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Dear Mohamad,

The measurement of anthocyanin in leaves is very easy. please find attached file.

Wishing you the best

Gitelson et al.;

2001 American Society for Photobiology 0031-8655/00 $5.00

Photochemistry and Photobiology, 2001, 74(1): 38–45

Optical Properties and Nondestructive Estimation of Anthocyanin

Content in Plant Leaves

¶ 

We use the following method for the determination of anthocyanin: extract the leaf sample with alcoholic HCl. Alcoholic HCl consist of 85% ethanol(95%)+15% HCl 1.5N. after the extraction,filtrate,centrifuge and then take 1ml of the clear solution and dilute it to 10ml.then read absorbance at 535nm. calculate anthocyanin concentration as follows:Anthocyanin (mgper100g fresh wt)= absorbance at 535nmx volume of exracrtion solutionx100/ wt of sample ingmx98.2

As you can see from the other posts, Spec based estimations are pretty simple. They might be suitable for your requirements, but they may not.

We typically extract flavonoids (including anthocyanins) from ~20-50 mg DW in 70% acidified methanol (70:30;  methanol:10% acetic acid), followed by a 90% acidified methanol extraction. The extracts get pooled and concentrated in a speed vac, then made up to a consistent final volume. An aliquot is then diluted in 0.1N HC;/methanol and the absorption at 530nm is measured.

Using Beers' law (A=ecl, where e is extinction coefficient, c concentration and l pathlength = 1cm) you can estimate the anthocyanin content. It is typical to use an extinction coefficient from a common anthocyanin (e.g. C3G) and express the data as mg/g DW anthocyanin (C3G equivalents). Make sure to account for your final volume of the extract, and the dilution factors.

Some comments/suggestions:

These assays do not only measure anthocyanins, they measure how much an extract absorbs at ~530nm. This is usually anthocyanin, so is a good estimation for anthocyanin content. But there can be other compounds that absorb at these wavelengths in some taxa, so be careful.

If you want reliable data, you should extract from dried ground tissue and express as anthocyanin (e.g. cyanidin-3- glucoside equivalents, or the anthocyanin you are using for the extinction coefficient) per mgDW. Fresh weight varies a lot.

Is your sample below the detection limit? You can increase the amount of sample/0.1N HCl, but don't exceed 10%.

If your sample doesn't turn pink (even faint) in 0.1N HCl, there isn't any anthocyanin in there... or at least, levels are far too low to report.

Have a look in good papers for their methods.

Cheers,

N

For large numbers of leaves or in vivo determinations, it is advisable to set up a green LED apparatus as in Van der Berg and Perkins http://www.uvm.edu/~pmrc/490305hs.pdf

Sims & Gamon 2002 is the best!

how about the measurement unit ? I mean should it be in mg/L or others ?

A530, the absorbance at 530 is sufficient to achieve comparisons among different leaves. The conversion in concentration relative to surface reuires a standard or the identification of the anthocyanin. https://jxb.oxfordjournals.org/content/51/347/1107.full

To calculate Anthocyanins try: (A530/cm2=A530-1/3 A657/total leaf area(cm2)

Muayed Fadhil Abbas Can i know the reference of this equation, please ?

Muayed Fadhil Abbas Do you have a reference for the method? thank you

I copied our lab protocol for spec assays. Ideally you'd use HPLC, but spec can be ok if you know the red compounds are anthocyanins, for example. And the flavonoid measurements will be affected by other UV absorbing compounds. But the correlation and trends are pretty good between spec and HPLC (though absolute quantities might be off - which isn't such an issue usually, because even with HPLC quantities are usually expressed as equivalents to one standard.)

As for trying to do this on leaf discs - that might be tough, you need a reasonable amount of compound to detect it reliably. But have a go.

Extraction and Quantification of Flavonoids and Anthocyanins from Absorbance Values using spectrophotometer

Weigh out 3 x 20 mg DW samples for each tissue into separate eppendorfs

(Weight used will depend on tissue and pigment content. Aim for a clearly coloured extract – if colour too faint then readings tend to be outside linear range)

Extract overnight in 1 ml 70:30 MeOH:Water (with 10% acetic acid in water i.e. if making 100 ml then 3 mls acetic acid)

(if useful can use more extraction solvent. This is a general method – adjust as appropriate)

Spin (3min x 10000rpm) Remove supernatant to new eppy. Resuspend pellet and do 2nd extraction over night in 90% MeOH (with 10% acetic acid in water)

Dry down first supernatant to 100-200μl. When 2nd extraction is spun down remove supernatant and add to original. Dry down to approx. 200μl, make up to 200μl with RO water if necessary. Add 800μl of MeOH. Mix . Spin (3min x 10000 rpm) to ensure any solid matter pellets out.

Estimate the total flavonoids (flavonols, flavones etc.) by reading absorption of 10-20 μl of extract in 1 ml of MeOH. Use quartz cuvettes.

Wavelength = 350 nm and E = 14300

Make sure absorbance is between 0.3 and 0.7 AU

Use Beer=s law to calculate concentration

A = EC l A/E = C

Then remember to multiply by the dilution factor

Convert to mg/ml by multiplying by 610 (MW for Rutin) Quantities determined as rutin equivalents

Convert to mg in 1g of sample by multiplying by (1000/wt (mg))

Similar procedure used to estimate the total anthocyanins. Use new aliquot of the same extract but measure in 0.1N HCL (in MeOH) at visible maximum (530 mm) and E = 35000. Absorbance of 0.1 is ok. Values of 0.01-0.02 is regarded as no anthocyanin present. (0.1N HCL in MeOH = 0.83ml conc HCL in 100 ml MeOH)

I do 2 readings for each extract and average them. Calculate quantities for each extract and then determine mean and SE for the 3 extracts from each sample

Use MW of 647 (Cyanin – cyanidin 3,5 diglucoside) for anthocyanin readings

Conc = Abs/35000 x dil factor (50 ul of extract in 950 ul of methanolic HCL gives a dilution factor of 20) x 647x 1000/mg of sample extracted = conc mg.g-1DW or FW

DW = dry weight can use fresh material too

Method described in

Strack D, Wray V (1989) Anthocyanins. In: Harborne JB (ed) Methods in Plant Biochemistry, vol 1. Academic Press, London, pp 325-356

(use of absorb. Coeff.)

Francis FJ (1982) Analysis of anthocyanins. In: Markakis P (ed) Anthocyanins as food colors. Academic Press, New York, pp 181-207

(use of absorb. Coeff. And general value for all anthocyanins)

Albert NW, Lewis DH, Zhang H, Irving LJ, Jameson PE, Davies KM (2009) Light-induced vegetative anthocyanin pigmentation in Petunia. Journal of Experimental Botany 60 (7):2191-2202. doi:10.1093/jxb/erp097

(flavonoids use 14300 for rutin, anthocyanins actually use 35000 as a generic coefficient for anthocyanins despite extinction coefficient used in this paper)

Bradley JM, Davies KM, Deroles SC, Bloor SJ, Lewis DH (1998) The maize Lc regulatory gene up-regulates the flavonoid biosynthetic pathway of Petunia. Plant Journal 13 (3):381-392

(use of 35000 to quantify anthocyanins)

Giusti MM, Rodriguez-Saona LE, Wrolstad RE (1999) Molar absorptivity and colour characteristics of acylated and non-acylated pelargonidin-based anthocyanins. Journal of Agricultural & Food Chemistry 47:4631-4637

(Extinction coefficients for anthocyanins)