Elisa please see the method in the attached paper. Cheers

Thanks a lot Bernardo!

Estimation of anthocyanins:

A known weight of fresh leaf tissues was soaked in 3 ml of acidified methanol (1% v/v HCl) for 24 h in darkness at 4°C with occasional shaking. About 2 ml of dist water and 4.8 ml of chloroform were mixed and added to the extract. The mixture was then centrifuged for 15 min at 5000 rpm. The absorbance of the upper phase was determined at 530 and 657nm. The concentrations of the anthocyanins as mg g-1 dry weight of the differently treated plants using the following equation:

Anthocyanins = [OD530 - 0.25 OD657] x TV/ [d wt x 1000]

OD = optical density; TV = total volume of the extract (ml); d wt = weight of the dry leaf tissue (g)

Please see this paper:

Gou, J.Y., et al., 2011, 23: 1512–1522.

Negative Regulation of Anthocyanin Biosynthesis in Arabidopsis by a miR156-Targeted SPL Transcription Factor.

Good luck!

Heng Lian

Please see the attached paper (Gou et al., Plant Cell, 2011)

Please see the attached paper (Michael M. Neff and Joanne Chory, Plant Physiology)

Please see the attached paper (Michael M. Neff and Joanne Chory, Plant Physiology, 1998).

Thanks you all!

hv a luk at this paper. access is free •

Wolfe K, Wu X, Liu RH. 2003. Antioxidant activity of apple peels. J. Agric. Food Chem. 51(3): 609-614. hope this helps u out. luk

We have used this method (see attachment, please)..

Hi Elisa. :) Here is a very simple method. ;)

Good luck.

Marta

Dear Elisa.

I've been using the method attached by Marta Nunes da Silva in wood species and it is working very well so far.

Good luck!

I could suggest the method described in attachment for wet analysis of anthocyanins in leaves in fruits. It is reliable enough and simple as well.

Hi Elisa,

You can try this method:

To measure anthocyanin according to Krizek et al (1993), 0.2g of leaves will homogenized with 3ml 1% HCL-methanol (99:1), the extract will be centrifuged at 18000 g for 30 min, at 4°C, the supernatant will leave overnight in dark place at 5°C. Anthocyanin content will measure at 550nm using spectrophotometer. The extinction coefficient for anthocyanin is 33000 cm-2mol-6.

Hi Elisa,

I guess this is redundant to the anwers above, but since I have not seen a real formula except the last (but different from here), I paste in the description in a thesis of one of my students (Yin Ruohe):

2.2.28 Photometric determination of anthocyanins

Extraction of anthocyanins of 21-DAG Arabidopsis plants was performed as following. 250 μl

of acidic methanol (1% HCl, w/v) was added to 50 mg of fresh plant material. The plant

material was homogenized on ice and then incubated at 4°C for 1h with moderate shaking.

The suspension was clarified by centrifugation (14,000 rpm, room temperature, for 5 min).

Absorption of the extracts at 530- and 657-nm wavelength was determined photometrically.

Quantification of anthocyanins was performed using the following equation: QAnthocyanins =

(A530 – 0.25* A657) X M-1, where QAnthocyanins is a corrected absorption value linearly correlated

with the amount of anthocyanins, A530 and A657 is the absorption at the indicated wavelengths

and M is the weight of the plant material used for extraction (g).

Good luck,

Tony

1% HCl-Methanol can be used and the OD can be measured at 600 nm

Buenas tardes Elisa,

Adjunto te envío una publicación mía donde medía contenido en antocianos totales en espárrago blanco, en un proyecto que trabajé sobre la problemática de la coloración rosa del espárrago blanco (que constituía una merma de calidad del producto y que se debía a la acumulación de dichos pigmentos). Espero que te pueda ser útil.

Saludos,

Borja

Upps, se me olvidó adjuntar la publi! Aquí va. Saludos. Borja

Extraction in cold acidified methanol is the common method. However, when I did anthocyanin extraction from grape berry skins I added liquid nitrogen to the mixture and froze everything and then ground it. This assures complete rupture of the cells and very high extraction rates.

Here is one :)

http://online.liebertpub.com/doi/abs/10.1089/jmf.2012.0151

Try the method in this paper

Article Ecophysiological adaptations of two halophytes to salt stres...

Please read the following file attached.

Thanks you all for your valuable help!

Here is a simple method usinf spectrophotometer

Hi

Here is a sample method.

Good luck

Content and Vacuole/Extravacuole Distribution of Neutral

Sugars, Free Amino Acids, and Anthocyanin in Protoplasts1

Anthocyanins were extracted from the oven-dried ground tissues by suspending in 10 ml of acidified methanol (methanol: water: HCl, 79: 20: 1, v/ v) and autoextracting at 0°C for 72 hours in dark with continuous shaking. The extracts were then centrifuged for 10 minutes at 5000 rpm and the absorbance was measured at 530 and 657 nm for each supernatant (Mirecki and Teramura, 1984). The absorbance readings at 530 nm (A530) were corrected for scattering using the absorbance readings at 657 nm (A657) using Rayleigh's formula as following:

Corrected A530 = A530 – 1/3 A657

Anthocyanins were calculated as the corrected absorbance (Lange et al., 1971; Mancinelli et al., 1975; Lindoo and Caldwell, 1978).

1- Extraction anthocyanin pigment from the dried ground tissues by suspending in 10 ml of acidified methanol (methanol: water: HCl, 79: 20: 1, v/ v) and extracting at 0°C for 72 hours in dark with continuous shaking.

2- Extracts were then centrifuged for 10 minutes at 5000 rpm and the absorbance was measured at 530 and 657 nm for each supernatant according to Mirecki and Teramura, 1984).

3- Readings the absorbance at wave length 530 nm as corrected for scattering using the absorbance readings at 657 nm using Rayleigh's formula as following: Corrected A530 = A530 – 1/3 A657.

4- Calculate the concentrations of anthocyanin as the corrected absorbance according to the methods described by Lange et al., 1971; Mancinelli et al., 1975; Lindoo and Caldwell, 1978).