Dear Azlan Nazeri,

From your question, I understand that you have a solubility issue with the dragon fruit peel. Secondly, your question is if IC50 of 0.52 mg/mL obtained from your DPPH assay is the median inhibitory concentration of dried Dragon fruit peel or that of the plant part in 70% ethanol?

1. Extraction:  The first part of your analysis was basically extraction which was dissolution of 2 mg of dried, pulverised peel in 10 mL in 70% ethanol. At this step of your analysis, obtaining an extract is the primary step, which includes macerating or soaking the powdered peel in for a number of hours to days [e.g. 24 hours to 72 hours (3 days) or a week]. You can refer to the methods published in journals (Muhit et al., 2010. Isolation and  Identification of compounds from the leaf extract of Dillenia Indica Linn.) described a maceration protocol. This is followed by concentrating your extract to dryness, which involves filtering the extract from the soaked pulverised plant material. Vacuum distillation is commonly used at a reduced pressure and a choice temperature (40 - 60 degree Celsius) can be used because your fruit peel may contain thermolabile constitutents (i.e. constituents that may be destroyed by heat if you concentrate your extract above 60 degree Celsius. The dried extract should be allowed to completely evaporate to dryness, in order to remove traces of the 70% ethanol to get a dried, hydroalcoholic extract.

2. Assay method: You did not cite your DPPH assay method but your concentration range (0.2 - 1 mg/mL) is good. Your stock solution which may be 2 mg/mL (as you might have initially intended) is still feasible provided you have sufficient extract .

Preparation of stock solution (2 mg/mL): Your extracting solvent, 70% ethanol is more polar than absolute ethanol, because of 30% water component; therefore you can ensure dissolution of your extract adding a few drops of 70% ethanol to your weighed extract (200 mg) in a beaker. Stirring gently and well with a spatula till there is total dissolution. This will be followed by transferring the dissolved extract into a 100 mL standard  volumetric flask and rinsing the spatula into the beaker with the ethanol concentration described in your selected assay.  The ethanol (described) should be transferred into the dissolved extract in the volumetric flask. Make up the volume of the cited solvent to the meniscus mark of the volumetric flask and proceed to derive your serial dilutions to get your concentration range.

*The IC50 derived is the median inhibitory concentration of the antiradical constituents in the hydroethanolic extract of Dragon fruit peel, which reduce DPPH radical to DPPH - H.

Cheers

Dear Omodeboyin Lawal 

Thanks for detail explaination. I'm really appreciate that. before that, I just use freeze dryer to remove water from dragon fruit peel since integrity of active compounds are still maintained. After that I just weighed, (20,40,60,80,100)mg in 10 ml 70% ethanol for each and concentration become (0.2,0.4,0.6,0.8 and 1) mg/ml. I never do macerating or soaking the powdered peel in for a number of hours to days [e.g. 24 hours to 72 hours (3 days) or a week] as you mentioned. is there any effect if my sample not fully dissolve during antioxidant activity test? I think with discard the sediment in hydroethanolic extract of Dragon fruit peel is ok. So 0.52mg/ml refer to the plant part in 70% ethanol to reduce by 50% the initial DPPH concentration, right?

Dear Azlan

For these kind of studies you can only use dissolved portion. and that is one of the drawback of these kind of studies. u can justify your antioxidant results on the basis of dissolved phenolics and terpenes.

Hi vishwanath

Do you have any experience for this case?what would you do for undissolved particle? Just discard and take out

Dear Azlan Nazeri,

I would have liked to answer your second question earlier, but I was unable to do so because I was very busy. Now, I will answer and I hope it will clarify any uncertainty concerning your DPPH assay on the Dragon fruit.

Your freeze-drying the Dragon fruit peel, only reduced the water content of the fruit and left behind the solid fruit peel with a myriad of constituents trapped in it. The constituents contained in the freeze-dried powder may contain constituents which range from fibre, sugars, vitamins to phenolic compounds, e.t.c. Extraction of the freeze-dried peel in a solvent would give you an extract which contains all phytochemicals that are soluble in the solvent.

When you measured 20 - 100 mg of the powdered fruit peel in the 70% ethanol; the solvent only solvated and extracted the hydroethanol-solubles into the solvent, while the powdered fruit peel settled at the bottom of your flask. 

The bottomline is, you need to extract phytochemicals from your dried fruit peel. The process of extraction requires a period. It may be even be within 24 hours but please refer to journals. Filtering the freeze-dried powder from the solvent (e.g. 70% ethanol) will follow this, and then concentrating your extract to dryness using a rotary evaporator at a temperature (40 - 60 degrees Celsius), plus ensuring a total evaporation of the solvent till there is an extract. Your extract may be resinous in nature or sticky or viscid, e.t.c. 

Afterwards, you can proceed with the DPPH assay by selecting a DPPH assay which describes ethanol in the method. Afterwards, dissolution of your extract when preparing a stock solution from the extract, may not pose much of a problem, provided you follow my previous answer on resolving dissolution. 

Best of luck.

dear Omodeboyin Lawal 

Thanks for detail explanation. actually after i  Filtering the freeze-dried powder from the solvent (e.g. 70% ethanol), I skip for this part which is  concentrating my extract to dryness using a rotary evaporator at a temperature (40 - 60 degrees Celsius), plus ensuring a total evaporation of the solvent till there is an extract. After filtering, i just directly analysis using DPPH assay without concentrate my extract, but I know this technique is not really accurate for my IC50 result.

Truly, I will recommend conducting the analysis again; and timing the extraction of your freeze-dried Dragon fruit powder.

A paper was published and it reported that extraction was done on the leaves of a plant, Perilla pankinensis for 2 hours. (Reference: Gulcin et al., 2005. Antiradical and antioxidant activity of total anthocyanins from Perilla pankinensis decne. Journal of Ethnopharmacology 101: 287 – 293). 

Another research study was conducted on the powdered plant, Black Cohosh for 12 hours. (Reference: Nuntanakorn et al., 2006. Polyphenolic Constituents of Actaea racemosa. Journal of Natural Products 69: 314 - 318.). The URL is:     http://pubs.acs.org/doi/pdf/10.1021/np0501031

Since Gulcin and co-researchers (above) cited doing a 2-hour extraction in a solvent with continuous stirring; it is advisible to time the extraction of your freeze-dried, Dragon fruit peel powder, by using any good publication you find as a guide.

I will also like to add that ensuring the purity of your solvent is important. You can do this by preparing your 70 % Ethanol accurately by adding 30 mL of distilled water to a 100 mL standard volumetric flask; and making up the volume to the meniscus mark on the standard flask with absolute ethanol, or 300 mL of distilled water to a 1L (1000 mL) standard volumetric flask and making up to the meniscus mark with absolute ethanol.  Alternatively, if you use redistilled solvents, ensure the ethanol supplied to you is distilled and measure the density to have any value in the range (0.7893 - 0.81 g/mL - Ref: Chemspider.com); before you prepare he 70% Ethanol.

I wish you all the best!