Flavonoids Analysis Service

What Are Flavonoids

Flavonoids represent a class of compounds primarily distinguished by their shared 2-phenylchromone nucleus structure. These compounds are the products of extensive natural selection in plants and are ubiquitously found in the roots, stems, leaves, flowers, and fruits of higher plant species. Flavonoids play a pivotal role in various physiological and biochemical processes, rendering them significant contributors to the prevention and treatment of human ailments, including cancer, anti-tumor effects, cardiovascular disease mitigation, anti-aging attributes, osteoporosis prevention, antiviral properties, and anti-inflammatory actions, to name a few. The diverse pharmacological activities of flavonoids have propelled them into the forefront of both domestic and international research, with wide-ranging applications in the realms of medicine, nutrition, and healthcare.

Classification of Flavonoids

Flavonoids (C6–C3–C6) can be classified in different subclasses (flavones, flavanones, flavonols, isoflavones, flavanonols, flavanols, chalcones and anthocyanins) according to the degree of unsaturation and degree of oxidation of the 3-carbon skeleton. Subclasses of flavonoids can be further differentiated on the basis of the number and nature of substituent groups attached to the rings. Flavonols and anthocyanins are the main subclasses of flavonoids present in plants. Many of these compounds are glycosylated, and some of these glycosyl derivatives are esterified with aromatic or aliphatic acids whose combinations yield a large variety of compounds. Aromatic acylation increases the stability of anthocyanins by intramolecular stacking of anthocyanins with polyphenols. Malonylation is one of the most common forms of aliphatic acylation of anthocyanins and is important for enhancing pigment solubility in water, protecting glycosyl moiety from enzymatic degradation, and stabilizing anthocyanin structures, what contributes to keep the color hue. Some bioactive properties of anthocyanins, including antioxidant activity, have been shown to be strongly modulated by acylation.

Evidence is emerging showing that consecutive enzymes of the phenylpropanoid and flavonoid biosynthesis are organized into macromolecular complexes that can be associated with endomembranes. Metabolic channeling in plant secondary metabolism enables plants to effectively synthesize specific natural products and thus avoid metabolic interference. The existence of cytochrome P450 monooxygenases (P450s)-related metabolons has been demonstrated: direct and indirect experimental data describe P450 enzymes in the phenylpropanoid, flavonoid, cyanogenic glucoside, and other biosynthetic pathways. Additional evidence for the channeling of intermediates between specific isoforms of phenylalanine ammonia lyase and cinnamate-4-hydroxylase has been provided using transgenic tobacco plants expressing epitope-tagged versions of two phenylalanine ammonia lyase isoforms (PAL1 and PAL2) and of cinnamate-4-hydroxylase. Moreover, the existence of a multienzyme complex has been proposed for the anthocyanin pathway in rice by yeast-two hybrid experiments.

More than 6000 different flavonoids have been identified, and surely this number will increase. The different flavonoids have diverse biological functions, including protection against ultraviolet (UV) radiation and phytopathogens, signaling during nodulation, male fertility, auxin transport, as well as the coloration of flowers as a visual signal that attracts pollinators. Flavonoids are also responsible for the display of fall color in many plants, which may protect leaf cells from photooxidative damage, enhancing the efficiency of nutrient retrieval during senescence. Flavonols are probably the most important flavonoids participating in stress responses; they are the most ancient and widespread flavonoids, having a wide range of potent physiological activities.

Our Flavonoids Quantification Service

Creative Proteomics harnesses the power of a state-of-the-art triple quadrupole liquid chromatography-mass spectrometry (LC-MS/MS) system renowned for its precision and stability. Our approach incorporates an electrospray ionization (ESI) source and leverages the multiple reaction monitoring (MRM) mode. This methodology has been meticulously developed to enable the simultaneous qualitative and quantitative assessment of numerous flavonoid compounds within a concise timeframe. Rigorous assessments of parameters such as linearity, precision, and limit of quantification have been conducted to ensure the reliability and alignment with research requirements of our results.

Our product has been meticulously designed with a focus on the fundamental metabolic pathways of flavonoids, encompassing well-known research compounds like quercetin, catechin, rutin, and kaempferol. This offering adeptly addresses the diverse testing requirements of a broad spectrum of clientele.

Flavonoid Detection List (Partial)

Workflow

Service Advantages

Comprehensive Coverage of Flavonoid Core Metabolic Pathways: This product is developed based on the core metabolic pathways of flavonoids and provides extensive coverage of the Flavonoid Biosynthesis, Isoflavonoid Biosynthesis, and Flavone and Flavonol Biosynthesis pathways according to the KEGG database.

Stringent Quality Control System: Our project employs internal standards for absolute quantification and utilizes quality control (QC) systems. All data results undergo manual verification to ensure the accuracy and reliability of qualitative and quantitative results.

Precise Quantification: We employ a triple quadrupole liquid chromatography-mass spectrometry system known for its stability. Utilizing the MRM mode and an ESI ion source, we have thoroughly examined parameters including linearity, precision, and limit of quantification. The developed detection method meets analytical requirements, providing reliable results.

Sample Requirements

Plant Tissues: Fresh samples require 2 grams, dry samples require 1 gram, and freeze-dried powder samples require 250 mg.

Case Study: LC-MS/MS-Based Investigation of Flavonoid Metabolic Pathways in Arabidopsis Seeds and Seedlings

Journal: BMC Plant Biology

Published: 2016

Methodology:

This study employed a rigorous approach for the precise qualitative and quantitative analysis of 12 flavonoids within various Arabidopsis seed and seedling genotypes. The analysis was performed using the LC-ESI-MS-QTRAP system operating in the MRM mode, incorporating isotopic internal standards. The scope of these flavonoid compounds encompassed several pivotal metabolic pathways, notably including the biosynthesis of quercetin to kaempferol or dihydrokaempferol, quercetin and isorhamnetin, anthocyanins, catechins, and proanthocyanidins. Furthermore, the LC-ESI-MS-QTOF system was utilized to detect and identify glycosylated flavonoids.

Results:

The investigation unveiled notable disparities in the modulation of kaempferol by the transcription factor HY5 (Elongated Hypocotyl 5) within the contexts of seeds and seedlings. In seedlings, the ubiquitin ligase COP1 (Constitutive Photomorphogenic 1) emerged as a principal regulatory player in the flavonoid biosynthesis pathway, whereas its influence in seeds was comparatively limited. These observations furnish valuable perspectives for the exploration of genetic variations across diverse ecotypes.

Flavonoid biosynthesis pathway and the concept of hydrolysis to reduce complexity of extracts.

Reference

1. Jaegle, B., Uroic, M.K., Holtkotte, X. et al. A fast and simple LC-MS-based characterization of the flavonoid biosynthesis pathway for few seed(ling)s. BMC Plant Biol 16, 190 (2016).