Anthocyanins Profiling Service

What are Anthocyanins?

Anthocyanins belong to the flavonoids class of the polyphenol. They are contributing to the colors such as red, blue, yellow and purple of many fruits and vegetables. In nature, anthocyanins predominantly exist as glycosides of polymethoxy and polyhydroxy derivatives of flavylium salts or 2-phenyl-benzopyryliurn. Though some anthocyanins can be found in lower plant like mosses and ferns, almost all anthocyanins are found in higher plants. Generally speaking, the anthocyanins found in flowers or ornamental plants are more complex than those in fruits. The anthocyanins in fruits are rather simple and there maybe one or two main pigments in each plant source. However, the pigments in flowers involve a highly regulated series of biochemical steps and undergo both polyglycosylations and polyacylations. Therefore, there are various kinds of anthocyanins in flowers contributing to the variety of shades or hues. In most of the fruits and vegetables, anthocyanins range from 0.1% up to 1.0% of dry weight.

The C ring of anthocyanins is fully unsaturated and there is a hydroxyl at position 3. Anthocyanins are the most oxidized form of flavonoids. The basic structure of anthocyanins is an aglycone or anthocyanidin. Sugars, which sometimes esterified, usually found at C3, C5, or C7. Among the 19 naturally occurring anthocyanidins, cyanidin, malvidin, petunidin, pelargonidin, peonidin, and delphinidin are the most common anthocyanidins identified in edible plants.

Because anthocyanins can undergo complex modification and structural transformations, it is a real challenge to develop the sensitive and reliable analysis method for anthocyanins. Also, it is difficult to differentiate anthocyanins from other flavonoids because they share similar structures and properties.

Comprehensive Anthocyanins Profiling in Creative Proteomics

Standard Anthocyanin Analysis

Identification of Anthocyanins: Utilizing High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS), we identify and profile the anthocyanin compounds present in your samples.

Quantification of Anthocyanins: We provide accurate quantification of individual anthocyanins in your samples, reporting concentrations in µg/mL.

Advanced Characterization

Structural Elucidation: Using Nuclear Magnetic Resonance (NMR) spectroscopy, we provide detailed structural information about anthocyanins, aiding in the identification of complex compounds and understanding their molecular configurations.

Acylation and Glycosylation Analysis: We analyze the degree of acylation and glycosylation of anthocyanins to understand their chemical modifications and effects on pigment stability and color.

Comprehensive Profile Analysis

Full Spectrum Analysis: For complex samples, we offer comprehensive profiling that includes the separation and identification of all anthocyanin components, providing a complete overview of the anthocyanin profile.

Comparison Studies: We perform comparative analyses between different samples or formulations to assess variations in anthocyanin content and composition.

Custom and Targeted Analysis

Custom Method Development: For specific research needs, we develop and validate custom analytical methods tailored to the unique requirements of your samples.

Targeted Quantification: If you require detailed analysis of particular anthocyanins, we offer targeted quantification services using optimized methods.

Quality Control and Assurance

Nutritional Analysis: We assess anthocyanin content as part of nutritional profiling to support product development and label claims.

Stability Testing: Evaluate the stability of anthocyanins under various conditions to understand their shelf-life and degradation patterns.

List of Anthocyanins We Can Analyze

Analytical Techniques for Anthocyanins Profiling

High-Performance Liquid Chromatography (HPLC)

• Waters Alliance HPLC System: Known for its reliability and precision, this system provides high-resolution separation of anthocyanins.
• Agilent 1260 Infinity II LC System: Offers advanced capabilities for separating and quantifying complex mixtures.
• Shimadzu Prominence UFLC: Provides high-speed and high-resolution separation, ideal for detailed anthocyanin analysis.

Application: HPLC is used to separate individual anthocyanin compounds from a mixture, allowing for detailed quantification and profiling based on retention times and peak areas.

Liquid Chromatography-Mass Spectrometry (LC-MS)

• Thermo Scientific Q Exactive Plus: This high-resolution mass spectrometer provides precise identification and quantification of anthocyanins.
• AB Sciex Triple Quad 5500: Offers high sensitivity and specificity for detecting low-abundance anthocyanins.
• Agilent 6545 Q-TOF LC/MS: Provides accurate mass measurements and detailed structural information of anthocyanins.

Application: LC-MS combines chromatographic separation with mass analysis, allowing for highly sensitive detection and structural elucidation of anthocyanins.

Spectrophotometry

• UV-Vis Spectrophotometer (PerkinElmer Lambda 365): Measures absorbance at specific wavelengths, such as 520 nm, to estimate total anthocyanin content.
• Agilent Cary 60 UV-Vis Spectrophotometer: Provides accurate absorbance measurements and is suitable for routine analysis of anthocyanin concentrations.

Application: Spectrophotometry is used for rapid estimation of total anthocyanin content based on their absorbance characteristics.

Nuclear Magnetic Resonance (NMR) Spectroscopy

• Bruker AVANCE III 600 MHz NMR: Provides detailed structural information through high-resolution NMR spectroscopy.
• JEOL JNM-ECZ 400: Offers comprehensive analysis of anthocyanin structures, including glycosylation and acylation patterns.

Application: NMR spectroscopy is used to elucidate the molecular structure of anthocyanins, including detailed information on their chemical modifications.

Sample Requirements for Anthocyanins Analysis

Living in extreme environments: a photosynthetic and desiccation stress tolerance trade-off story, but not for everyone.

Journal: Authorea Preprints

Published: 2023

Background

Plants rely on environmental conditions for growth and have evolved mechanisms to tolerate extreme abiotic stresses. These include synthesizing solutes, antioxidants, and anti-freeze proteins to manage drought, freezing, and salinity. Studies on species like Arabidopsis thaliana, halophytes, and resurrection plants have expanded understanding of stress tolerance mechanisms.

A key challenge is the trade-off between stress tolerance and productivity, where resources invested in tolerance mechanisms can reduce photosynthesis. This trade-off, often influenced by nitrogen allocation, has been observed across various traits and in crops selected for yield at the expense of stress resistance.

Chile's extreme environments, like the Atacama Desert and Surire Salar, offer a unique setting to study extremophiles. A screening of 21 species from these areas identified three Prosopis species—P. tamarugo, P. alba, and P. chilensis—for their ability to balance stress tolerance with photosynthetic capacity. Further analysis aims to understand the mechanisms behind this balance.

Materials & Methods

Experimental Design and Study Site

Photosynthetic capacity and desiccation tolerance of 21 species were evaluated in two field campaigns at the Atacama Desert and Surire Salar. Detailed measurements were conducted on Prosopis tamarugo, P. chilensis, and P. alba, focusing on gas exchange, chlorophyll fluorescence, and physiological responses under full sun exposure.

Xylem Water Potential Measurements

Xylem water potential was measured using a pressure chamber in excised branches to assess plant water status.

Desiccation Tolerance Test

Desiccation tolerance was tested on excised leaves under varying humidity, with recovery measured through photosystem II efficiency (Fv/Fm).

Gas Exchange and Chlorophyll Fluorescence

Gas exchange and chlorophyll fluorescence were measured to generate A N-C i curves and calculate parameters like carboxylation velocity (V cmax) and electron transport rates.

Photosynthetic Pigments Quantification

Photosynthetic pigments were extracted and quantified using HPLC to assess chlorophyll, carotenoids, and xanthophyll cycle pigments, including the de-epoxidation state (DEPS).

Antioxidant and Radical Scavenging Analysis

Total phenols and flavonoids were quantified, and radical scavenging activity was measured using the DPPH method.

Primary Metabolism Analysis

Leaf samples were analyzed for primary metabolites (glucose, fructose, sucrose, starch), amino acids, cell wall components, and lipids to study metabolic adjustments.

Untargeted Metabolomics Analysis

Untargeted metabolomics analysis was performed using UPLC-MS to identify and quantify metabolites. Differentially accumulated metabolites (DAM) were analyzed for pathway enrichment using MetaboAnalyst v5.0.

Statistical Analysis

One-way ANOVA and multiple comparisons were used to assess species differences, with metabolite analysis performed using volcano plots and pathway enrichment.

Results

Trade-off Between Desiccation Tolerance and Photosynthetic Capacity

A screening of 21 species from the Atacama Desert and Surire Salar revealed an inverse relationship between desiccation tolerance (measured as recovery of Fv/Fm) and photosynthetic capacity (Amax). Most species showed a trade-off between these traits, except Prosopis tamarugo, which had both high desiccation tolerance (69.21%) and high Amax (19.2 μmol CO2 m-2 s-1), making it an outlier in the analysis.

Physiological Performance of Prosopis tamarugo

Further analysis of P. tamarugo, compared to its congeneric species (P. chilensis and P. alba), showed significant differences in photosynthetic parameters under natural conditions. P. tamarugo had the highest values of Amax, water use efficiency (WUEi), electron transport rate (ETR), and photochemical quenching (qP), despite lower xylem water potential, indicating a superior ability to maintain photosynthetic activity under stress.

Leaf Pigment Content Differences

Differences in leaf pigment content were observed among Prosopis species. P. tamarugo had the highest levels of chlorophyll a, b, lutein, and β-carotene, which likely contributed to its high photosynthetic capacity. P. chilensis uniquely showed detectable zeaxanthin and the highest de-epoxidation state (DEPS) of the xanthophyll cycle.

Antioxidant Performance

Antioxidant capacity, measured by flavonoid content and ROS scavenging, was significantly higher in P. tamarugo and P. chilensis compared to P. alba. This suggests that higher antioxidant capacity is associated with desiccation tolerance.

Metabolic Balance

Untargeted metabolomic analysis revealed 228 metabolites with significant differences among Prosopis species. P. tamarugo had unique metabolic profiles enriched in tryptophan and anthocyanin pathways. Notably, P. tamarugo showed a lower content of amino acids but no significant difference in total protein, and specific osmoprotectants such as mannitol were highly accumulated.

Osmoprotectant Accumulation

Osmoprotectant metabolites varied across species. P. tamarugo accumulated higher levels of mannitol, a non-nitrogen osmoprotectant positively correlated with Amax and chlorophyll content. In contrast, P. alba had higher levels of nitrogen osmoprotectants, which negatively correlated with photosynthetic capacity.

Untargeted metabolomics analysis in leaves of three Prosopis species

Significant differences in metabolite relative contents among Prosopis species

Chemical classification and metabolic pathway analysis of significant accumulated metabolites

Reference

1. Gajardo, Humberto A., et al. "Living in extreme environments: a photosynthetic and desiccation stress tolerance trade-off story, but not for everyone." Authorea Preprints (2023).