SOP: Estimation of Total Anthocyanins
GIFSQ — Global Institute for Food Safety & Quality
Principle
Anthocyanins are extracted with ethanolic HCl (95% ethanol–1.5 N HCl, 85 : 15) and the colour is measured at the wavelength of maximum absorption. The total anthocyanin content is calculated using the molecular extinction (ε max) values given in the source’s Table 4-1, or by establishing the average extinction coefficient for the pigments present. The procedure given is for cranberries; with suitable modifications it may be used for other plant materials.
Apparatus required
- Blender
- Glass-stoppered bottles, 500-ml
- Buchner funnel; Whatman No. 1 filter paper
- Fine porosity sintered glass funnel or polyvinylchloride (Polyvic) millipore filter
- Volumetric flasks, 500-ml
- Spectrophotometer
Reagents
a. Ethanolic HCl: 95% ethanol–1.5 N HCl (85 : 15).
Procedure
Extraction
Blend 100 g of the fruit with 100 ml of ethanolic HCl in a blender at full speed
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Transfer to a 500-ml glass-stoppered bottle, using approximately 50 ml of ethanolic HCl for washing the blender jar
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Store overnight in a refrigerator at 4 °C
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Filter on a Whatman No. 1 paper using a Buchner funnel; wash the bottle and the residue on the filter paper repeatedly with ethanolic HCl until approximately 450 ml of extract is collected
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Transfer the extract to a 500-ml volumetric flask and make to volume
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To prepare the extract for spectrophotometric measurement, filter about 25 ml through a fine porosity sintered glass funnel or a Polyvic millipore filter
Determination
Dilute a small aliquot of the filtrate with ethanolic HCl to yield optical density (OD) measurements within the optimum range of the instrument
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Store in the dark for 2 hr and measure the colour at the absorption maximum (533 nm in the case of cranberry extract)
Establishment of the extinction coefficient
Extract the pigments from the plant material, purify, separate into individual pigments chromatographically, and crystallize or precipitate with ether by any one of the procedures in SOP: Extraction, Purification and Separation of Anthocyanin Pigments
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Prepare stock solutions of the pigments in ethanolic HCl; dilute aliquots of the stock solution to 25 ml or a suitable volume with ethanolic HCl; allow to stand for 2 hr in the dark and measure the colour in triplicate at the wavelength of maximum absorption
Alternative procedure: identify the anthocyanins present in the extract (by the grape procedure in SOP: Extraction, Purification and Separation of Anthocyanin Pigments, or by SOP: Separation and Estimation of Individual Anthocyanins); from the source’s Table 4-1 note the extinction coefficient of the pigments present and find the average (as in the source’s Table 4-2 for cranberry); extract a known weight of the plant material with the solvent used for determining the extinction coefficient, make to a known volume, find the absorption maxima of the extract and note the optical density at the wavelength of maximum absorption
Calculation
In ethanolic medium, the peak absorption of cranberry anthocyanins is: cyanidin glycosides — 535 and 536 nm; peonidin glycosides — 532 nm. Values at 535 nm are used in the calculations of total anthocyanin content.
Total OD per 100 g of berry = [OD × V₂ × V₁ × 100] ÷ [v × W]
The E value for a 1% solution (i.e. 10 mg per 1 ml) at 535 nm is 982; therefore the absorbance of a solution containing 1 mg per ml is 98.2.
Total anthocyanin content, mg/100 g of berry = Total OD per 100 g ÷ 98.2
Where,
- OD = absorbance measured at the absorption maximum (535 nm for cranberry)
- V₂ = volume made up of the extract for colour measurement (ml)
- V₁ = total volume to which the extract was made up (ml)
- v = ml of the extract taken for dilution before colour measurement
- W = weight of the sample taken (g)
To illustrate: 100 g of berry was macerated, filtered, washed and made to 500 ml as given under extraction; 2 ml of the extract was made to 100 ml with ethanolic HCl, the absorbance of which was 0.4 at 535 nm —
Total absorbance per 100 g of berry = (0.4 × 100 × 500 × 100) ÷ (2 × 100) = 10,000
Total anthocyanin content = 10,000 ÷ 98.2 = 101.8 mg/100 g of berry
Precautions
- Allow the diluted extract to stand in the dark for 2 hr before measuring the colour.
- Dilute the filtrate so the OD falls within the optimum range of the instrument.
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.
- Fuleki, T. & F.J. Francis, J. Food Sci., 33, 72 (1968).