I am doing an optimization of ginger extract using doe software, firstly I extract the ground ginger with 20ml solvents of 60%, 70% and 80% ethanol using the ultrasound water bath in different temperature, afterwards, i filtered the extract from conical flask with cotton, then i collect it in a centrifuge tube and put for centrifugation at 5100rpm, 10 mins, then i filter again using whatmann filter paper, the supernatant is collected to do tpc, tfc and dpph assay, is this way accessible or it is wrong? Is rotary evaporator a must before tpc, tfc and dpph assay? please advice me.
Hi there - Your procedure looks good! The analyses of TPC, TFC and DPPH can be done using the collected supernatants, as long as you know their concentrations (i.e., weight of ginger samples, volumes of solvents used in each extraction). You will need them later to calculate the values. However, you will want to take a known volume of the supernatant and run it down using a rotary evaporator so that you get the extraction yields for further use. Good luck with the work! .. NK
@Nisit thank you so much for you confirmation. For the extract yield, I have done it using the second time filtration, I measured what is left on the filter paper after letting it dry naturally under ambient temperature, is this accessible?
Jing Yi Tan According to your procedure, what is left on the filter paper after 2nd filtration would be either the plant matrix that got through the cotton filter, or something that precipitated from the extracted solution after centrifugation, which are not what you want. The extract yield should be calculated from the supernatant which contained the dissolved components. Taking a small, known volume and dry it, take the weight of what is left and back calculate to the total solvent volume used to get the whole extract amount. Times 100 and divided by the weight of the original Ginger material, and you get the extract yield (%).
Nisit Kittipongpatana , thank you again, but i have few more questions, so sorry as there isn't a lot of journals about this and i am still as clear as mud. So i use the supernatant after second filtration to do tpc, tfc assay is ok? does the solvent (I'm using 60%,70% or 80% ethanol as solvents) in it interfere the tpc and tfc assays? As i am told to use rotary evaporator before the assays. Is there any journals that use similar methods as i am? and the extract yield is important for what calculations afterwards?
Jing Yi Tan I assumed you were using 60,70,80% ethanol to see which one worked the best. Each of them will give you different yield and, maybe, different chemical profile/compositions, which will affect the test (TPC, TFC, DPPH) results. By using the original extracted solutions, the results you get will reflect the effect of the solvents. You will need to do controls using each solvent in each test and subtract the absorbance from that of the sample before final calculation. As for the extract yield, because you do the analysis using the (extract) solution, you would want to know how much of the (dry) extract is actually in it, thus taking a small (separate) portion of the extract solution and dry it to take the weight. TPC and TFC are usually reported in the unit of (standard) equivalent ug/mg of extract.
A link to an article that used supernatant part for the analyses.
Article Total phenolics, flavonoids and antioxidant properties of th...
though the authors reported the values as g of plant material used.
@Nisit yes Dr, I use three different solvent concentration to test the optimum concentration for ginger extraction. In my understanding, means that I will have to prepare 3 blank solutions using (60,70 and 80% ethanol solvents) and measure the absorbance according to the sample extract solvent concentration (et. Run 1, 200mg, 60c, 60%ethanol ; so I measure the absorbance with blank solution using 60% ethanol and change the blank with other concentration) in uv spectrometer right? Do this means that I have to do 3 different standard curve?
Extract yield can I do like this? Say I take 2ml of the crude extract and drop it on pre-weighed filter paper, then measure again after drying at ambient temperature, or better using rotavap? But for all my extraction I use only 20ml of solvents no matter how much quantity of my ginger. (200mg ginger/20ml solvent, 400mg /20ml, 600mg/20ml)
In this calculation right?
2ml to dry and weighed =x
(X/ori ginger material) x100% = extract yield
Jing Yi Tan Yes, three blank solutions for each test. And they have to go through the same protocol as the sample (all reagents and conditions as per the test, so the difference in the absorbance recorded was due to the compound(s) in the extract). Using a microplate for holding all the samples and blanks would be ideal for UV abs measurement. Only one standard curve for each test is needed because what will be compared (and calculated) is the absorbance.
For small scale, using your method of drying the extract on a filter paper is o.k., as long as the sample is all absorbed on it. Use an analytical balance, as the weight difference will probably be small.
For the calculation, because the original volume was 20 ml and you only take 2 ml, so to back calculate you need to time 10 to the X to obtain the extract dry weight of the sample. This number can then be divided by the material weight used, and times 100 to get the % yield.
Nisit Kittipongpatana Thank you so much Dr ! now i have a clear picture of all of it
Hi, Nisit, how are you?
When doing quantitative measurement of an extract, don't forget to consider its water content.
@Nisit
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