Analyzing Antioxidant Properties of Plants

hello friends in this video i'm going to talk about how to analyze the antioxidant properties of the various plant so before moving into the antioxidant essay let me just give you an explanation about what is antioxidant what are free radicals and what chemicals are we going to use so for example first let me give you example with our own body then let me go to the plants plant system so in our body because of due to exposure of various toxic chemicals it may be adulteration in the food or food or it may be due to cosmetics or it may be due to smoking or it may be drinking or due to exposure to some harmful radiation when there are so many biochemical pathways that is undergoing in our body so as a result as a byproduct what happens there are certain unstable molecules which are having an unpaired electron in the outermost orbit are released these are called as free radicals so the name free suggests that the outermost orbit has an unpaired electron so we all know that when there is an unpaired electron that radical will be highly unstable and it is highly harmful for the body so it is also been suggested that because of this free radicals it may lead to arthritis skin inflammation cell toxicity or even to cancer susceptibility so these are all the disadvantages because of the free radicals that are produced in our body and not only that this free radicals are like chain reaction if one free radicals are produced they go and knock the other cell another atom and similarly electron is lost and that again becomes free radical now two free radicals are formed these two free radicals goes and hits again another two cells or another two atoms and again four free radicals are formed these four free radicals goes and hits and again eight free radicals are formed it is like a chain reaction so one becomes two two becomes four four becomes eight sixteen like that and it goes on so when this happens what happens it leads to various disorders in mankind or in animal beings similarly same thing happens in the plant system also so certain plants when they're exposed to certain radiation or whenever you're doing some experiments related to some stress physiology when you're giving some heavy metal treatment for the plant when there are so many metabolic pathways that are happening in the plant system as a result as a byproduct these free radicals are released even in our body there are so many free radicals are released due to various metabolic reasons that is one of the major cause for your aging of the skin for the wrinkles for all those things so to counter that what we want is we want to stop the activity of the free radical that process is called as free radical scavenging so what happens is there should be some molecule that goes and stops the action of free radical i said it is a chain reaction so there should be some molecule that goes and stops so how will this reaction stop i said that it will be having an unpaired electron in the outermost orbital so if i just give one electron that becomes paired electron and stability will be there so the unstable molecule becomes highly stable now i need to introduce some compound that can donate an electron for these antioxidant for these free radicals so that they become stable and did not cause any damage and these molecules which donate an electron and which control these free radicals are called as antioxidants so antioxidant what do you mean by oxidant or oxidation loss of electron these people will donate an electron so they are called as antioxidants so what have what is the function of antioxidant so you would have heard many advertisements for green tea and all those things it reduces your belly fat it gives bloats it grows to the skin your skin becomes brightening when you are drink the green tea all those things green they are rich in antioxidants so because they have high they have a compound called as tocopherols these tocopherols are nothing but antioxidant molecule so what is an antioxidant so whenever a free radical is produced in a body these antioxidants will absorb them they will give an electron they will donate an electron and they will reduce their activity and they will make them stable as a result whatever the damage effects are caused by by the free radicals they are reduced so this activity is called as free radical scavenging activity they scavenge they absorb all the free radicals that are produced in their body so that is the complete scenario background explanation for what are antioxidants what are free radicals and what is free radical scavenging activity now if you see these antioxidants are present in all the plants some some shows high antioxidant property like your green tea some shows very less antioxidant property now if you are doing some experiment with any plant and if you want to know what is the antioxidant capacity of my plant we have to do one experiment and that experiment is called as antioxidant assay by dpph method what is this dpph it is an organic chemical compound so i have a dbph the complete name is 2 comma 2 diphenyl 1 picryl hydrazil you get in two forms 2 comma 2 and one comma one so some people use two comma two some people use one comma one so it depends on which chemical you have in your laboratory and which protocol or which paper you are referring to so what is this dpp h diphenyl picrine hydrozye it is a crystalline purple color compound it is a stable free radical so i said right the free radicals are produced in the body so it is one such free radical so now what we are going to do is and this is a purple crystal color crystal form and it is insoluble in water so you have to use organic solvents like ethanol or methanol you can use so normally i use methanol because ethanol it's costly also and it evaporates very soon so if you're not managing it properly it evaporates and there is change in the concentration the concentration alters so that is why we most pro most we prefer methanol methanolic solution of dpp hb user and some people use 0.1 millimolar some people use 8 mg that is 0.008 percent so i have fixed 0.004 percent so what is 0.004 percent it is nothing but 4 mg of this db ph dissolved in 100 ml of methanol will give me 0.004 percent so like this if you want 50 ml so accordingly you calculate it will become 2 mg in 50 ml methanol so this is the first to the agent that we need and very very important thing is so it is light sensitive always whenever you're weighing it or whenever you're doing this experiment try to do it in as much as dark condition it is possible so you can't con cover the entire lab with or something so at least that space that you're working make sure that it is dark and also whenever you're preparing this dp ph prepare it in a volumetric flask or in glass beaker and cover it with aluminum foil surround it with aluminum foil two to three layers and even the top bottom so make sure it is completely dark and also at least whenever you're doing this experiment maintain the dark condition if not completely at least some uh try to not expose it to direct light so that is very important thing so that is about your dp ph now what is the entire principle of this so i said you this dpph is a free radical if your plant is having any antioxidant molecules those antioxidant molecules will come and react with this dpph which is a free radical and they will reduce it so this dpph is a purple color crystal so when you dissolve it in methanol you will get beautiful purple colored solution you will get and when you add your extracts to this plant extract here we use ethanol extract methanol extract so i have used methanol extract so i have made another video how to prepare methanolic extracts of plant sample for those who are having any doubt or you don't know how to prepare this methanolic extract or any plant extract matter i will give you the link in the description please do go and watch this video how to prepare extract plant extracts using different solvent so now i will take this plant extract methanolic extract and i will add it to this dbph solution now what will happen all the antioxidant molecules that are present in my plant extract will react with this db ph and they will reduce them this is a free radical the antioxidants what i said antioxidants will give an electron because the free radicals will be having an unpaired free unpaired electron in the outermost shell so they will donate an electron and they will reduce them dpph will be reduced to form d p p h so this is the chemical formula so this will be purple in color so when you have the reduction it will become either gold and color or it will become colorless and that solution we are going to take the absorbance we are going to take absorbance at pi 17 nanometer because this dp ph has an absorption maxima at around 550 to 520 nanometer some papers they were taken at 517 518 520 also they're taken so with the paper you are following stick on to that i am sticking it to 517 nanometer so once i take the absorbance then i will come to know how much dpph has been reduced so there is a formula absorbance of control minus absorbance of sample divided by absorbance of sample into 100 that gives me the percentage of antioxidant activity of my plant sample so i will save the formula again make a note out of it absorbance of control minus absorbance of sample divided by absorbance of sample into 100 into 100 is because to convert into percentage so for example let me say 60 is the antioxidant activity like that you can take various plant extracts or various plants and you can find which plant is having the highest antioxidant capacity normally citrus fruits will be having like lemon gowa gooseberry all these are having high antioxidant activities chili capsicum all those things so this is the complete principle and the background explanation that you need whenever you're doing this experiment so i will say the protocol and all those things a bit wide and one more thing i wanted to say you this you have to compare with the standard antioxidants that are available you don't know whether your plant is very high antioxidant you need something to compare right so we will take some standard like how if you see for carbohydrate estimation or protein estimation we would have plotted standard graph here we are not plan plotting any standard graph we are just comparing the activity without with my plant extract to that of the standard antioxidants so what are standard antioxidants there are certain chemical compounds like ascorbic acid and one more chemical compound called as vht butylated hydroxy tauline butylated hydroxytaolin and one more compound called as bha butylated hydroxy anisole so these are some of the cocopherols so these are all the standard antioxidants bht butylated hydroxytolin butylated hydroxy anisole ascorbic acid tocopherols whichever is available in your laboratory you can take that and you can take in different concentration and see follow the same procedure and find the antioxidant activity of those standards then you will come to know your plant extract is having more antioxidant capacity or your standards are having more antioxidant capacity generally these antioxidants are used to prepare drugs like a vitamin c tablet or so many drugs they have prepared and they're also used in food industry so if you see some take any food packets and see they will add like preservatives what are preservatives preservatives are nothing but to maintain a particular food fresh to avoid spoilage of the food so there are some preservatives like bht bha they're all they're added to diary products or let it be any edible products in food industries in cosmetics they are all added to as a preservative so these are the industrial applications so now you will get an idea when you do the antioxidant activity of your plant extracts and the antioxidant activity of your standard that is available in the market which is more antioxidant whether the plant or whether your standards so that will give you clear pictures so always you have to do the antioxidant activity with using both you have to do to come back which is best your plans are good or your standards are good so this is the procedure this is the principle behind it so i will just give you summarize the entire thing so now what are free radicals free radicals are produced in the body uh no living organism as a result of many metabolism that is occurring in our body and these are highly toxic it is like a chain reaction they will be having an unpaired electron in the outer most shell they go and knock another atom that goes and knocks another atom so it is a chain reaction which leads to cell damage and finally this leads to various metabolic disorders like rheumatism arthritis inflammation cancer aging so many uh there are so many disorders that are produced because of this uh free radicals now how do we control this free radical so there's a phenomenon called as free radical scavenging where the this is unstable free radicals are they brought down to stability how they are brought down to stability so one electron is donated by these chemical compounds these chemical compounds which are called as antioxidants anti means against so these antioxidants they donate an electron and they make highly unstable free radicals into a stable compound that's what happens their damage the cost that the damage that is caused by these free radicals are controlled so this is about the antioxidant so the property of antioxidants now if you're taking any plant and you want to know what is the antioxidant molecule that is present what is the percentage of antioxidant we will follow this method called as antioxidant i say by dpph dpph is nothing but it is a free radical which is stable it is light sensitive and it is insoluble in water soluble in ethanol and methanol so it is nothing but diphenyl picturing hydrazil so now what i'm going to do is now i'll save the protocol we will prepare 0.004 percent of methanolic dpph that is i will take 4 mg of dpph i will dissolve it in methanol so i will resolve it in methanol so that will give me 0.004 percentage of dpph methanolic solution first thing that is done second thing you need your plant extracts to analyze now what we are going to do is one plant x that i am taking and i will take it in different concentration i will take 20 microliter 40 microliter 60 microliter 80 microliter 100 microliter 200 microliter it depends on your so it's like trial and error method so some people will take 500 150 200 some will take 100 200 300 400 500 so it is just like trial and error method for starting with i am taking like this 20 40 60 80 100 200 micro liter i am going to take it in a test tube or in a pen of tubes anything you can take it why we are taking it in different concentration because i want to know at which concentration my plant is having the highest antioxidant capacity you may say that if i peep it out more it will be having more antioxidant right so if i take 100 microliter definitely 100 microliter will be having more antioxidant than 20 microliter i agree but after some stage the percentage becomes stable it will not change so for example if 80 microliter is giving me the highest antioxidant activity even if i take 500 microliters the antioxidant activity will not become high it will be almost similar to it for example let's say let me say 70 is the antioxidant activity for 80 microliter if i take 500 microliter also it will give 90 percent 100 above 100 it can't go so we will make different gradual concentrations to see at which concentration or at which lower concentration high antioxidant activity is seen that is the entire thing so we will take a different antioxidant concentration of this plant extracts and so there are two methods you can do after this after this you can directly add your dpph methanolic solution or you can dilute it with adding methanol and then you can add dpph so i will just explain you both but then i'm doing and showing you i will just do one method so now we will take 20 40 60 80 100 microliter of plant extracts now i will make up the solution using methanol up to 2 ml or 3 ml i so for example if i added 20 microliter i will make it up by adding 980 microliter of methanol to make up the total volume to 1 ml or similarly i will make it up to using 2 ml of methanol so similarly using various concentrations i can make it up to using 1 ml or 2 ml or 3 ml methanol and then finally i will add 1 ml of methanolic db ph solution and then we will incubate it in room temperature for 30 minutes and then we will take the absorbance at 570 nanometer and then we will calculate it according to the formula that i said that i mentioned previously this is one method second method is what we can do is we will take 20 microliter 40 60 80 100 microliters of the plant extract to that itself directly i will add 4 ml of 0.004 percentage of methanolic dpph solution 4 ml i will add and then 30 minutes incubation i will leave and i will take the absorbance reading so this is all the entire protocol i will do it and show it to you so whenever i'm saying anything make a note out of it because i'm not showing you any protocols for you so that later you can note it down so as i'm saying just note it down so now okay one more thing i wanted to say you is whenever we are taking doing absorbance using spectrophotometer or using any microplate method we need something called as blank so here the blank will be just methanol just your methanol will be your blank you will not add any bpps to it or any plant extract you will just take 4 ml or 5 ml of methanol that becomes your blank and one more thing there's something called as control i said you the formula is absorbance of control minus absorbance of sample divided by absorbance of control into 100. so what is this control so this control is your db ph solution in methanol so without adding any plant extract so it is that will be your control so what will be there there will be no plant extracts to reduce it so it will be purplish it will be purplish in color so and it will be showing the high absorbance since there are no antioxidants i'm not adding any plant extracts to the control so what we'll be having is so it'll be having i absorbance will be dark purple in color so that will be your control so control is methanol and dbph that is your control and your blank is methanol and the sample is whatever i will say to the protocol and apart from this you're going to plot a standard positive graph we call it as using togopherols or using ascorbic acid using tbh or utbt or butylated pht using bht or bha so whichever is available in your lab you can use that and you can do the antioxidant activity of this so that you can analyze whether your plant extract is having more or these that is naturally that is available in the form of chemicals in the industries which is having the highest antioxidant activity so this is the complete principle procedure everything i've discussed now i will just demonstrate over you and show so i will just let me prepare all the solutions and then i will show you how to do this procedure yeah friends now we will start the actual protocol analysis as i was saying there are many protocols in many ways that many people have conducted so stick on to one protocol like i said there are two methods or many more methods to do so i will just give you one method you can either follow this this will give you a good result now what we are going to do is we are going to take the methanolic extract of the plant sample and i am going to take this in different concentrations i am going to take 20 microliter 40 microliter 60 microliter 80 microliter 100 and 200 microliter in 5 6 different concentrations i am going to take and then i am going to make it up using methanol so if you don't want 20 40 you can go with 50 100 150 200 or if you want even more higher concentrations 100 200 300 you can watch just a trial and error method so if this method is not working then i have to change the protocol i have to change the concentration i have to increase it or decrease it so it's all trial and trial and error method so i am going to take 20 40 60 80 100. so here i have test tubes i have marked each concentrations over here i have labeled it and 20 microliter i am going to take and then i am going to make it up to 3 ml using methanol so if i'm taking 20 microliter then i'm going to make it up by adding 2980 microliter of methanol similarly if i'm taking 40 microliter of plant extract then i'm going to add 209 2960 microliter of methanol and then i'm going to make it up to 3 ml once after i make it up to 3 ml then i'm going to add 1 ml of dp ph solution so i said you that dbph it is light sensitive so i prepared and i kept it in the dark always prepare the solution cover it with aluminium foil like this label it and store it in a dark and cool place don't expose it to sunlight it is a light sensitive chemical so be very careful when you're doing this dpph reaction and also it is very costly so one gram costs about 10 000 rupees so just make sure you are just preparing it has to be freshly prepared so what is the necessity just prepare that much don't prepare excess and waste it is very costly chemical and also it is very much heat and light sensitive so always cover it all the four sides with aluminum foil and keep it in dark place and also as much as possible try to do this experiment in dark conditions may be weighing or adding all those things you can do it in bad condition so this is all the protocol my dear friends so you are going to take different concentrations of your plant extract so i have given a separate video how to prepare their methanolic plant extracts here i have prepared my plant sample various methanolic extracts i have prepared so i will give you the link in the description how to prepare methanol extract or any solvent method for that and you can see that uh video and prepare methanolic extract of plant sample you can actually prepare ethanolic extract also i am following methanolic extract and different concentrations i'm going to prepare it out and then i'm going to make it up to 3 ml adding methanol then i'm going to add 1 ml of dp ph solution 0.004 mg i have taken i mean 0.004 grams i have taken in 100 ml that is nothing but zero point double zero four percent it is on almost equal to zero point one millimolar like that so there are different concentrations again so whichever paper you're following follow that protocol so according to my paper i'm following this protocol so without much extension let me start i'm going to take leaf extract i have taken so here i have kept methanol is ready everything is ready you need methanol to make it up and your plant samples yes so i'm going to take 20 microliter so make sure when you're preparing the values are proper don't make any pipetting errors so first 20 microliters and in the second tube 40 microliters this is 20 and 20 more 40 microliters then third one will be 60 microliters 60 microliters and 80 microliters fourth one and 100 microliters 100 and the last one is 200 100 more yes it is done so plant extract has been over now i i'm going to make it up using methanol so using the methanol i'm going to make it up to 3 ml so if you have taken 100 200 300 accordingly calculate it and then make it up to 3 ml so since i have taken 20 microliter for the first one i'm going to add 2980 microliter for the second one two thousand nine sixty microliter two thousand nine forty similarly i'm going to add methanol and i'm going to make it up make up the total volume to three ml so yeah friends now i've made it up if you can see so this is 20 microliter i made it up to 3 ml similarly this is 40 you can see the concentration is increasing it is becoming more darker and darker green and the last one is 200 microliter is being the complete green you can see the concentration so you should find the color difference like this whenever you're doing serial concentration so this is my plant extract on the other side we should also do the positive thing i was talking about there are many things like ascorbic acid butylated hydroxytolamidulated hydroxy antisole all those things so i have taken the dilated hydroxyl towline bh3 we call it as so this bht is also insoluble in water so i've taken methanol as a solvent and then i have made a stock so this is my stock so this is 10 mg per ml stock i have made so i have taken 0.1 gram of bhd butylated hydroxytolate it is a white crystalline form and i have dissolved using methanol and made it up to 10 ml so this is 10 mg per ml stock from this again same concentrations i'm going to pip it out like how i did my plant samples same 20 micrometer 40 microliter 60 microliter 80 microliter 100 microliter 200 microliter and then again i'm going to add methanol and then i'm going to make it up to 3ml so same thing this is called as positive graph so we need to compare our plant extract how much effective it is how much antioxidant activity is having when compared to industrially available in the market there are some compounds whether it is as effective as that or less effective or more effective so we need some comparison so we are just doing this so let me prepare all these things and make it up to 3ml and then i will show you how to proceed so yes even the phd related hydroxytolane also i have prepared into serial concentrations and i made it up to 3ml using methanol now what is the formula formula says absorbance of control minus absorbance of sample divided by absorbance of control into 100. so what is this control so this control will be 3 ml of methanol and 1 ml of dpph solution so there will be no having no plant extract or nothing it is without the plant extract that will be our control so this has to be having an higher absorbance when compared to the other thing so i have said to the principle and all those things because this uh dpph is a free radical they get reduced by the presence of this plant extracts when the antioxidants present in the plant extracts and the color gets changed from purple to oranges or yellow sometimes even colorless so if there are more antioxidants all the dpph gets reduced and it becomes colorless solution if there are less antioxidants only few dpph gets reduced and you will get a golden color or orangish lowest color you will get so based on that we will come to know so this solution this is my control where i'm having 3 ml of methanol and i'll be just adding 1 ml of dpph since there are no antioxidants this will remain as purplish on itself and it will give me greater absorbance the absorbance value will be high and whenever we are doing spectrophotometric we need something called as blank to make it auto zero so for that black use just methanol so whatever solvents you you're using if you're using ethanol your blank will be just ethanol if uh since i have used methanol my blank will be just methanol now everything is ready now i'm going to add dp ph to it so i'm going to add 1 ml of dpph and then i'm going to incubate it in the dark for 20 to 30 minutes and then i'm going to take the absorbance at 517 nanometer so as soon as i add this dpph you can find it is purple in color but as soon as i add the reaction will occur and it will become orangish color so one ml i'm going to add for everything so you can find it is pinkish color so now i'm adding in 20 micro tutorial plant extract so it has just retained slight pinkish color you can find similarly i'm going to add for all the cubes including the positive thing so you can see the color change i'll show you after 30 minutes then you can find it beautiful transformations you can find see as i am adding you can find the reaction unlocker and reduction reaction occurs and the color changes that is the beauty of this experiment so you can see this so green it is dark dark green it is because it is 200 microliter but just as soon as i add this the color changes so why we have taken different concentrations we will come to know which concentration gives me the best percentage so yes i have added for everything now let me add for the control also control will be just methanol 3ml methanol and one mldb ph solution so it will have that purple color pinch will be there so you can see that purplish color so now let me let us add it to the standards also php see bht lower concentration that pinkish color is there but in higher concentration if i add since bh3 is an antioxidant it will reduce all the dpp edge and the color will change can you see so the pinkish is disappearing slightly and it's becoming colorless so if you leave it after 30 minutes and observe it will be completely colorless and also you need to shake vigorously and keep it in dark for incubation so after you add shake it vigorously after 5 10 minutes again you can shake it and keep it in incubation in dark place so the color is already changed for the higher concentration it has already become colorless yes so this one if it is any remaining keep it in the dark again for further experiments so you can see here this is my 40 micro and 200 microliter and this is my 20 microliter the pink color is still there while here the pink color has been disappeared that what is the reason behind it so whatever dph was there it got reduced and it has become colorless or light yellowish color so now we will keep this in dark for incubation and then after 20 or 30 minutes we'll note down the time and we will take the absorbance at 517 nanometer and using the formula we have to calculate the percentage of antioxidant activity so i will show you after 30 minutes so the color difference and i'll take the absorbance reading and then i will see you how to calculate it so friends 30 minutes has been over incubation i will show you how the color change has been occurred you can notice it so beautifully that so this is my control you can see the control how it is pinkish in color so we have just added methanol and dpph so there are no antioxidants to reduce it so it has retained its purple or pinkish color while if you see this bht 20 micro liter it has been slight a lowish or greenish in color but when you see this 200 microliter has completely become colorless this is because whatever dpph that we have added this vht acts as an antioxidant and it gets reduced into dpp hh and it forms a lowish or colorless solution like this so you can see 100 microliter has been there has been color change so you can compare these two and find this is in case of your standards in case of a positive graph now when we move on to plant extract in plant extracts 20 microliter has light pinkish you can find it completely does not reduce because 20 microwater has lesser concentration of antioxidants so it has that pinkish color has is been there still but on the other hand if you see the other higher concentrations i you can find it over here the pinkish color has completely disappeared so here the antioxidants that are present in the plant sample reacts with this and reduced this into and it has formed this yellowish color so now we will go take the absorbance at 570 nanometer and then using that formula we can calculate to find the percentage of antioxidant activity in my plant samples so friends this is the formula that we are going to apply to calculate the antioxidant activity absorbance of control minus absorbance of sample divided by absorbance of control into hundred this android is to represent it in the percentage form and this is my absorbance of this is the absorbance of my control and this is the absorbance of my sample so this i will show you one so for this absorbance of control 0.11 0.118 minus absorbance of sample 0.082 divided by 0.118 into 100 so if i do this calculated 0.118 minus 0.082 divided by 0.118 into 100 so if i do in 200 i will get 30.5 percent so this is for 20 microliters similarly this is for the other microliters so i have got the antioxidant activity is 30 so like this you can whatever absorbance you get for your standard graph as well as for your plant sample using this formula you can calculate the all the percentage of the antioxidant activity so this is how we calculate the antioxidant essay using dpph method so thank you for your patience watching if you like this video video do give a thumbs up and subscribe to my channel for more biology related video thank you

hello friends in this video i&#39;m going to talk about how to analyze the antioxidant properties of the various plant so before moving into the antioxidant essay let me just give you an explanation about what is antioxidant what are free radicals and what chemicals are we going to use so for example first let me give you example with our own body then let me go to the plants plant system so in our body because of due to exposure of various toxic chemicals it may be adulteration in the food or food or it may be due to cosmetics or it may be due to smoking or it may be drinking or due to exposure to some harmful radiation when there are so many biochemical pathways that is undergoing in our body so as a result as a byproduct what happens there are certain unstable molecules which are having an unpaired electron in the outermost orbit are released these are called as free radicals so the name free suggests that the outermost orbit has an unpaired electron so we all know that when there is an unpaired electron that radical will be highly unstable and it is highly harmful for the body so it is also been suggested that because of this free radicals it may lead to arthritis skin inflammation cell toxicity or even to cancer susceptibility so these are all the disadvantages because of the free radicals that are produced in our body and not only that this free radicals are like chain reaction if one free radicals are produced they go and knock the other cell another atom and similarly electron is lost and that again becomes free radical now two free radicals are formed these two free radicals goes and hits again another two cells or another two atoms and again four free radicals are formed these four free radicals goes and hits and again eight free radicals are formed it is like a chain reaction so one becomes two two becomes four four becomes eight sixteen like that and it goes on so when this happens what happens it leads to various disorders in mankind or in animal beings similarly same thing happens in the plant system also so certain plants when they&#39;re exposed to certain radiation or whenever you&#39;re doing some experiments related to some stress physiology when you&#39;re giving some heavy metal treatment for the plant when there are so many metabolic pathways that are happening in the plant system as a result as a byproduct these free radicals are released even in our body there are so many free radicals are released due to various metabolic reasons that is one of the major cause for your aging of the skin for the wrinkles for all those things so to counter that what we want is we want to stop the activity of the free radical that process is called as free radical scavenging so what happens is there should be some molecule that goes and stops the action of free radical i said it is a chain reaction so there should be some molecule that goes and stops so how will this reaction stop i said that it will be having an unpaired electron in the outermost orbital so if i just give one electron that becomes paired electron and stability will be there so the unstable molecule becomes highly stable now i need to introduce some compound that can donate an electron for these antioxidant for these free radicals so that they become stable and did not cause any damage and these molecules which donate an electron and which control these free radicals are called as antioxidants so antioxidant what do you mean by oxidant or oxidation loss of electron these people will donate an electron so they are called as antioxidants so what have what is the function of antioxidant so you would have heard many advertisements for green tea and all those things it reduces your belly fat it gives bloats it grows to the skin your skin becomes brightening when you are drink the green tea all those things green they are rich in antioxidants so because they have high they have a compound called as tocopherols these tocopherols are nothing but antioxidant molecule so what is an antioxidant so whenever a free radical is produced in a body these antioxidants will absorb them they will give an electron they will donate an electron and they will reduce their activity and they will make them stable as a result whatever the damage effects are caused by by the free radicals they are reduced so this activity is called as free radical scavenging activity they scavenge they absorb all the free radicals that are produced in their body so that is the complete scenario background explanation for what are antioxidants what are free radicals and what is free radical scavenging activity now if you see these antioxidants are present in all the plants some some shows high antioxidant property like your green tea some shows very less antioxidant property now if you are doing some experiment with any plant and if you want to know what is the antioxidant capacity of my plant we have to do one experiment and that experiment is called as antioxidant assay by dpph method what is this dpph it is an organic chemical compound so i have a dbph the complete name is 2 comma 2 diphenyl 1 picryl hydrazil you get in two forms 2 comma 2 and one comma one so some people use two comma two some people use one comma one so it depends on which chemical you have in your laboratory and which protocol or which paper you are referring to so what is this dpp h diphenyl picrine hydrozye it is a crystalline purple color compound it is a stable free radical so i said right the free radicals are produced in the body so it is one such free radical so now what we are going to do is and this is a purple crystal color crystal form and it is insoluble in water so you have to use organic solvents like ethanol or methanol you can use so normally i use methanol because ethanol it&#39;s costly also and it evaporates very soon so if you&#39;re not managing it properly it evaporates and there is change in the concentration the concentration alters so that is why we most pro most we prefer methanol methanolic solution of dpp hb user and some people use 0.1 millimolar some people use 8 mg that is 0.008 percent so i have fixed 0.004 percent so what is 0.004 percent it is nothing but 4 mg of this db ph dissolved in 100 ml of methanol will give me 0.004 percent so like this if you want 50 ml so accordingly you calculate it will become 2 mg in 50 ml methanol so this is the first to the agent that we need and very very important thing is so it is light sensitive always whenever you&#39;re weighing it or whenever you&#39;re doing this experiment try to do it in as much as dark condition it is possible so you can&#39;t con cover the entire lab with or something so at least that space that you&#39;re working make sure that it is dark and also whenever you&#39;re preparing this dp ph prepare it in a volumetric flask or in glass beaker and cover it with aluminum foil surround it with aluminum foil two to three layers and even the top bottom so make sure it is completely dark and also at least whenever you&#39;re doing this experiment maintain the dark condition if not completely at least some uh try to not expose it to direct light so that is very important thing so that is about your dp ph now what is the entire principle of this so i said you this dpph is a free radical if your plant is having any antioxidant molecules those antioxidant molecules will come and react with this dpph which is a free radical and they will reduce it so this dpph is a purple color crystal so when you dissolve it in methanol you will get beautiful purple colored solution you will get and when you add your extracts to this plant extract here we use ethanol extract methanol extract so i have used methanol extract so i have made another video how to prepare methanolic extracts of plant sample for those who are having any doubt or you don&#39;t know how to prepare this methanolic extract or any plant extract matter i will give you the link in the description please do go and watch this video how to prepare extract plant extracts using different solvent so now i will take this plant extract methanolic extract and i will add it to this dbph solution now what will happen all the antioxidant molecules that are present in my plant extract will react with this db ph and they will reduce them this is a free radical the antioxidants what i said antioxidants will give an electron because the free radicals will be having an unpaired free unpaired electron in the outermost shell so they will donate an electron and they will reduce them dpph will be reduced to form d p p h so this is the chemical formula so this will be purple in color so when you have the reduction it will become either gold and color or it will become colorless and that solution we are going to take the absorbance we are going to take absorbance at pi 17 nanometer because this dp ph has an absorption maxima at around 550 to 520 nanometer some papers they were taken at 517 518 520 also they&#39;re taken so with the paper you are following stick on to that i am sticking it to 517 nanometer so once i take the absorbance then i will come to know how much dpph has been reduced so there is a formula absorbance of control minus absorbance of sample divided by absorbance of sample into 100 that gives me the percentage of antioxidant activity of my plant sample so i will save the formula again make a note out of it absorbance of control minus absorbance of sample divided by absorbance of sample into 100 into 100 is because to convert into percentage so for example let me say 60 is the antioxidant activity like that you can take various plant extracts or various plants and you can find which plant is having the highest antioxidant capacity normally citrus fruits will be having like lemon gowa gooseberry all these are having high antioxidant activities chili capsicum all those things so this is the complete principle and the background explanation that you need whenever you&#39;re doing this experiment so i will say the protocol and all those things a bit wide and one more thing i wanted to say you this you have to compare with the standard antioxidants that are available you don&#39;t know whether your plant is very high antioxidant you need something to compare right so we will take some standard like how if you see for carbohydrate estimation or protein estimation we would have plotted standard graph here we are not plan plotting any standard graph we are just comparing the activity without with my plant extract to that of the standard antioxidants so what are standard antioxidants there are certain chemical compounds like ascorbic acid and one more chemical compound called as vht butylated hydroxy tauline butylated hydroxytaolin and one more compound called as bha butylated hydroxy anisole so these are some of the cocopherols so these are all the standard antioxidants bht butylated hydroxytolin butylated hydroxy anisole ascorbic acid tocopherols whichever is available in your laboratory you can take that and you can take in different concentration and see follow the same procedure and find the antioxidant activity of those standards then you will come to know your plant extract is having more antioxidant capacity or your standards are having more antioxidant capacity generally these antioxidants are used to prepare drugs like a vitamin c tablet or so many drugs they have prepared and they&#39;re also used in food industry so if you see some take any food packets and see they will add like preservatives what are preservatives preservatives are nothing but to maintain a particular food fresh to avoid spoilage of the food so there are some preservatives like bht bha they&#39;re all they&#39;re added to diary products or let it be any edible products in food industries in cosmetics they are all added to as a preservative so these are the industrial applications so now you will get an idea when you do the antioxidant activity of your plant extracts and the antioxidant activity of your standard that is available in the market which is more antioxidant whether the plant or whether your standards so that will give you clear pictures so always you have to do the antioxidant activity with using both you have to do to come back which is best your plans are good or your standards are good so this is the procedure this is the principle behind it so i will just give you summarize the entire thing so now what are free radicals free radicals are produced in the body uh no living organism as a result of many metabolism that is occurring in our body and these are highly toxic it is like a chain reaction they will be having an unpaired electron in the outer most shell they go and knock another atom that goes and knocks another atom so it is a chain reaction which leads to cell damage and finally this leads to various metabolic disorders like rheumatism arthritis inflammation cancer aging so many uh there are so many disorders that are produced because of this uh free radicals now how do we control this free radical so there&#39;s a phenomenon called as free radical scavenging where the this is unstable free radicals are they brought down to stability how they are brought down to stability so one electron is donated by these chemical compounds these chemical compounds which are called as antioxidants anti means against so these antioxidants they donate an electron and they make highly unstable free radicals into a stable compound that&#39;s what happens their damage the cost that the damage that is caused by these free radicals are controlled so this is about the antioxidant so the property of antioxidants now if you&#39;re taking any plant and you want to know what is the antioxidant molecule that is present what is the percentage of antioxidant we will follow this method called as antioxidant i say by dpph dpph is nothing but it is a free radical which is stable it is light sensitive and it is insoluble in water soluble in ethanol and methanol so it is nothing but diphenyl picturing hydrazil so now what i&#39;m going to do is now i&#39;ll save the protocol we will prepare 0.004 percent of methanolic dpph that is i will take 4 mg of dpph i will dissolve it in methanol so i will resolve it in methanol so that will give me 0.004 percentage of dpph methanolic solution first thing that is done second thing you need your plant extracts to analyze now what we are going to do is one plant x that i am taking and i will take it in different concentration i will take 20 microliter 40 microliter 60 microliter 80 microliter 100 microliter 200 microliter it depends on your so it&#39;s like trial and error method so some people will take 500 150 200 some will take 100 200 300 400 500 so it is just like trial and error method for starting with i am taking like this 20 40 60 80 100 200 micro liter i am going to take it in a test tube or in a pen of tubes anything you can take it why we are taking it in different concentration because i want to know at which concentration my plant is having the highest antioxidant capacity you may say that if i peep it out more it will be having more antioxidant right so if i take 100 microliter definitely 100 microliter will be having more antioxidant than 20 microliter i agree but after some stage the percentage becomes stable it will not change so for example if 80 microliter is giving me the highest antioxidant activity even if i take 500 microliters the antioxidant activity will not become high it will be almost similar to it for example let&#39;s say let me say 70 is the antioxidant activity for 80 microliter if i take 500 microliter also it will give 90 percent 100 above 100 it can&#39;t go so we will make different gradual concentrations to see at which concentration or at which lower concentration high antioxidant activity is seen that is the entire thing so we will take a different antioxidant concentration of this plant extracts and so there are two methods you can do after this after this you can directly add your dpph methanolic solution or you can dilute it with adding methanol and then you can add dpph so i will just explain you both but then i&#39;m doing and showing you i will just do one method so now we will take 20 40 60 80 100 microliter of plant extracts now i will make up the solution using methanol up to 2 ml or 3 ml i so for example if i added 20 microliter i will make it up by adding 980 microliter of methanol to make up the total volume to 1 ml or similarly i will make it up to using 2 ml of methanol so similarly using various concentrations i can make it up to using 1 ml or 2 ml or 3 ml methanol and then finally i will add 1 ml of methanolic db ph solution and then we will incubate it in room temperature for 30 minutes and then we will take the absorbance at 570 nanometer and then we will calculate it according to the formula that i said that i mentioned previously this is one method second method is what we can do is we will take 20 microliter 40 60 80 100 microliters of the plant extract to that itself directly i will add 4 ml of 0.004 percentage of methanolic dpph solution 4 ml i will add and then 30 minutes incubation i will leave and i will take the absorbance reading so this is all the entire protocol i will do it and show it to you so whenever i&#39;m saying anything make a note out of it because i&#39;m not showing you any protocols for you so that later you can note it down so as i&#39;m saying just note it down so now okay one more thing i wanted to say you is whenever we are taking doing absorbance using spectrophotometer or using any microplate method we need something called as blank so here the blank will be just methanol just your methanol will be your blank you will not add any bpps to it or any plant extract you will just take 4 ml or 5 ml of methanol that becomes your blank and one more thing there&#39;s something called as control i said you the formula is absorbance of control minus absorbance of sample divided by absorbance of control into 100. so what is this control so this control is your db ph solution in methanol so without adding any plant extract so it is that will be your control so what will be there there will be no plant extracts to reduce it so it will be purplish it will be purplish in color so and it will be showing the high absorbance since there are no antioxidants i&#39;m not adding any plant extracts to the control so what we&#39;ll be having is so it&#39;ll be having i absorbance will be dark purple in color so that will be your control so control is methanol and dbph that is your control and your blank is methanol and the sample is whatever i will say to the protocol and apart from this you&#39;re going to plot a standard positive graph we call it as using togopherols or using ascorbic acid using tbh or utbt or butylated pht using bht or bha so whichever is available in your lab you can use that and you can do the antioxidant activity of this so that you can analyze whether your plant extract is having more or these that is naturally that is available in the form of chemicals in the industries which is having the highest antioxidant activity so this is the complete principle procedure everything i&#39;ve discussed now i will just demonstrate over you and show so i will just let me prepare all the solutions and then i will show you how to do this procedure yeah friends now we will start the actual protocol analysis as i was saying there are many protocols in many ways that many people have conducted so stick on to one protocol like i said there are two methods or many more methods to do so i will just give you one method you can either follow this this will give you a good result now what we are going to do is we are going to take the methanolic extract of the plant sample and i am going to take this in different concentrations i am going to take 20 microliter 40 microliter 60 microliter 80 microliter 100 and 200 microliter in 5 6 different concentrations i am going to take and then i am going to make it up using methanol so if you don&#39;t want 20 40 you can go with 50 100 150 200 or if you want even more higher concentrations 100 200 300 you can watch just a trial and error method so if this method is not working then i have to change the protocol i have to change the concentration i have to increase it or decrease it so it&#39;s all trial and trial and error method so i am going to take 20 40 60 80 100. so here i have test tubes i have marked each concentrations over here i have labeled it and 20 microliter i am going to take and then i am going to make it up to 3 ml using methanol so if i&#39;m taking 20 microliter then i&#39;m going to make it up by adding 2980 microliter of methanol similarly if i&#39;m taking 40 microliter of plant extract then i&#39;m going to add 209 2960 microliter of methanol and then i&#39;m going to make it up to 3 ml once after i make it up to 3 ml then i&#39;m going to add 1 ml of dp ph solution so i said you that dbph it is light sensitive so i prepared and i kept it in the dark always prepare the solution cover it with aluminium foil like this label it and store it in a dark and cool place don&#39;t expose it to sunlight it is a light sensitive chemical so be very careful when you&#39;re doing this dpph reaction and also it is very costly so one gram costs about 10 000 rupees so just make sure you are just preparing it has to be freshly prepared so what is the necessity just prepare that much don&#39;t prepare excess and waste it is very costly chemical and also it is very much heat and light sensitive so always cover it all the four sides with aluminum foil and keep it in dark place and also as much as possible try to do this experiment in dark conditions may be weighing or adding all those things you can do it in bad condition so this is all the protocol my dear friends so you are going to take different concentrations of your plant extract so i have given a separate video how to prepare their methanolic plant extracts here i have prepared my plant sample various methanolic extracts i have prepared so i will give you the link in the description how to prepare methanol extract or any solvent method for that and you can see that uh video and prepare methanolic extract of plant sample you can actually prepare ethanolic extract also i am following methanolic extract and different concentrations i&#39;m going to prepare it out and then i&#39;m going to make it up to 3 ml adding methanol then i&#39;m going to add 1 ml of dp ph solution 0.004 mg i have taken i mean 0.004 grams i have taken in 100 ml that is nothing but zero point double zero four percent it is on almost equal to zero point one millimolar like that so there are different concentrations again so whichever paper you&#39;re following follow that protocol so according to my paper i&#39;m following this protocol so without much extension let me start i&#39;m going to take leaf extract i have taken so here i have kept methanol is ready everything is ready you need methanol to make it up and your plant samples yes so i&#39;m going to take 20 microliter so make sure when you&#39;re preparing the values are proper don&#39;t make any pipetting errors so first 20 microliters and in the second tube 40 microliters this is 20 and 20 more 40 microliters then third one will be 60 microliters 60 microliters and 80 microliters fourth one and 100 microliters 100 and the last one is 200 100 more yes it is done so plant extract has been over now i i&#39;m going to make it up using methanol so using the methanol i&#39;m going to make it up to 3 ml so if you have taken 100 200 300 accordingly calculate it and then make it up to 3 ml so since i have taken 20 microliter for the first one i&#39;m going to add 2980 microliter for the second one two thousand nine sixty microliter two thousand nine forty similarly i&#39;m going to add methanol and i&#39;m going to make it up make up the total volume to three ml so yeah friends now i&#39;ve made it up if you can see so this is 20 microliter i made it up to 3 ml similarly this is 40 you can see the concentration is increasing it is becoming more darker and darker green and the last one is 200 microliter is being the complete green you can see the concentration so you should find the color difference like this whenever you&#39;re doing serial concentration so this is my plant extract on the other side we should also do the positive thing i was talking about there are many things like ascorbic acid butylated hydroxytolamidulated hydroxy antisole all those things so i have taken the dilated hydroxyl towline bh3 we call it as so this bht is also insoluble in water so i&#39;ve taken methanol as a solvent and then i have made a stock so this is my stock so this is 10 mg per ml stock i have made so i have taken 0.1 gram of bhd butylated hydroxytolate it is a white crystalline form and i have dissolved using methanol and made it up to 10 ml so this is 10 mg per ml stock from this again same concentrations i&#39;m going to pip it out like how i did my plant samples same 20 micrometer 40 microliter 60 microliter 80 microliter 100 microliter 200 microliter and then again i&#39;m going to add methanol and then i&#39;m going to make it up to 3ml so same thing this is called as positive graph so we need to compare our plant extract how much effective it is how much antioxidant activity is having when compared to industrially available in the market there are some compounds whether it is as effective as that or less effective or more effective so we need some comparison so we are just doing this so let me prepare all these things and make it up to 3ml and then i will show you how to proceed so yes even the phd related hydroxytolane also i have prepared into serial concentrations and i made it up to 3ml using methanol now what is the formula formula says absorbance of control minus absorbance of sample divided by absorbance of control into 100. so what is this control so this control will be 3 ml of methanol and 1 ml of dpph solution so there will be no having no plant extract or nothing it is without the plant extract that will be our control so this has to be having an higher absorbance when compared to the other thing so i have said to the principle and all those things because this uh dpph is a free radical they get reduced by the presence of this plant extracts when the antioxidants present in the plant extracts and the color gets changed from purple to oranges or yellow sometimes even colorless so if there are more antioxidants all the dpph gets reduced and it becomes colorless solution if there are less antioxidants only few dpph gets reduced and you will get a golden color or orangish lowest color you will get so based on that we will come to know so this solution this is my control where i&#39;m having 3 ml of methanol and i&#39;ll be just adding 1 ml of dpph since there are no antioxidants this will remain as purplish on itself and it will give me greater absorbance the absorbance value will be high and whenever we are doing spectrophotometric we need something called as blank to make it auto zero so for that black use just methanol so whatever solvents you you&#39;re using if you&#39;re using ethanol your blank will be just ethanol if uh since i have used methanol my blank will be just methanol now everything is ready now i&#39;m going to add dp ph to it so i&#39;m going to add 1 ml of dpph and then i&#39;m going to incubate it in the dark for 20 to 30 minutes and then i&#39;m going to take the absorbance at 517 nanometer so as soon as i add this dpph you can find it is purple in color but as soon as i add the reaction will occur and it will become orangish color so one ml i&#39;m going to add for everything so you can find it is pinkish color so now i&#39;m adding in 20 micro tutorial plant extract so it has just retained slight pinkish color you can find similarly i&#39;m going to add for all the cubes including the positive thing so you can see the color change i&#39;ll show you after 30 minutes then you can find it beautiful transformations you can find see as i am adding you can find the reaction unlocker and reduction reaction occurs and the color changes that is the beauty of this experiment so you can see this so green it is dark dark green it is because it is 200 microliter but just as soon as i add this the color changes so why we have taken different concentrations we will come to know which concentration gives me the best percentage so yes i have added for everything now let me add for the control also control will be just methanol 3ml methanol and one mldb ph solution so it will have that purple color pinch will be there so you can see that purplish color so now let me let us add it to the standards also php see bht lower concentration that pinkish color is there but in higher concentration if i add since bh3 is an antioxidant it will reduce all the dpp edge and the color will change can you see so the pinkish is disappearing slightly and it&#39;s becoming colorless so if you leave it after 30 minutes and observe it will be completely colorless and also you need to shake vigorously and keep it in dark for incubation so after you add shake it vigorously after 5 10 minutes again you can shake it and keep it in incubation in dark place so the color is already changed for the higher concentration it has already become colorless yes so this one if it is any remaining keep it in the dark again for further experiments so you can see here this is my 40 micro and 200 microliter and this is my 20 microliter the pink color is still there while here the pink color has been disappeared that what is the reason behind it so whatever dph was there it got reduced and it has become colorless or light yellowish color so now we will keep this in dark for incubation and then after 20 or 30 minutes we&#39;ll note down the time and we will take the absorbance at 517 nanometer and using the formula we have to calculate the percentage of antioxidant activity so i will show you after 30 minutes so the color difference and i&#39;ll take the absorbance reading and then i will see you how to calculate it so friends 30 minutes has been over incubation i will show you how the color change has been occurred you can notice it so beautifully that so this is my control you can see the control how it is pinkish in color so we have just added methanol and dpph so there are no antioxidants to reduce it so it has retained its purple or pinkish color while if you see this bht 20 micro liter it has been slight a lowish or greenish in color but when you see this 200 microliter has completely become colorless this is because whatever dpph that we have added this vht acts as an antioxidant and it gets reduced into dpp hh and it forms a lowish or colorless solution like this so you can see 100 microliter has been there has been color change so you can compare these two and find this is in case of your standards in case of a positive graph now when we move on to plant extract in plant extracts 20 microliter has light pinkish you can find it completely does not reduce because 20 microwater has lesser concentration of antioxidants so it has that pinkish color has is been there still but on the other hand if you see the other higher concentrations i you can find it over here the pinkish color has completely disappeared so here the antioxidants that are present in the plant sample reacts with this and reduced this into and it has formed this yellowish color so now we will go take the absorbance at 570 nanometer and then using that formula we can calculate to find the percentage of antioxidant activity in my plant samples so friends this is the formula that we are going to apply to calculate the antioxidant activity absorbance of control minus absorbance of sample divided by absorbance of control into hundred this android is to represent it in the percentage form and this is my absorbance of this is the absorbance of my control and this is the absorbance of my sample so this i will show you one so for this absorbance of control 0.11 0.118 minus absorbance of sample 0.082 divided by 0.118 into 100 so if i do this calculated 0.118 minus 0.082 divided by 0.118 into 100 so if i do in 200 i will get 30.5 percent so this is for 20 microliters similarly this is for the other microliters so i have got the antioxidant activity is 30 so like this you can whatever absorbance you get for your standard graph as well as for your plant sample using this formula you can calculate the all the percentage of the antioxidant activity so this is how we calculate the antioxidant essay using dpph method so thank you for your patience watching if you like this video video do give a thumbs up and subscribe to my channel for more biology related video thank you

Transcript for:Analyzing Antioxidant Properties of Plants

Transcript for:
Analyzing Antioxidant Properties of Plants