular, studies using experimental animals have been exponential. But most of the results find no correlation with data obtained in humans, as Dr. Singla pointed out. In this context, animal models have been extensively employed in studies on carcinogenesis and coronary artery disease (atherosclerosis). Overwhelming evidence for animal carcinogenicity has been obtained with certain procarcinogens (for example 7,12-dimethylbenz[a]anthracene, an angular polynuclear aromatic hydrocarbon). But we cannot administer these procarcinogens (molecules that require metabolic activation) to humans and experiment with them. In vitro pharmacological studies constitute a crucial part in the assessment of efficacy, proof of concept, etc.

In vitro studies showed that cromolyn (disodium cromoglycate, a chromone which shares the first two rings [phenylchromone] of the flavonoid, quercetin), a drug used for preventing the symptoms of asthma, inhibited the assembly of an active NADPH oxidase and prevented oxygen radical generation (and oxygen radical induced tissue damage) in neutrophils.  Another study, however, described the exacerbation of ascorbate-ADP initiated lipid peroxidation in washed rat liver microsomes by cromolyn. Such inconsistencies are sometimes observed .

Dear Mahesha,

The following (you would have read this) is an intriguing!

What scientific idea is ready for retirement?

According to Dr Azra Raza (see link below), the mouse models of cancer (animal models and billions!).

http://www.theguardian.com/science/2014/jan/12/what-scientific-idea-is-ready-for-retirement-edge-org   

Bear in mind that when contemplating potential effects of radical-scavenging antioxidants on the body, we are not deaing with "drug-receptor" interactions.  We are just dealing with chemistry in a very complex chemical environment.

It is interesting discussion!

@ Dr. Chitan and Dr. Richard,

I absolutely agree with your comments. So we may conclude like, in vitro antioxidant study is more dependent on the "chemistry" of the antioxidant molecule and. But in vivo studies gives an idea on its "biology" or "chemical biology"

The in vitro antioxidant study of herbal material seems an important characteristics of a plant, and yet phytochemists do not seem serious enough about an unequivocal assessment of this characteristics, so that the comparisons among different plants and different laboratories are hardly possible. As if chemists / biochemists / phytochemists seriously avoided any "confrontation" on this important subject matter (to later falsely claim that such investigations are a waste of time and money). More info to this effect is contained in the attached two papers.

Very interesting discussion

Please if someone can answer this question , it will help me

Thank you

https://www.researchgate.net/post/What_about_alpha_amylase_concentration_for_the_enzyme_inhibition_test#56463d1b5cd9e3b6308b45d8

Teresa:

You are clearly demonstrating in a sophisticated way that different extracts from many different Thymus specimens and species exhibit different levels of antioxidant activity in a particular in vitro assay.  However, these results could not meaningfully be compared in the same way with results from, say, a selection of Salvia species, or from various parts of Silybum marianum because these different plants contain different families of antioxidant molecules.

You surely have to have an end-use in mind as you endeavour to discover which part of which Thymus species collected at which time of the year provides the best antioxidant effect.  So, how do you think you can now apply the antioxidant activity you have found?  I can imagine a use as a natural antioxidant in foods.  But vitamin C and vitamin E already do a good job in this application.

And do your in vitro results predict in any way the likely effects of these extracts if administered in a standard way to a "standard human"?  I think not.

This surely answers Mahesha's original question?

Respected Researchers,

I would also like to add a notion to the ongoing discussion. In vitro antioxidant assays usually never extrapolates the same findings at in vivo level. But they are clearly an indicator of drug standardization. The values of in vitro antioxidant potential can also be linked indirectly to the shelf life of the said extracts. And in case of batch-to-batch variation control, in vitro antioxidant assays might also play an important role as one can standardize a given extract on the basis of its antioxidant fingerprints and whenever the drug is made for the second batch, these antioxidant fingerprint profile can be matched up and if found in standardized range, then it definitely provides an indication that the prepared extract is homologous to the previous batch, without any significant variation. 

Nice, totally agreed!!!

Thank you Teresa Kowalska for sharing a common thought. :)

Dear Matthew,

Theoretical arguments appear to have constituted a marked basis for framing Dr. Linus Pauling’s recommendations for vitamin C. Finalization of recommendations appears to be predicated on cross-species comparisons and evaluation, evolution centered incisive deliberations and arguments, and insights gleaned thereof, the concept of biochemical individuality, and the anticipated quantity of ascorbate that would be ingested in diets from raw plant foods. Pauling estimated that goat the weight of a man makes about 800 mg of ascorbate a day. The suggested high dose of ascorbate was to be consumed in portions of 3 or 4 equal doses (usually up to 2 g per dose), during the course of the day, and not the ingestion of a high quantity such as 10 g in a single dose. These higher amounts, a bolus, were parenterally administered to cancer patients. A minimum optimal daily intake of about 2 g of ascorbate was recommended with the insistence of a minimal concentration to the tune of at least 200-250 mg/day for each individual. He is said to have remarked (cf Linus Pauling Institute) in one of his radio interviews in 1974 that “the first 250 mg is more important than any later 250 mg. The first 250 mg leads you up to the level where the blood is saturated. You can achieve a higher volume in the blood by a larger intake, but you get much better improvement for the first 250 mg than for additional grams.” This is consistent with the complete absorption of a dose of 200 mg of ascorbate in humans as reported years later. Pauling argued for a higher RDA of ascorbate in 1974, and wrote that the (the then) RDA of vitamin C, 45 mg/d, was merely sufficient to prevent scurvy, and therefore this RDA be named “Minimum Dietary Allowance”.

The  National Cancer Institute (NCI) had funded studies on the possible role of high dose ascorbate in the treatment of cancer patients at the Mayo clinic; the route of ascorbate administration was pro-oral. Negative results obtained in this trial were eventually used (predominantly in a negative way) to discredit the findings of Cameron and Pauling (and to disparage Professor Pauling) who had used parenteral administration of ascorbate and adduced substantive evidence for potential benefits. Considering retrospectively, discrepant results obtained on the effect of vitamin C in cancer patients appear to be owing to differences in the routes of administration of ascorbate, based on insights that might be gleaned from more recent studies. Padayatti et al (2004) in Bethesda, MD (NIDDK, Clinical Center, NIH),  reported pharmacologic plasma concentrations of ascorbate following parenteral administration of the vitamin (confer with posted information in this tread).   

Dear Chithan-

This has been a most popular thread! As a graduate of Oregon State (Dr. Pauling's alma mater) and as former postdoc at NCI, I really appreciate your comments above. I hope that you, as well as my former students, will forgive my off-the-top-of-my-head hyperbole regarding Vitamin C dosage. I am happy for whatever small contribution I was able to make to my students' liberal arts education and to this conversation.

Good health to you!

MB

Dear Matthew,

Thank you! I am deeply touched by your graceful comments with humility. I did not perceive your answer here on vitamin C dosage in the way that you have now expressed; in fact, I must say that your contribution provided impetus for my answer. I also concur with you on the issue of food derived antioxidant substances. After reading your earlier comment on the dose of vitamin C, I revisited Dr. Pauling's reviews and Oregon State archives. I think that you are fully correct about the vitamin C dose supported by Dr. Pauling. It seems  that he had raised the issue of what happens to a major part of ingested ascorbate in view of the lack of 100% elimination in urine of unabsorbed ascorbate. The notion that excess ascorbate (unabsorbed) would be eliminated through the kidneys did not satisfy Dr. Pauling, it would seem! He is regarded to have ingested large doses of ascorbate and estimated its elimination!!

It is wonderful to learn that you are a graduate of Oregon State. It must indeed be gratifying! Oregon State appears to loom “larger than life” with the indelible aura of Dr. Pauling, one of the founders of modern day chemistry, and the “father of molecular biology” (per Dr. F.HC. Crick).  Dr. Jacques Monod (of Jacob and Monod fame) in 1960 emphasized that sickle cell anemia is simultaneously genetic and molecular, and “is a milestone in the development of our discipline.” ‘‘From now on,’’ he wrote, ‘‘some of the most important problems of pathology fall under the jurisdiction of Molecular Biology”, the new discipline. Pauling was acknowledged as the “father of molecular biology” by Dr. F.H.C. Crick; his discovery of sickle cell anemia in 1949 as a “molecular disease” opened the way toward examining genetically acquired mutations at a molecular level.  Pauling’s findings on sickle cell anemia led to the reality that a protein bore a relationship to a single gene and to the delineation of a functional gene with its one-to-one relationship of gene to protein.  Pauling L, Itano HA, Singer SJ, Wells IC. Sickle cell anemia: a molecular disease. Science 1949; 110:543-548.

Pauling was included in the list of the 20 greatest scientists of all time by the magazine, New Scientist, with Albert Einstein being the only other scientist from the twentieth century on the list. Dr Gautam R. Desiraju, the author of the Millennium Essay in the journal, Nature, characterized Pauling as one of the greatest thinkers and visionaries of the millennium, along with Galileo, Newton and Einstein.  He is considered the greatest chemist since Lavoisier.

I would be glad to share with you later an episode of  a lecture by Dr. Pauling at a cancer conference in the U.S. when he was 91 or 92, at which juncture the story of theoretical considerations, cross-species comparison and evolutionary arguments related to the development of a recommended everyday dose of vitamin C for every individual unfolded.  

Dr.  Pauling used the term, “molecular medicine” in 1949. He had emphasized that dietary requirements of vitamin C and of other B vitamins may vary among individuals and highly stressed biochemical individuality.  In 1956, Dr. Roger Williams, a pioneer in vitamins and nutrition often credited with popularizing the term “biochemical individuality,” wrote the book, “Biochemical Individuality: The Basis for the Genetotrophic Concept “(McGraw-Hill, 1998). Williams postulated that requirements for nutrients differ widely in different individuals, and that some persons may require much higher amounts of certain nutrients than others do for appropriate and optimal functioning, based upon their unique biochemistry. He wrote, “Individuality in nutritional needs is the basis for the genetotrophic approach and for the belief that nutrition applied with due concern for individual genetic variations, which may be large, offers the solution to many baffling health problems.”

The potential of ascorbate in combating tissue oxidative damage is well known. Oxidative damage has been implicated in connective tissue disorders. Pauling advanced the potential role of ascorbate in the maintenance of connective tissue architecture (intercellular matrix) and  assessed vitamin C in relation to glycosaminoglycans or proteoglycans, collagen matrix and prolyl hydroxylase [2-oxoglutarate-dependent dioxygenase].  We now hear much  about HIF (hypoxia inducing factor) and HIF-prolyl hydroxylase in cancer cells. Dr. Pauling  placed a great deal of emphasis on vitamin C and the modifications of L-lysine and proline residues in collagen. Hydroxy-lysine and hydroxy-proline occur in tissue collagen post-translationally, and these modified amino acids located in the helical domain of collagen become glycosylated. Delays in triple helix folding of collagen result in over modification of lysine residues by excessive hydroxylation and the over glycosylation of the resulting hydroxy-lysine residues. These over modifications perturb the triple helical structure of collagen (the famed coiled coil structure of collagen, worked out by Drs. G.N. Ramachandran and G. Kartha)  and contribute to the severity of osteogenesis imperfecta, a heritable connective tissue disorder.

Ascorbate has been considered to inhibit oxidative damage in the development of atherosclerosis. G.C. Willis in Montreal in 1953 reported that parenteral ascorbate was effective in inhibiting atherosclerosis of cholesterol feeding in guinea pigs.  Scurvy can be developed only in a few animals, and guinea pig is one of them; guinea pig is suitable for atherosclerosis studies. Ascorbate deficiency in guinea pigs leads to atherosclerosis, irrespective of whether the scurvy is acute or chronic. Atherosclerosis of scurvy occurred at normal cholesterol levels and without the deposit of lipid in the reticuloendothelial system. This form of the diseases is considered to closely simulate the human form of the disease. Parenteral ascorbate greatly   retards atherosclerosis produced by cholesterol feeding in guinea pigs. It was concluded that massive doses of parenteral ascorbate might be of therapeutic value in the treatment of atherosclerosis and in the prevention of thrombosis.    G. C. Willis, M.D., Montreal. AN EXPERIMENTAL STUDY OF THE INTIMAL GROUND SUBSTANCE IN ATHEROSCLEROSIS. Can Med Assoc J.1953; 69: 17–22.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1822858/pdf/canmedaj00682-0019.pdf

Very best...

CK

Hello Dear researchers

I totally agree with Mrs Teresa Kowalska. Mr. Richard Schmidt is very correct when he said vitamin C is already doing every thing better as antioxidant molecule. But from there to say that "adding random new antioxidant wonder-molecules to our diet is probably a bad thing" is not correct for me because we (human beings) are eating those molecules from our daily diet for several centuries. So I thing the best statement could have been "adding random SYNTHETIC antioxidant wonder-molecules to our diet is probably a bad thing. Thus coming back to the question, I thing researchers should continue checking for in vitro antioxidant activity (vitamin C equivalent) of natural products because this could eventually leads us to the discovery of a new natural vitamin C-like molecule from our wonderful nature.

Kind Regards!

NOBOSSE

Hi,

The molecules do not need to be synthetic to be toxic, the most efficienty poisons known to man are natural. It is really annoying to see this claim of "natural is all good" in a scientific discussion. 

About antioxidants there has been an number of clinical assays using natural vitamin antioxidants that have had to be stopped before the planned term because of increased mortality of participants, see e.g. J Natl Cancer Inst 1996;88:1550–9 or 2004; 96: 1729-1731.or JAMA 2003;290:476–85.

There is most often no positive effect demonstrated of the antioxidant supplements,  frequently increased risks, and exceptionally benefits (eye health). What is beneficial is eating an balanced diet with a lot of fruit and vegetables, not "antioxidants". The NIH has a good summary with relevant references: https://nccih.nih.gov/health/antioxidants/introduction.htm. Many researchers, including some of the ealry proponents of antioxidants, are now publishined articles and reviews reconsidering the antioxidants, see Critical reviews in food science and nutrition 2012:52:162 -171 or Nutrition 2014 :30: 791-793

Hi,

It is well known that the molecules do not need to be synthetic to be toxic as "all substances are poisons. There is none which is not a poison; the right dose differentiates a poison and a remedy" (Paracelsus, 1493 - 1541). But could a "safe" vitamin C from a fruit spontaneously become "toxic" just because it has been extracted, concentrated and administrated at the right dose? I think no!

The studies #J Natl Cancer Inst 1996;88:1550–9 and 2004; 96: 1729-1731 were carried out not with "natural vitamin antioxidants" !!! but with beta-carotene, retinyl palmitate and retinol manufactured by Hof... So I would like to see the results if  a concentrated fraction of carotenoids from carrots for example have been used.

In the field of antioxidants, the fact is that intake of dietary antioxidants from foods improve health status; but to check how this occurs, researchers leave aside those dietary antioxidants and use pure synthesized compounds from industry; then come back and say "this does not work".

So, rather than discredit this field of research, we should try to standardize protocols and then start large scale studies USING DIETARY ANTIOXIDANTS.

Regards!

NOBOSSE.

The following may be of historical interest.

Stich et al, in 1976, published a paper in the journal, Nature, on the mutagenicity of ascorbic acid.   

Stich HF,  Karim J, Koropatnick J, Lo L (1976) Mutagenic action of ascorbic acid. Nature (London) 260:722–724.

They indicated that ascorbic acid metabolites can be mutagenic for mammalian cells.  Ascorbic acid and metal complexes were also assessed (the prooxidant effect of such complexes is obvious).   An increased production of ascorbic acid metabolites could be a key factor in aging, according to the intrinsic mutagenesis theory of aging (Sir Frank Macfarlane Burnet, The Nobel Prize in Physiology or Medicine 1960).  

Burnet, M (1974) Concepts of autoimmune disease and their implications for therapy. Perspect Biol Med 10:141–151.

http://muse.jhu.edu/login?auth=0&type=summary&url=/journals/perspectives_in_biology_and_medicine/v010/10.2.burnet.html

Testing the mutagenic action of substances in order to identify potential procarcinogens and carcinogens had exploded following the development of (the Ames test) the Salmonella mutagensesis test (detection of carcinogens as mutagens in the Salmonella/microsome test) by Ames et al and McCann and Ames (1973-1976).  The number of tested compounds has been momentous, including, perhaps, a bewildering array of environmental chemicals, occupational hazards and natural products (including food components and xenobiotics). 

The extraordinary and preeminent safety of ascorbate in humans has been well known.

Mutagenicity of antioxidants such as ascorbic acid, quercetin, and scores of other flavonoids (see http://dx.doi.org/10.1016/0165-1161(78)90020-1) is almost certainly caused by their propensity to auto-oxidise in in vitro systems.  And that in turn happens because it is extremely difficult to produce an in vitro system that does not contain de-compartmentalised iron, this iron acting as a Fenton catalyst in the generation of reactive oxygen species.  It is these reactive oxygen species that are [probably] the mutagens. This kind of reaction only occurs in vivo where there is tissue damage and inflammation, and explains why chronic inflammation associated with tissue injury can lead to tumour formation in a clinical situation.

Yes, these auto-oxidation reactions can occur with a "bewildering array of environmental chemicals", and this is because these chemicals typically contain hydrogen atoms with low bond-dissociation energy, i.e. they can act as antioxidants.  As I pointed out in my very first contribution to this discussion (see above), just about anything and everything can be shown to have antioxidant activity if you choose an appropriate test method.  So, readers should be able to deduce that in vitro mutagenic activity is probably a predictable feature of any free-radical scavenging antioxidant.  The surprising thing to me is that those who carry out such tests do not seem to undertstand this simple "Fenton chemistry".

The aspect of mutagenicity (paper by H. Stich et al on ascorbate and mammalian mutagenicity) was merely mentioned here in light of Dr. Nobosse’s comments above (Paracelsus and the issue of toxicity). I was not trying to make a point on mutagenicity here.

It is known that flavonoids possessing vicinal hydroxyl groups can autoxidize in aqueous media at biologically relevant pH and radicals subsequently generated can cause strand scission of DNA. This could explain the observed effects of these compounds on the frequency of chromosomal aberrations caused in cultured cells exposed to these flavonoid constituents. Bacterial mutagenicity studies on plant flavonoids, which have generally employed pharmacologic concentrations of the compounds, are known to have yielded highly complex, inconsistent and contradictory results. Certain studies have reported weak mutagenic activity (or minimal mutagenic activity) of some (hydroxylated) flavonoids. In the investigations of MacGregor and Jurd, and of MacGregor and Wilson, the same flavonoid entity showed different (differential) mutagenic effects in different tester strains of Salmonella typhimurium. The mutagenic response was reported to be  greatly dependent on the absence of excision repair and the presence a certain plasmid. Other authors have reported the mutagenic effects of different flavonoids tested in Salmonella typhimurium strains, TA 98, TA 100 and TA 102; quercetin was mutagenic only in TA 98. Taxifolin (dihydroquercetin, a pentahydroxy compound), a flavonone in which the middle ring, the heterocyclic C-ring of the flavone, lacks a 2,3-double bond (saturation of this bond) was only weakly mutagenic in tests with Salmonella typhimurium strains. Taxifolin possesses vicinal hydroxyl groups (amenable for autoxidation and redox cycling) in its B-ring (3’-4’). At lower concentrations, flavonoids showed no mutagenicity in some studies.

MacGregor JT, Wilson RE. Flavone mutagenicity in Salmonella typhimurium:dependence on the pKM101 plasmid and excision-repair deficiency. Environ MolMutagen. 1988;11(3):315-22. PubMed PMID: 3281825.  MacGregor JT, Jurd L. Mutagenicity of plant flavonoids:structural requirements for mutagenic activity in Salmonella typhimurium. Mutat Res. 1978 Dec;54(3):297-309. PubMed PMID: 368618. 

http://dx.doi.org/10.1016/0165-1161(78)90020-1

Flavone (unsubstituted flavone, with no hydroxyl groups) showed mutagenicity (after metabolic activation) in one of the three strains of Salmonella typhimurium tested for mutagenicity.  Presumably, metabolic activation (by the mixed function oxygenases in the microsomal preparation, S-9 mix used with the tester strain) of the compound yielded a catecholic flavonoid.  Among monohydroxylated flavones (3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone), the presence of the hydroxyl groups only resulted in minor changes in mutagenic response. 

http://www.ncbi.nlm.nih.gov/pubmed/22565478  

Quercetin and another flavonoid, kaempferol (with one hydroxyl group in its B-ring), were studied for their mutagenic effects on cultured mammalian cells. They produced point mutations only at pharmacologic concentrations.

Production of oxygen radicals through pH dependent autoxidation of quercetin has been linked to in vitro mutagenic response in Salmonella typhimurium tester strains (reverse mutation test). Certain studies have reported the inhibition of the mutagenic effect of quercetin in Salmonella typhimurium TA98 by addition of metal salts (Mn, Cu, Fe, etc.). Catalytic oxidation of quercetin was suggested as a reason for this inhibition. Further, ascorbate and superoxide dismutase promoted the mutagenic activity of quercetin in the absence of the mammalian-microsome [post-mitochondrial fraction (S9)] metabolic activation system. Scavenging superoxide radicals (and consequent inhibition of the autoxidation of quercetin) was proposed to account for the observed higher mutagenic activity of quercetin in the presence of the S9 system.

http://www.ncbi.nlm.nih.gov/pubmed/3918257

In case quercetin (also other flavonoids) is able to elicit its full complement of mutagenic response by its autoxidation (in presence of transition metals) in solution (and radical formation), then the critical question of the contribution of the S9 mix (cofactor-supplemented mammalian microsomal preparation from the livers of rodents treated with P450 enzyme inducers) to the elicitation of mutagenicity of quercetin (and of other flavonoids) in Salmonella arises. Is the  S9 mixture (metabolic activation system containing hemoproteins) then, not of any consequence at all for the mutagenic potential of quercetin? The corollary may be the tacit assumption that the mutagenic response of polyhydroxylated flavonols, flavones and methylated flavonoids (which might be oxidatively demethoxylated by P450 dependent demethylases in the S9 mixture), and related molecules in the Salmonella mutagenicity system is an artifact!

Flavonoids, at similar concentrations, have shown both mutagenic and antimutagenic effects on mammalian cells. Stich et al, who investigated the mutagenic effect of ascorbate in mammalian cells, have studied the action of transition metals on the genotoxicity (clastogenic effects) of simple phenols, phenolic acids and cinnamic acids in mammalian cells, and reported that the addition of an S9 mixture or the transition metals, Cu2+ and Mn2+, increased the chromosome-damaging activity in some phenolics and suppressed it in others.

The action of transition metals on the genotoxicity of simple phenols, phenolic acids and cinnamic acids.

http://www.sciencedirect.com/science/article/pii/0304383581901518

Flavonoids are pleiotropic. They exert biphasic (and often paradoxical) and ambivalent effects in vitro. These compounds (generally at micromolar concentrations) exert diverse pharmacologic activities in vitro (often nonspecific) and show promiscuity in the broad range of their actions.  Quercetin has been reported to inhibit topoisomerase II, a nuclear enzyme, which controls the topology of DNA during replication and recombination.  This inhibition by quercetin (and also by the topoisomerase II inhibitory drug, etoposide), is considered to stabilize double strand breaks in DNA and elevate the risk of chromosomal abnormalities.

Interestingly, structural requirements [example, vicinal hydroxyl groups, 3’,4’, in the B-ring, a free hydroxyl group at the C3 position in the C-ring (heterocyclic ring), a double bond at the C2-C3 position in the C-ring, a keto group at the C-4 position in the C-ring, etc.) of flavonoids for Salmonella mutagenicity appear to be generally similar to those for enzyme inhibition (example, protein kinase C, iodothyronine deiodinase), immune cell modulation, cell activation, antioxidant activity, antiinflamrmatory, and histamine release-inhibiting activities,etc.  In the flavone (benzo-gamma-pyrone) structure, A (the first ring) and B (the second ring) are aromatic, while the middle ring, C, is heterocyclic.

Studies examining the mutagenic response of flavonoids in the Ames Salmonella/microsome mutagenicity assay (Salmonella reverse mutation assay with different tester strains) showed ambivalent results. Quercetin glycones/glycoconjugates (containing galactose and arabinose moieties; they have vicinal hydroxyl groups, 3’4-OH in their B-ring), (+)-catechin (contains vicinal hydroxyl groups, 3’4-OH in its B-ring) and certain other hydroxylated flavones were not mutagenic to Salmonella typhimurium TA98 strain with and without S9 (microsomal metabolic activation system containing cofactor supplements and cytochrome P450). Amentoflavone was reported to be highly mutagenic in Salmonella typhimurium mutagenic tests (+S9; -S9). This flavone, a biphenol (a carbon-carbon dimer of the dietary flavone, apigenin), contains 5- and 7- hydroxyl groups in its A-ring (flavone skeleton), and a single hydroxyl group (4’-OH) in its B-ring  (aromatic ring); this B-ring (with 4’-OH) is not subject to hydroxylation by S9 mixture because cytochrome P450 mixed-function-oxygenases oxidize an unsubstituted aromatic ring (example, benzene) to an epoxide (arene oxide), which can be converted to a (dihydrodiol) and catechol by the mediation of epoxide hydrolase. Amentoflavone was mutagenic in the absence of the S9 mixture. Since it does not possess vicinal hydroxyl (or para hydroxyl) groups one may infer that it causes mutagenicity without metabolic activation (without autoxidation).

2-Phenylphenol or o-phenylphenol is readily metabolized in vivo by mouse, rat and humans. Microsomal cytochrome P450-dependent redox cycling of o-phenylphenol and its in vitro genotoxicity have been well documented.  This compound yields a hydroquinone (2,5- phenylhydroquinone) by para hydroxylation mediated by rat liver microsomes (P450).  2-Phenylphenol,  guaiacol, phenol, cresols, eugenol and several other phenols were also negative in Salmonella/microsome mutagenicity assay.  Guaiacol (O-methoxyphenol, methylcatechol) is a substrate for peroxidase. Microsomal cytochrome P450 possesses peroxidase activity, and can be expected to metabolize this compound. Eugenol (4-allylguaiacol), a cinnamate derivative of the shikimate pathway found in clove oil, is oxidized by rat hepatic microsomal P450 to an epoxide, which is rapidly converted to dihydrodiol/catechol metabolites (that can undergo one electron oxidation and generate  semiquinones, thus facilitating them to elicit mutagenicity).  Benzene, as far as I know, is negative in Salmonella/microsome mutagenicity assay. Benzene is biotransformed into an arene oxide, and further metabolized to dihydrodiol/catechol (which can undergo autoxidation) by rat liver microsomal oxygenases in S9 mixture (P450).

With regard to radicals generated by the autoxidation of flavonoids and related compounds, the question arises as to the probability of these radicals generated in solution (medium) reaching intracellular targets of polynucleotide chains (DNA) in order to bring about the induction of reverse mutations with different mutation mechanisms, such as base-pair substitution and frameshift mutations (depending on different Salmonella typhimurium tester strains). 

The points surmised in the above paragraphs may be considered in inferring and drawing conclusions that the mutagenicity of quercetin (and of related compounds with antioxidant activity) in Salmonella typhimurium is due to the generation of oxygen radicals through pH dependent autoxidation of quercetin in solution.  

Bilion of years existing free radicals on Earth affect many metabolic ways. Many diseases are influenced by free radicals and antioxidants. Absorption of antioxidants in gastrointestinal tract depends on many factors. More than 100 of diseases or states are influenced by free radicals and antioxidants. There is doubt that the research is useful.

Dear Mahesha,

The following may of interest in connection with your question of the significance of in vitro antioxidant assays.

Enhancement of lipid peroxidation and its amelioration by vitamin E in a subject with mutations in the SBP2 gene.  November 2015. The Journal of Lipid Research, 56, 2172-2182.  http://www.jlr.org/content/56/11/2172.full

The incorporation of selenium into proteins is primarily as selenocysteine (Sec), which is the 21st amino acid donor to an elongating polypeptide chain to be encoded by the UGA codon. Sec insertion sequence-binding protein 2 (SBP2) is essential for the biosynthesis of Sec-containing proteins (selenoproteins). A series of such proteins, including glutathione peroxidases, operate in the removal of hydroperoxides, thus preventing oxidative damage. Till now, nine families with mutations in the SBP2 gene have been discovered. Patients with mutations in SBP2 develop abnormalities in thyroid hormone levels, which are regulated by the selenoproteins, iodothyronine deiodinases. Accumulated evidence strongly indicates the elevated levels of lipid peroxidation and oxidative stress in subjects harboring SBP2 mutations, leading to several disorders.

The levels of lipid peroxidation products, composition of blood cells, and biochemical profiles are reported in this investigation in an individual with mutations in the SBP2 gene (the ninth patient to be reported in the world). Results indicate an increase in free radical-mediated oxidative damage in this case. Treatment of this patient with vitamin E (α-tocopherol acetate, 100 mg/day) for 2 years lowered lipid peroxidation product levels to those of control subjects. Of salience, the results clearly suggest the effectiveness of vitamin E administration in abating elevated lipid peroxidation.

The authors draw attention to the negative results obtained with antioxidant supplementation, including that of vitamin E, in many large-scale clinical, intervention trials, and pinpoint the importance (and judiciousness) of “treating the right subject at the right time and for the right duration” in order to spot or cognize the beneficial effects of vitamin E in impairing lipid peroxidation in various diseases that involve free radical-mediated oxidative damage. 

An article entitled "Radical rethink" recently published in Chemistry World 12(12): 58-61 (2015) addresses the original question posted by Mahesha. It is accessible at http://www.rsc.org/chemistryworld/2015/11/free-radicals-reactive-oxygen but unfortunately is not open access.

The final three paragraphs read as follows:

‘Free radicals are important signalling molecules for biology,’ Melov concludes. ‘The challenge is to dissect out what is optimal versus what is pathology. Therein lies the problem.’

In the meantime, the antioxidant industry remains in rude health. Billions of dollars are reaped selling useless supplements, in the face of overwhelming evidence against their effect. 

‘The idea you can get more effective compounds from a pill than you would from eating fresh fruits and vegetables is completely unfounded,’ says Davies. But, this premise is underpinned by the folksy simplicity and impressive terminology of ‘good antioxidants versus bad free radicals’, making it remarkably popular. The science is more muddled: reactive species can be good or bad, depending on the context and dose. 

Richard, I share your views AND your frustration.  I work with many Integrative Medicine clinicians and these entrenched views are very difficult to reverse.  When I explain the roles of ROS as essential to the intra-cellular signalling processes, I am met with the iciness that comes from criticising a person's religion.  

In my estimation, we are about 20-25 years away from redressing the balance.  Such is the power of Big Health Food, which is every bit as culpable as the Big Pharma they love to denigrate.

To me a concerning aspect of the issues is the fact that nutrigenomically-active compounds like Brassica-derived sulforaphane activate transcription factor Nrf2 by sending a weak pro-oxidant signal - strong enough to activate the Nrf2-ARE complex but not strong enough to alter the redox status.  When a supra-physiological quantity of one or more synthetic antioxidant vitamins appears in the cell, the weak pro-oxidant signal is masked.  The result?  The cell does not detect the signal to upregulate its endogenous defences associated with Nrf2.

This 2009 paper by Ristow et al. on the adverse effects of vitamins C and E on the expected beneficial response to exercise illustrates this point.  Antioxidants prevent health-promoting effects of physical exercise in humans

http://www.pnas.org/content/106/21/8665

Exercise induced radical production has been believed as a paradox.  One looks at exercise as a healthy act. And yet, it could have a propensity to cause deleterious effects on tissues and cellular constituents. This may be an acute effect (analogous to an acute inflammatory response). Other observed (often transient) responses to exercise training such as dehydration, muscle damage, substrate depletion and inflammation are similar to the response of radical production (and their possible damaging effects).  A recovery phase then follows, which is of benefit. The above responses might subtly affect homeostasis.  Free radicals have been considered to have physiologic effects on exercise adaptation processes, thus conferring adaptive advantages in the body in confronting future disturbances. Radicals are believed to be necessary for exercise-induced hypoxia adaptation and muscle contraction. Exercise training was shown to increase antioxidant defenses (increases in the activities of superoxide dismutase and of glutathione peroxidase) nearly 3 decades ago. Crucial transcription factors implicated in the upregulation of antioxidant enzymes have been reported to be dependent on the enhanced production of oxidants as well as the presence of oxidized or unfolded cellular proteins. If exercise provoked oxidative changes in tissues constitute integral components of redox regulation interference with antioxidants at the adaptive phase (of exercise) might not be expected to confer any benefits (and might even vitiate the exercise adaptation process). 

In  so far as antioxidants are concerned, whether they are from  vegetables /fruits/ nuts/ oils, in vitro examination ideally should  follow up with in vivo testing. If the animal experimentation is not feasible (due to restrictions) at least cell/ tissue cultures could be used.  Very often the isolated pure compounds may give the results in a test tube atmosphere and not in the biological systems. Sole dependence on in vitro systems (that use chemical reactions)  may not be useful all the time. 

Yes, you are right, In vitro antioxidant activity of random plants not required all the time. Further studies on the same plant or phytochemical, like hepatoprotection, anti inflammatory, anti aging, diabetic complications, and cancer etc., will give a better understanding of antioxidant molecules (Their role in disease condition). 

Random screening of plant fractions or extracts for antioxidant activity is of no use, just waste of time and money.

I might be wrong. 

This is the very reason that the USDA removed its ORAC tables from its website in 2010.  They realised that these tables were being inappropriately used by supplement companies to promote their products along the lines of "My ORAC is higher than your ORAC".   The reality is that cells, by upregulating gene expression,  produce their own redox-modulating compounds; many are enzymes capable of quenching literally millions/billions of reactive species per second.  Compare that to the limited quenching ability of direct-acting antioxidant vitamins like ascorbate.  In my opinion, we need to discard the notion that cells need as many so-called 'antioxidants' as possible.  Cells respond to stressors in their environment and it is these stressors that signal an induction of expression of the appropriate protective genes.

The reality is that cells, by upregulating gene expression,  produce their own redox-modulating compounds; many are enzymes capable of quenching literally millions/billions of reactive species per second.  Compare that to the limited quenching ability of direct-acting antioxidant vitamins like ascorbate.  In my opinion, we need to discard the notion that cells need as many so-called 'antioxidants' as possible.  Cells respond to stressors in their environment and it is these stressors that signal an induction of expression of the appropriate protective genes.

Furthermore, we need to discard the notion that cells need as many so-called 'antioxidants' as possible.  Cells respond to stressors in their environment and it is these stressors that signal an induction of expression of the appropriate protective genes.  There is evidence that excessive ingestion of antioxidant vitamins masks the stressor signals needed for the cell to register a threat.  This may explain why large-scale studies on the potentially preventive effect of vitamin E on CVD, cancer and Type 2 diabetes have failed to show benefit.  More so, some studies showed increased mortality with supraphysiological doses of vitamin E.

Excessive ingestion of antioxidant vitamins have been shown to mask the stressor signals needed for the cell to register a threat.  This may explain why large-scale studies on the potentially preventive effect of vitamin E on CVD, cancer and Type 2 diabetes have failed to show benefit.  More so, some studies showed increased mortality with supraphysiological doses of vitamin E.

Maybe it's time for those of us at the coal-face of the research to be more vocal in influencing the message 'Big Supplement' uses to build its business.  Consumers are the losers!

And another point that makes in vitro antioxidant assays a waste of time and money is that the antioxidants in the plant extracts are NOT what the cells encounter: they are modified e.g. glucuronidated when crossing the intestinal barrier, many of them are barely bioavailable and what is circulating are microbiota metabolite sproduced in the gut (valeric acid and co for phenoilics), etc.

The nutritional message should be clear: eat many diverse fruit and vegetables, this is what epidemioloy supports (plus exercise, less red meat etc). Not supplements, antioxidants or not. Remember also that vitamin C is a vitamin for its role as precursor of collagen, not antioxidant. .

Catherine, I agree with your sentiments.  I would go even further by saying that we need to move away from the notion that cells require antioxidants for their optimal function.  I prefer to refer to 'cellular defences' instead of 'antioxidant status'.  Redox status is just one element associated with the cell's endogenous defence mechanisms.

Industry won't change its attitude to the very lucrative 'antioxidant' supplement market but it is surely up to us as scientists to start changing the dialogue - and this means changing the terminology we use when we speak - and publish!

Great comments....Christine. Well, lets not forget that the industries has to sell their products, services and above all IDEAS (howsoever it may appear). We should understand this and ignore it and not take it to our heart. We know what is right and what is wrong and carry on with our work.

Jai:  Sounds like you believe it is OK for healthcare enterprises to be operated unethically ... parting the gullible and uninformed from their cash. In my work as a pharmacist in the UK, I am obliged to deliver my services in a manner that is in the best interests of patients and indeed the "worried well".

Dear Richard: Thats exactly what I am saying industries sometime do work away (infact very far away) from ethics only because they and their medical practitioners need to survive. I would go one step ahead that people like you, Catherine, Christine or even me should give the right advise to any body who would ask us for our personal opinion. 

The balance between free radicals and antioxidants is very important. The increase of free radicals  or decrease of antioxidants must be teated.  Aldehydes, DNA mutants. etc. lead to many various diseases and to their progression. The problem is which antioxidants, how many, how often, how much, their combnination.

Part of the reason that some supplement companies are 'getting away with' their dodgy products backed by even more dodgy science is that clinicians are not equipped to evaluate the claims made for a product - nor are they equipped to evaluate the scientific references attached to the claims.  

If you study a so-called Technical Data Sheet for many supplements (even those claiming to be 'Practitioner Only' companies), you will frequently find a list of references that do NOT substantiate the claims being made - in vitro and animal data and human data for different raw materials with different characteristics.  Another common failing is in using studies where the findings have not reached statistical significance.  

Perhaps the most common issue is that multi-ingredient formulae can contain a list of 'popular' ingredients but the doses are much lower than any clinical trials would indicate for a response.

When challenged, the company may claim a synergistic effect from the combination of ingredients - but yet there is no evidence to show this.

Nothing will change until clinicians are well-enough trained in evaluating a formulation that they demand a better standard from their suppliers.

Depends on variety of sample used for it and the purpose behind assays. Completely agree with @Christine Houghton.

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