Anthocyanins: Sources, Structures, Analysis Methods & Applications

Sources of Anthocyanins

Anthocyanins, the vibrant pigments responsible for the striking colors seen in many fruits, vegetables, and flowers, are derived from the flavonoid pathway in plants. These water-soluble compounds are synthesized in various plant tissues, including leaves, stems, fruits, and flowers, predominantly in response to environmental cues such as light exposure, temperature fluctuations, and nutrient availability. The accumulation of anthocyanins in plant tissues serves multiple purposes, including attracting pollinators, deterring herbivores, and protecting against environmental stresses such as UV radiation and pathogen attacks. The diversity of anthocyanin sources spans a wide range of botanical species, with each species exhibiting unique profiles of anthocyanin pigments, contributing to the rich tapestry of colors found in nature. Common sources of anthocyanins include berries such as blueberries, strawberries, and blackberries, as well as red cabbage, grapes, eggplants, and certain varieties of flowers like roses and pansies.

Structure and Classification of Anthocyanins

Structure of anthocyanins

Anthocyanins are composed of an anthocyanidin (the aglycone or sugar-free molecule) and one or more sugar moieties (sugar molecules). The anthocyanidin consists of two aromatic rings (Ring A and Ring B) connected by a three-carbon bridge, which forms a heterocyclic ring (Ring C). The structure of the anthocyanidin determines the color of the pigment, and the sugar moieties and any additional groups attached to the anthocyanidin core can further modify the color.

Classification of anthocyanins

Anthocyanins can be classified based on the type of anthocyanidin, the number and type of sugar moieties, and the presence and type of any additional groups.

• Based on the Type of Anthocyanidin: There are six common types of anthocyanidins – cyanidin, delphinidin, pelargonidin, peonidin, petunidin and malvidin. These differ in the number and position of hydroxyl groups and methoxyl groups on the anthocyanidin structure.
• Based on the Number and Type of Sugar Moieties: Anthocyanins can be monoglycosides (one sugar moiety), diglycosides (two sugar moieties) or triglycosides (three sugar moieties). The sugar moieties can be types like glucose, galactose, arabinose, etc.
• Based on the Additional Groups: Additional groups can be attached to the anthocyanin molecule, such as acyl groups (acetylated, coumaroylated, caffeoylated, malonylated etc.) or alkyl groups.

The structure and classification of anthocyanins are important in determining their color, stability, and health properties. Most importantly, variations in the anthocyanin structures give rise to the vast array of colors found in the plant kingdom.

Types of commonly occurring anthocyanins reported in the plant system (Kaur et al., 2021).

What is The Role of Anthocyanin?

Anthocyanin is one of the important pigments that determine the color of flowers, fruits and seed coats of angiosperms. It is found in the vesicles of plant epidermal cells and appears orange, red to blue. Anthocyanins also play an important role in plant resistance to stress.

Anthocyanins are bioflavonoids, and the main physiologically active functions of flavonoids are free radical scavenging ability and antioxidant ability. Studies have proven that anthocyanins are effective antioxidants and potent free radical scavengers. The antioxidant performance of anthocyanins is 50 times higher than that of VE and 20 times higher than that of VC. Purple sweet potato anthocyanin products have scavenging and inhibiting effects on -OH, H₂O₂, and other reactive oxygen species, especially the scavenging ability of -OH is stronger than that of ascorbic acid, and the scavenging effect is dose-dependent with the concentration.

With the development of science and technology, people pay more and more attention to the safety of food additives, the development and utilization of natural additives has become a general trend in the development and use of additives. Anthocyanins can be used in food not only as nutritional fortification agents, but also as food preservatives instead of synthetic preservatives such as benzoic acid, and can be used as food coloring agents in normal beverages and food, which meets the total requirements of people for natural, safe and healthy food additives.

How is Anthocyanin Measured?

Analyzing anthocyanins presents unique challenges due to their structural complexity, diverse chemical properties, and susceptibility to degradation. Therefore, a variety of analytical methods have been developed to accurately detect, quantify, and characterize these compounds in complex biological matrices. Anthocyanin analysis methods can be broadly categorized into traditional spectrophotometric techniques, chromatographic methods, advanced analytical techniques such as mass spectrometry and nuclear magnetic resonance spectroscopy, and biological assays. Each method offers distinct advantages and limitations, allowing researchers to choose the most suitable approach based on the specific requirements of their study.

Spectrophotometric methods

Spectrophotometric techniques, such as UV-visible spectroscopy, are commonly used for rapid and cost-effective quantification of anthocyanins in plant extracts and food products. These methods rely on the measurement of absorbance at specific wavelengths corresponding to the maximum absorption of anthocyanin pigments, typically around 520-550 nm. By comparing the absorbance of samples to standard anthocyanin solutions, the concentration of anthocyanins can be determined. However, spectrophotometric methods may lack specificity, especially in complex mixtures containing other pigments or compounds with overlapping absorbance spectra.

Chromatographic methods

Liquid chromatography (LC) and gas chromatography (GC) are powerful separation techniques widely used for anthocyanin analysis. High-performance liquid chromatography (HPLC) coupled with UV-visible or diode array detection allows for the separation and quantification of anthocyanins based on their retention times and absorbance spectra. Additionally, HPLC coupled with mass spectrometry (MS) provides enhanced sensitivity and specificity, enabling the identification of individual anthocyanin compounds in complex mixtures. Gas chromatography, although less commonly used for anthocyanin analysis due to their polarity and thermal instability, can be coupled with MS for specific applications.

High-performance liquid chromatography-mass spectrometry (HPLC-MS)

HPLC-MS is considered the gold standard for anthocyanin analysis due to its high sensitivity, selectivity, and ability to provide structural information. This technique combines the separation power of HPLC with the detection capabilities of MS, allowing for the identification and quantification of anthocyanins at trace levels in various samples. HPLC-MS facilitates the analysis of complex mixtures, structural elucidation of anthocyanin compounds, and determination of their fragmentation patterns.

Nuclear magnetic resonance (NMR) spectroscopy

NMR spectroscopy is a non-destructive analytical technique used for structural elucidation and quantitative analysis of anthocyanins. By measuring the interactions between nuclei in anthocyanin molecules and an external magnetic field, NMR spectroscopy provides valuable information about molecular structure, chemical environment, and composition. NMR is particularly useful for confirming the presence of specific functional groups, determining stereochemistry, and elucidating complex molecular arrangements in anthocyanin compounds.

Capillary electrophoresis (CE)

Capillary electrophoresis (CE) is a rapid and efficient separation technique based on the migration of charged analytes in a capillary under the influence of an electric field. CE offers high resolution and short analysis times, making it suitable for the separation and quantification of anthocyanins in complex samples. CE techniques such as capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) have been successfully applied for anthocyanin analysis, providing valuable information about compound purity, charge, and molecular weight.

Biological methods

Biosensors

Biosensors are analytical devices that utilize biological components, such as enzymes or antibodies, to selectively detect and quantify anthocyanins in biological samples. These methods offer rapid detection, high specificity, and low detection limits, making them suitable for on-site analysis and real-time monitoring of anthocyanin levels in plants, foods, and beverages.

Immunoassays

Immunoassays are analytical techniques based on the specific binding between antibodies and antigens, enabling the quantitative analysis of anthocyanins in complex matrices. Enzyme-linked immunosorbent assay (ELISA) and fluorescence immunoassay are commonly used immunoassay formats for anthocyanin detection, offering high sensitivity and specificity.

Data Processing and Analysis

Standard Curve Method

The standard curve method is a widely used approach for quantitative analysis of anthocyanins based on external calibration standards. In this method, known concentrations of anthocyanin standards are prepared and analyzed using the chosen analytical technique, such as spectrophotometry or chromatography. A calibration curve is then constructed by plotting the absorbance or peak area of the standards against their respective concentrations. By extrapolating the absorbance or peak area of unknown samples onto the calibration curve, the concentration of anthocyanins in the samples can be determined. The standard curve method offers simplicity, accuracy, and reproducibility, making it suitable for routine quantitative analysis of anthocyanins in various samples.

Multivariate Statistical Analysis

Multivariate statistical analysis encompasses a range of techniques used to analyze large datasets with multiple variables. In the context of anthocyanin analysis, multivariate statistical methods such as principal component analysis (PCA), partial least squares regression (PLS), and cluster analysis can be employed to extract meaningful information from complex datasets. These methods allow researchers to identify patterns, correlations, and trends in anthocyanin composition, distribution, and abundance across different samples or experimental conditions. Multivariate statistical analysis enhances data interpretation, facilitates sample classification, and provides insights into the factors influencing anthocyanin content and variability.

Applications of Anthocyanin Analysis

In Food Industry

In the food industry, the analysis of anthocyanins serves various crucial purposes, from ensuring product quality and authenticity to enhancing nutritional value and consumer appeal. One primary application lies in the quantification of anthocyanin content in food products, such as fruits, vegetables, beverages, and processed foods. This quantitative analysis aids in verifying label claims regarding the presence and concentration of anthocyanins, providing consumers with accurate information about the bioactive compounds present in the products they consume.

Moreover, the data obtained from anthocyanin analysis facilitates product development and optimization processes. Food manufacturers can use this information to formulate products with consistent anthocyanin levels, ensuring uniform color intensity and antioxidant properties across different batches. Additionally, understanding the stability of anthocyanins during processing and storage enables the development of preservation techniques that maintain their bioactivity and sensory attributes over time.

Furthermore, anthocyanin analysis plays a vital role in exploring the potential health benefits of these compounds and their applications in functional foods. By quantifying anthocyanin levels in various food matrices, researchers can investigate the correlation between anthocyanin intake and health outcomes, such as reduced risk of chronic diseases like cardiovascular disease and cancer. This knowledge informs the development of functional foods fortified with anthocyanins to promote health and well-being.

In Agriculture

In the agricultural sector, the analysis of anthocyanins contributes to crop management practices, breeding programs, and the development of novel cultivars with enhanced nutritional and agronomic traits. Quantitative analysis of anthocyanin content in crops, such as fruits, vegetables, and grains, provides valuable insights into plant physiology, stress responses, and fruit quality.

Anthocyanin analysis helps breeders select plant varieties with desirable traits, such as vibrant coloration, improved flavor, and increased antioxidant activity. By correlating anthocyanin levels with traits related to plant health and productivity, breeders can identify markers for selecting high-performing cultivars through conventional breeding or biotechnological approaches.

Furthermore, understanding the factors influencing anthocyanin accumulation, such as environmental conditions, soil nutrients, and cultural practices, allows farmers to optimize growing conditions to maximize anthocyanin production and crop yield. This optimization not only enhances the visual appeal and marketability of agricultural products but also contributes to their nutritional value and economic value.

Moreover, anthocyanin analysis supports research on the role of these compounds in plant defense mechanisms, responses to abiotic and biotic stresses, and interactions with beneficial microorganisms. By elucidating the physiological functions of anthocyanins in plants, researchers can develop sustainable agricultural practices that harness the potential of these compounds to improve crop resilience, yield stability, and overall agricultural sustainability.

In Pharmaceutical Industry

In the pharmaceutical industry, the analysis of anthocyanins plays a pivotal role in the discovery, development, and quality control of herbal medicines, dietary supplements, and nutraceuticals. Anthocyanin analysis enables researchers to quantify the concentration of these bioactive compounds in medicinal plants, herbal extracts, and finished products, ensuring consistency and efficacy in therapeutic applications.

Quantitative determination of anthocyanin content provides valuable information for standardization and quality assurance, allowing manufacturers to meet regulatory requirements and ensure product safety and efficacy. By establishing reliable analytical methods for anthocyanin analysis, pharmaceutical companies can verify the authenticity, purity, and potency of herbal products, guarding against adulteration and contamination.

Furthermore, anthocyanin analysis supports pharmacological research on the bioactivity, pharmacokinetics, and mechanisms of action of these compounds. By elucidating the therapeutic potential of anthocyanins in various disease conditions, researchers can develop evidence-based formulations and dosage regimens for treating or preventing health disorders, such as cardiovascular disease, diabetes, and neurodegenerative diseases.

Additionally, anthocyanin analysis aids in exploring novel delivery systems and formulation strategies to improve the bioavailability and stability of these compounds in pharmaceutical formulations. By optimizing the formulation parameters, such as excipients, dosage forms, and administration routes, researchers can enhance the therapeutic efficacy and patient compliance of anthocyanin-based medications, opening new avenues for drug development and personalized medicine.

Reference

1. Kaur, S., Sharma, N., Kapoor, P., et al. (2021). Spotlight on the overlapping routes and partners for anthocyanin transport in plants. Physiologia Plantarum, 171(4), 868-881.