Anthocyanin extraction method

The extraction of anthocyanins is the first step in the determination of both total and individual anthocyanins in any type of plant tissue. The choice of an extraction method is of great importance in the analysis of anthocyanins and largely depends on the purpose of the extraction, the nature of the anthocyanins, and the source material. A good extraction procedure should maximize anthocyanin recovery with... 

Hence better extraction methods seem necessary, but this -problem is not simple. The laws of extraction are complex in the juice there is a simultaneous dissolution of phenolic compounds from the skins and seeds and their precipitation onto yeasts and other solid particles in suspension. Finally, the unstable anthocyanins are partially destroyed at this stage. 

This is the classical method of extracting anthocyanins from plant materials. This procedure involves maceration or soaking of the plant material in methanol containing a small concentration of mineral acid (e.g., HC1). Methanol extraction is a rapid, easy, and efficient method for anthocyanin extraction. However, a crude aqueous extract with several contaminants is obtained, and methanol evaporation can result in hydrolysis of labile acyl linkages, which is aggravated by the presence of HC1. 

Due to the increasing demands of anthocyanin extracts together with the market and prize competition, blending and adulteration of berry products appears to become attractive. Consequently, detailed analysis of anthocyanins is necessary to prove the content, composition and source of anthocyanins. Traditional methods for the determination of the "total anthocyanin" content by UV/VIS spectroscopy are nowadays augmented by HPLC with UV-detection and mass spectrometry. Only the latter techniques fulfill the current need for reliable results in terms of content and identity of individual anthocyanins. Based on this information, the real versus the labeled amount of anthocyanins, the identity of the... 

Consequently, detailed knowledge of the chemical properties of anthocyanins occurring in berries and of analytical methods used are important to judge the quality of anthocyanin extracts from berries. The following overview introduces the chemistry of berry anthocyanins and their occurrence in individual berries, addresses all relevant topics of anthocyanin analysis, substantiate the need to replace simple by sophisticated methodology and proposes a sequence analytical methods for quantitative and qualitative analysis under routine conditions. 

The first step of each analysis is the extraction of anthocyanins. The majority of extraction methods are comprised of polar organie solvents, mostly as mixture with strong acids. Polar, organic solvents seeure complete dissolution of the anthocyaiuns. A strong acidic pH is important in terms of stability and of stable UVA71S spectral properties. 

For specific purposes (pH-differential UVA/ IS method, see below) aqueous solution/extraction is used. Although this method likely underestimates the anthocyanin content in fresh berries several authors reported similar results for aqueous solution/extraction compared to the methods described before for dried anthocyanin extracts [38], Aqueous extraction with diluted HCl can also be considered if further purification/ enrichment steps are planned. Also for technical scale extraction, aqueous extraction comprising sulphur dioxide, acetic acid and water has been described [124],... 

Yang Y, Zhao S, Xu Y, Yu Z (2012) Optimization and comparison of three methods on anthocyanins extraction from blackcurrant (Ribes nigrum L.) using RSM. Adv Mater Res... 

The solvent extraction is the most used method for phenolic compoimds obtainment from plant tissue. The main factor is the phenolics solubility, which depends on its chemical structure. Plant materials may contain different concentrations of phenolic acids, phenylpropanoids, anthocyanins and tannins. It is possible to occur interactions between phenolics and other plant components, such as carbohydrates and proteins which form complexes responsible for insolubility. Besides that, the polarity of solvent affects the solubility and therefore, it is eonsidered difficult to develop a extraction method suitable for all plant phenolics [18]. 

Among the most common methods are those which use acidic methanol (1% HCl) as extracting solvent at room temperature [Vareed et al., 2006]. Jayaprakasam et al. (2005) used acidified water (pH = 3) to isolate anthocyanins from Cornus officinalis and Cornus mas fruits, Moldovan et al. (2014) reported the extraction of pigments from frozen Cornelian cherries in acetone while Pantelidis et al. (2007) used air dried fruits (at 55°C) and 50% aqueous methanol as solvent. In the anthocyanins extraction from Cornus alba Sibirica the plant material was extracted with methanol containing 0.2% trifluoroacetic acid [Bjoroy et al., 2007]. 

The quantity of anthocyanins and tannins found in wine depends first of all on their concentration in the grape crop. Ripe grapes are the first condition for obtaining rich and colored wines. However, only a fraction of the phenolic compound potential of the grape is found in wine. Their concentration depends not only on the ease of phenolic compound extraction but also on the extraction methods used. The phenolic compound concentrations of various components of grape clusters and wine have been compared. Approximately 20-30% of the phenolic potential of grapes is transferred to... 

Another practice, that does not produce negative effects on health but is considered as a food adulteration, is represented by the blending of wines with a wine very rich in anthocyanins ( Rossissimo ) or by adding anthocyanins extracted by black rice, in order to naturally increase the colour of red wines. However, the study by Ferrari et al. [66] deserves to be mentioned here because it not only compared two different techniques (Fourier transform near-infrared and H NMR spectroscopies), but also applied a series of multivariate classification methods. In particular, PLS-DA was used as... 

Timberlake and Bridle (1971) and later Wrolstad and Durst (1998) compared the acetone-chloroform method of extraction to acidified methanol and, depending on the material used, the anthocyanin recoveries were comparable or up to 30% higher with the use of acetone. This procedure proved efficient for a wide range of anthocyanin-rich plants to achieve high recoveries with minimal degradation. - ... 

The optimized RPLC UV-Vis ESI MS method for all typical blue colourants (indi-goids, hematein, tannins, anthocyanins and selected flavonoids) was used for the identification of dyes extracted from a thread taken from an Italian tapestry of unknown origin from the collection of the National Museum in Warsaw (Poland). It was found that to obtain the red-blue colour of the fibre a mixture of dyestuffs was probably used. The presence of indigotin, tannic and ellagic acid (at m/z 301, NI), as well as carminic acid, suggested the use of indigo and cochineal. Reseda luteola could also have been used due to the presence of luteolin and apigenin. 

Natural colours include annatto, anthocyanins, beetroot red (betalaines), caramel, carotenoids, cochineal and lac pigments, flavanoids, chlorophylls and tumeric. There is a trend towards encapsulating natural colours for food use, but this is not yet reflected in the extraction techniques described in the published analytical methods. Lancaster and Lawrence (1996) described the extraction and... 

Novel pyranoanthocyanins have also been isolated and identified in blackcurrant (Ribes nigrum) seed using HPLC, 2D NMR and ES-MS. Blackcurrant seeds were extracted with acetone-water (70 30, v/v) and the components of the extract were separated in a polyamide column followed by HPLC-DAD. The new pigments were finally separated in an MCI-HP20 column. The chemical structures of anthocyanins 1-2 and the novel pyranoanthocyanins 3-6 with the pyrano[4,3,2-de]-l-bcn/opyrylium core structure are shown in Fig. 2.110. It was stated that the analytical method developed separated well the novel pyranoanthocyanins [245],... 

The anthocyanin composition of flowers has also been frequently investigated by chromatographic methods. Acylated anthocyanins and flavonols have been extracted from the flowers of Dendrobium cv. Pompadour and separated by TLC and RP-HPLC techniques. Fresh flowers (approximately. 1 kg) were extracted with 101 of methanol-acetic acid-water (4 1 5, v/v), and the extract was concentrated and analysed by preparative and analytical RP-HPLC using ODS columns (150 X 19 mm i.d. and 250 X 4.6 mm i.d.) at 40°C. The gradient consisted of methanol-water-acetic acid in various volume ratios. Flow rates were 4 and 1 ml/min for preparative and analytical separations, respectively. Cyanidin 3- (6-malonylglucoside)-7,3 -di(6-synapilglucoside) and the demalonyl derivative were detected in the flowers [261],... 

The anthocyanin profile of the flowers of Vanda (Orchidaceae) was investigated with a similar technique. Flowers (2 kg) were extracted with 101 of methanol-acetic acid-water (9 l 10,v/v) at ambient temperature for 24 h. The extract was purified by column chromatography, paper chromatography, TLC and preparative RP-HPLC. Analytical HPLC was carried out in an ODS column (250 X 4.6 mm, i.d.) at 40°C. Gradient conditions were from 40 per cent to 85 per cent B in 30 min (solvent A 1.5 per cent H3P04 in water solvent B 1.5 per cent H3P04, 20 per cent acetic acid and 25 per cent ACN in water). The flow rate was 1 ml/min and analytes were detected at 530 nm. The chemical structures of acylated anthocyanins present in the flowers are compiled in Table 2.90. The relative concentrations of anthocyanins in the flower extracts are listed in Table 2.91. It can be concluded from the results that the complex separation and identification methods (TLC, HPLC, UV-vis and II NMR spectroscopy, FAB-MS) allow the separation, quantitative determination and identification of anthocyanins in orchid flowers [262],... 

A novel pigment has been isolated from the petals of Rosa hybrida with complex chromatographic techniques and the structure was elucidated with spectroscopic methods and high resolution fast-atom bombardement mass spectrometry, lH NMR, and FTIR. Anthocyanins were extracted from 7.9 kg of petals of Rosa hybrida cv. M me Violet with 80 per cent aqueous ACN containing 0.1 per cent TFA. The extract was purified in a Sephadex LH-20 column, and the fraction eluted with 80 per cent ACN was further fractionated in a HP-20 column using water, 15, 20 and 30 per cent ACN as mobile phases. The last fraction was lyophilized and separated by preparative RP-HPLC using an ODS column (50 X 5 cm i.d.). Solvents were 0.5 per cent aqueous TFA (A) and water-methanol... 

A correlation may be established between the concentration of oxidized lipids and the TEARS value, expressed as MDA equivalents, in uM units. Correction is due in some cases for the interference by dyes or other factors. For example, the presence of anthocyanins in red cabbage leaves or turbiditjf causes overestimation of lipid hydroperoxides in plant tissue by the TEARS method. TEARS was used to assert the level of endogenous peroxides in hypo- and hyperthyroidism, both conditions being characterized by low lipid and lipoprotein plasma levels and enhanced oxidative metabolism . In a procedure for determination of TEARS in edible oils, the sample is placed in a centrifuge at 12000 g before measuring at 532 nm (e = 1.56 x 10 M cm ) . A usual procedure for determination of TEARS in certain complex matrices involves steam distillation of the aldehydes responsible for the value, instead of extraction. In nitrite-cured meats, excess nitrite may cause nitrosation of MDA, thus interfering with distillation. To avoid this interference sulfanilamide is added, which is converted to a diazonium salt and... 

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