Extraction of Anthocyanins
Anthocyanins, natural compounds found in plants, are prone to degradation and loss of biological activity. Factors such as light, temperature, pH, oxygen, enzymes, and humidity affect their stability. To extract anthocyanins, different strategies are employed based on their complexity and desired chemical characteristics. For instance, aqueous/organic mixtures are often used, with the organic fraction playing a significant role. Conversely, more hydrophilic solvents or aqueous mixtures are preferred for extracting anthocyanins. Maintaining the ionized state of the compounds is crucial, achieved by adding inorganic or organic acids to the aqueous phase.
Ethanol/methanol:water mixtures (70-95:30-5) acidified with hydrochloric acid, formic acid, or citric acid are commonly employed for extracting anthocyanins from plant, food, and agricultural samples. Stronger and more lipophilic organic solvents, aided by ultrasound and temperature, are used for acid-resistant homogenization of matrixes like plant seeds. Liquid matrixes such as red wine, fruit pomace, and fruit juice are treated with acidic alcohol solutions.
Extraction temperature and pH also impact anthocyanin recovery. Preheating the plant material deactivates polyphenol oxidases and aids in cell disruption, increasing extraction yield. The extraction process is repeated in multiple rounds based on extractability and anthocyanin content. Optimal temperatures and pH levels vary for each batch.
While traditional solvents generate hazardous waste, efforts have been made to develop eco-friendly extraction alternatives. Natural Deep Eutectic Solvents (NADES) have emerged as a novel green extraction technique, offering biodegradability, sustainability, low toxicity, affordability, and ease of handling. NADES-based anthocyanin extraction methods have been optimized, showing comparable efficiency to traditional solvents but with better yields and reduced environmental impact.
Purification of Anthocyanins
Effective purification methods ensure better characterization of compounds, thereby ensuring more reliable spectral information. The extracted material contains various compounds such as sugars, organic acids, amino acids, and proteins, which can potentially compromise the stability of anthocyanins. After extraction, the first step in the purification process is to separate the extract rich in green and red pigments, depending on the matrix (fruit, leaves, or liquid biological samples).
The initial removal of strong chlorophyll components is necessary, followed by the purification of the matrix rich in anthocyanins (fruit, flowers, or liquid biological samples). Once chlorophyll is eliminated, purification is typically performed through different chromatographic steps, utilizing different stationary phases. For aqueous extracts, the earliest methods involved the absorption of anthocyanins on solid-phase extraction (SPE) resins such as C18 cartridges and Sephadex matrices to eliminate more polar non-retained byproducts.
With advancements in more extensive adsorption purification techniques, silica gel, Amberlite IRC 80, 237 Amberlitte XAD-7HP, and DOWEX 50WX8 have been studied for their higher adsorption and desorption capacities for anthocyanins.
In certain cases, high-speed countercurrent chromatography (HSCCC) is employed to separate specific components of anthocyanins from food. HSCCC is well-known for its low pressure, high flow rate, wide solvent compatibility, and cheaper stationary phases. It allows the separation and purification of compounds after enriching the crude extract on an XAD-7 resin column. It has been observed that HSCCC two-phase systems composed of n-butanol/ethyl acetate/0.5% acetic acid (3:1:4) and 0.2% trifluoroacetic acid/n-butanol-tert-butyl methyl ether/acetonitrile (6:5:1) are good mixtures for chromatographic separation, as confirmed by HPLC and NMR analysis.
Isolation and Identification of Anthocyanins
Reverse-phase liquid chromatography (RP-LC) is the most commonly used method for separation. Different laboratories often employ different parameter conditions to optimize the separation of anthocyanins, making it difficult to establish a single standard procedure.
Another detection technique is UV-Vis, which uses UV and visible light beams through a flow cell. It includes a sensor that detects changes in absorbance as the analyte passes through the column. UV provides information not only on glycosylation but also on the acylation degree in sugar molecules. UV-Vis spectra provide a wealth of information, especially for acylated anthocyanins, offering characteristic fingerprints in UV-Vis spectra. However, UV-Vis spectroscopy alone may not be sufficient for effective identification of anthocyanin molecules. Due to the similarity in spectra produced by two or more anthocyanin molecules, relying solely on UV-Vis may lead to incorrect detection.
Qualitative and quantitative methods to evaluate anthocyanins (Teng et al., 2020)
Currently, anthocyanin chromatographic analysis primarily relies on high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) methods, combined with UV-visible spectrophotometry and/or mass spectrometry detection. The normal conditions for HPLC involve a C18 column, a detection wavelength of 520 nm, and a solvent system of acetonitrile with pH adjusted by phosphoric acid, formic acid, or trifluoroacetic acid.
The sensitivity and simplicity of HPLC make it a popular analytical method in the qualitative and quantitative studies of anthocyanins and their complexes. Common 5 μm particle size HPLC columns are still widely used in most analytical laboratories, providing good selectivity and resolution for anthocyanins and complex mixtures of anthocyanins. UHPLC, compared to HPLC, offers shorter separation times and therefore lower solvent consumption.
Purified compounds from different chromatographic separations (silica gel, cation exchange, and/or reverse phase) can be structurally characterized using nuclear magnetic resonance (NMR), high-resolution and tandem mass spectrometry (HRMS/MSn), and Fourier-transform infrared spectroscopy (FT-IR). MS/MSn analysis aids in identifying specific fragmentation pathways. For example, anthocyanins can be differentiated from their glycosides (flavonols) by mass spectrometry-induced loss of the sugar moiety after MS/MS fragmentation. Conversely, glycosides are sensitive to cross-ring cleavage, particularly on ring C, producing distinct oxonium fragments.
In other anthocyanin studies, particularly when determining isomers in complex samples, ion mobility mass spectrometry (IM-MS) has emerged as a high-throughput and sensitive analytical method. IM-MS operates by separating gaseous ions based on size, shape, and charge. Typically, LC-MS is used for further analysis and identification of compounds separated by IM-MS at the compound level. Over the years, MS/MS databases have greatly aided in identifying plant compounds or detected metabolites, especially when complete structure determination is not possible with NMR and X-ray crystallography. Hence, tandem mass spectrometry (MS/MSn) combined with HPLC or UHPLC is a powerful tool. MS/MSn can analyze different molecular transformations and fragmentation and compare them with data from the literature and the latest mass spectrometry databases. Currently, LC-MS has proven to be a powerful analytical tool for the identification of flavonoid glycosides, especially anthocyanins, and is widely applied in routine research.
Quantitative analysis of anthocyanins and flavonols (Meng et al., 2019).
References
- Teng, Zhaojun, et al. "Qualitative and quantitative methods to evaluate anthocyanins." EFood 1.5 (2020): 339-346.
- Meng, Xiangyu, et al. "Functional differentiation of duplicated flavonoid 3-O-glycosyltransferases in the flavonol and anthocyanin biosynthesis of Freesia hybrida." Frontiers in plant science 10 (2019): 1330.
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