Optimization of a Sustainable Protocol for the Extraction of Anthocyanins as Textile Dyes from Plant Materials

Anthocyanins are the largest group of polyphenolic pigments in the plant kingdom. These non-toxic, water-soluble compounds are responsible for the pink, red, purple, violet, and blue colors of fruits, vegetables, and flowers. Anthocyanins are widely used in the production of food, cosmetic and textile products, in the latter case to replace synthetic dyes with natural and sustainable alternatives. Here, we describe an environmentally benign method for the extraction of anthocyanins from red chicory and their characterization by HPLC-DAD and UPLC-MS. The protocol does not require hazardous solvents or chemicals and relies on a simple and scalable procedure that can be applied to red chicory waste streams for anthocyanin extraction. The extracted anthocyanins were characterized for stability over time and for their textile dyeing properties, achieving good values for washing fastness and, as expected, a pink-to-green color change that is reversible and can therefore be exploited in the fashion industry.     

Methods of analysis for anthocyanins in plants and biological fluids

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. They are responsible for most of the red, blue, and purple colors of fruits, vegetables, flowers, and other plant tissues or products. The analysis of anthocyanins is complex as a result of their ability to undergo structural transformations and complexation reactions. In addition, they are difficult to measure independently of other flavonoids, as they have similar structural and reactivity characteristics. Anthocyanins are generally extracted with weakly acidified alcohol-based solvents, followed by concentration (under vacuum), and purification of the pigments. Paper and/or thin-layer chromatography and UV-Vis spectroscopy have traditionally been used for the identification of anthocyanins. Capillary zone electrophoresis, a hybrid of chromatography and electrophoresis, is gaining popularity for the analysis of anthocyanins; however, liquid chromatography (LC) has become the standard method for identification and separation in most laboratories and may be used for both preparative and quantitative analysis. LC with mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are possibly the most powerful methods for the structural elucidation of anthocyanins available, to date. At present, the most satisfactory method for mixture analysis is the multistep method of separation, isolation, and quantification by LC with peak identification by MS and high-field NMR.     

Environmental Significance of Anthocyanins in Plant Stress Responses

Abstract— Anthocyanins are water-soluble pigments found in all plant tissues throughout the plant kingdom. Our understanding of anthocyanin biosynthesis and its molecular control has greatly improved in the last decade. The adaptive advantages of anthocyanins, especially in non-reproductive tissues, is much less clear. Anthocyanins often appear transiently at specific developmental stages and may be induced by a number of environmental factors including visible and UVB radiation, cold temperatures and water stress. The subsequent production and localization of anthocyanins in root, stem and especially leaf tissues may allow the plant to develop resistance to a number of environmental stresses. This article reviews the environmental induction of anthocyanins and their proposed importance in ameliorating environmental stresses induced by visible and UVB radiation, drought and cold temperatures.     

Functional Characterization of an Anthocyanin Dimalonyltransferase in Maize

Anthocyanins are pigments with appealing hues that are currently being used as sources of natural colorants. The interaction of acylation on the stability of anthocyanin molecules has long been known. Maize is an abundant source of malonylglucoside and dimalonylglucoside anthocyanins. The enzyme Aat1 is an anthocyanin acyltransferase known to synthesize the majority of acylated anthocyanins in maize. In this paper, we characterize the substrate specificity and reaction kinetics of Aat1. It was found that Aat1 has anthocyanin 3-O-glucoside dimalonyltransferase activity and is only the second enzyme of this type characterized to this date. Our results indicate that Aat1 can utilize malonyl-CoA; succinyl-CoA and every anthocyanin 3-O-glucoside tested. Results of this study provide insight into the structure–function relations of dimalonyltransferases and give a unique insight into the activity of monocot anthocyanin acyltransferases.     

Anthocyanins Recovered from Agri-Food By-Products Using Innovative Processes: Trends,Challenges, and Perspectives for Their Application in Food Systems

Anthocyanins are naturally occurring phytochemicals that have attracted growing interest from consumers and the food industry due to their multiple biological properties and technological applications. Nevertheless, conventional extraction techniques based on thermal technologies can compromise both the recovery and stability of anthocyanins, reducing their global yield and/or limiting their application in food systems. The current review provides an overview of the main innovative processes (e.g., pulsed electric field, microwave, and ultrasound) used to recover anthocyanins from agri-food waste/by-products and the mechanisms involved in anthocyanin extraction and their impacts on the stability of these compounds. Moreover, trends and perspectives of anthocyanins’ applications in food systems, such as antioxidants, natural colorants, preservatives, and active and smart packaging components, are addressed. Challenges behind anthocyanin implementation in food systems are displayed and potential solutions to overcome these drawbacks are proposed.     

Light-stress-induced pigment changes and evidence for anthocyanin photoprotection in apples

Fruit of two apple (Malus domestica Borkh.) cultivars, differing in their ability to produce anthocyanin pigments when exposed to sunlight, have been studied using reflectance spectroscopy. Comparison of the spectra shows that apple anthocyanins in vivo possess a symmetric absorption band at 500-600 nm with a maximum near 550 nm. Anthocyanins considerably increase light absorption by apples. In on-tree-ripening Zhigulevskoe apples, accumulating high amounts of anthocyanin pigments, chlorophyll contents in sunlit and shaded sides of the fruits are found to be similar. In contrast, frequently considerably lower chlorophyll content is estimated in sunlit compared with shaded sides of Antonovka apples exhibiting low potential for anthocyanin formation. Sunlight also brings about an increase of carotenoid content over that of chlorophylls and accumulation of substances responsible for light absorption in the range 350-400 nm. The rates of high-light-induced chlorophyll bleaching in red zones of fruit containing anthocyanins are considerably lower than those in green zones and decrease with an increase in the pigment content. Anthocyanins show more stability to irradiation than chlorophylls. A protective function of anthocyanins against both light-induced stress in, and damage to, apples is suggested. It is proposed that anthocyanins function as an effective internal light trap filling the chlorophyll absorption gap in the green-orange part of the visible spectrum.     

Anthocyanins as antimicrobial agents of natural plant origin

Anthocyanins are particularly abundant in different fruits, especially in berries. The beneficial effects of these compounds for human health have been known from at least the 16th century. Despite the great number of papers devoted to the different biological effects exerted by anthocyanins only a limited number of studies is focused on the antimicrobial activity of these compounds. Anthocyanin content of berry fruits varies from 7.5 mg/100 mg fresh fruit in redcurrant (Ribes rubum) up to 460 mg/100 g fresh fruit in chokeberry (Aronia melanocarpa). After consumption, anthocyanins are intensively metabolized, mainly in the intestines and liver. Glucorination, methylation and sulfation are the most typical metabolic reactions. Antimicrobial activity of crude extracts of plant phenolic compounds against human pathogens has been intensively studied to characterize and develop new healthy food ingredients as well as medical and pharmaceutical products. However, there is very little information available about the antimicrobial activity of the pure anthocyanins. In the last part of this review we present the collection of papers describing the anthocyanin profiles of different fruits (mainly berries) and the antimicrobial properties of the identified compounds. Generally, anthocyanins are active against different microbes, however Gram-positive bacteria usually are more susceptible to the anthocyanin action than Gram-negative ones. Mechanisms underlying anthocyanin activity include both membrane and intracellular interactions of these compounds. Antimicrobial activity of berries and other anthocyanin-containing fruits is likely to be caused by multiple mechanisms and synergies because they contain various compounds including anthocyanins, weak organic acids, phenolic acids, and their mixtures of different chemical forms. Therefore, the antimicrobial effect of chemically complex compounds has to be critically analyzed.     

Conversion of Organic Dyes into Pigments: Extraction of Flavonoids from Blackberries (Rubus ulmifolius) and Stabilization

The blackberry’s color is composed mainly of natural dyes called anthocyanins. Their color is red–purple, and they can be used as a natural colorant. Anthocyanins are flavonoids, which are products of plants, and their colors range from orange and red to various shades of blue, purple and green, according to pH. In this study, the chemical composition of an extract obtained from blackberries was defined by LC-ESI/LTQOrbitrap/MS in positive and negative ionization mode. Furthermore, we investigated the adsorption process of blackberry extract using several inorganic fillers, such as metakaolin, silica, Lipari pumice, white pozzolan and alumina. The pigments exhibit different colors as a function of their interactions with the fillers. The analysis of the absorption data allowed the estimation of the maximum adsorbing capacity of each individual filler tested. Through thermogravimetric measurements (TGA), the thermal stability and the real adsorption of the organic extract were determined.     

Purification and antioxidant capacity analysis of anthocyanin glucoside cinnamic ester isomers from Solanum nigrum fruits

In a recent study, anthocyanins, which have a strong free radical‐scavenging activity, were examined for their potential to effectively prevent cancer. However, clinical trials are limited by the purity of the anthocyanin. Multiple methods are used to extract and purify anthocyanins. Based on previous work on Solanum nigrum, which is a widely distributed plant, in this study, DM130 macroporous resin, Sephadex LH20, and a C18 column were used to separate cis–trans anthocyanin isomers. These anthocyanins constitute the majority of total S. nigrum anthocyanins. The results showed that this “DM130‐LH20‐C18 system” can be used to obtain a cinnamic acid‐derived cis–trans anthocyanin, petunidin‐3‐(p‐coumaroyl)‐rutinoside‐5‐glucoside, with a purity of 98.5%, for effective quantitation. In order to determine the antioxidant ability of the petunidin‐3‐(p‐coumaroyl)‐rutinoside‐5‐glucoside cis–trans isomers, three ordinary methods were adopted. The maximum antioxidant ability of the cis–trans anthocyanin was dozens of times higher than that of vitamin C.     

Alternative Extraction and Downstream Purification Processes for Anthocyanins

Anthocyanins are natural pigments displaying different attractive colors ranging from red, violet, to blue. These pigments present health benefits that increased their use in food, nutraceuticals, and the cosmetic industry. However, anthocyanins are mainly extracted through conventional methods that are time-consuming and involve the use of organic solvents. Moreover, the chemical diversity of the obtained complex extracts make the downstream purification step challenging. Therefore, the growing demand of these high-value pigments has stimulated the interest in designing new, safe, cost-effective, and tunable strategies for their extraction and purification. The current review focuses on the potential application of compressed fluid-based (such as subcritical and supercritical fluid extraction and pressurized liquid extraction) and deep eutectic solvents-based extraction methods for the recovery of anthocyanins. In addition, an updated review of the application of counter-current chromatography for anthocyanins purification is provided as a faster and cost-effective alternative to preparative-scale HPLC.     

Anthocyanins as Key Phytochemicals Acting for the Prevention of Metabolic Diseases: An Overview

Anthocyanins are water-soluble pigments present in fruits and vegetables, which render them an extensive range of colors. They have a wide distribution in the human diet, are innocuous, and, based on numerous studies, have supposed preventive and therapeutical benefits against chronic affections such as inflammatory, neurological, cardiovascular, digestive disorders, diabetes, and cancer, mostly due to their antioxidant action. Despite their great potential as pharmaceutical applications, they have a rather limited use because of their rather low stability to environmental variations. Their absorption was noticed to occur best in the stomach and small intestine, but the pH fluctuation of the digestive system impacts their rapid degradation. Urine excretion and tissue distribution also occur at low rates. The aim of this review is to highlight the chemical characteristics of anthocyanins and emphasize their weaknesses regarding bioavailability. It also targets to deliver an update on the recent advances in the involvement of anthocyanins in different pathologies with a focus on in vivo, in vitro, animal, and human clinical trials.     

Visualization of anthocyanin species in rabbiteye blueberry Vaccinium ashei by matrix-assisted laser desorption/ionization imaging mass spectrometry

Anthocyanins are naturally occurring compounds that impart color to fruits, vegetables, and plants, and are believed to have a number of beneficial health effects in both humans and animals. Because of these properties, pharmacokinetic analysis of anthocyanins in tissue has been performed to quantify and identify anthocyanin species although, currently, no methods exist for investigating tissue localization of anthocyanin species or for elucidating the mechanisms of anthocyanin activity. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of a wide range of biomolecules. To investigate whether anthocyanin species could be identified and visualized by IMS, we performed matrix-assisted laser desorption/ionization (MALDI)-IMS analysis, by tandem mass spectrometry (MALDI-IMS-MS), of ten anthocyanin molecular species in rabbiteye blueberry (Vaccinium ashei). The distribution patterns of each anthocyanin species were different in the exocarp and endocarp of blueberry sections. Anthocyanin species composed of delphinidin and petunidin were localized mainly in the exocarp. In contrast, those species composed of cyanidin, peonidin, and malvidin were localized in both the exocarp and the endocarp. Moreover, MALDI-IMS analysis of anthocyanidins in a blueberry section indicated that the distribution patterns of each anthocyanidin species were nearly identical with those of the corresponding anthocyanins. These results suggested that the different distribution patterns of anthocyanin species in the exocarp and endocarp depended on the aglycone rather than on the sugar moieties. This study is the first to visualize anthocyanin molecular species in fruits.     

Stability of anthocyanins extracted from grape skins

Summary The extractability and stability of anthocyanins from the skins ofVitis vinifera were determined at different pH values. Anthocyanins were extracted using acetone, partitioned with chloroform and pre-purified by solid-phase extraction (SPE). They were analysed by RP-HPLC, and the kinetic parameters of decomposition were calculated. The total monomeric anthocyanin content was determined by spectrophotometry. Anthocianins were well separated by RP-HPLC. The efficiency of extraction depended strongly on the pH of the extracting agent and on the character of the pigment to be extracted. The amount of anthocyanins decreased with increasing duration of storage, more so at elevated temperatures. Presented at Balaton Symposium '01 on High-Performance Separation Methods Siófok, Hungary, September 2–4, 2001.     

Determination of anthocyanins in the urine of patients with colorectal liver metastases after administration of bilberry extract

Anthocyanins possess cancer chemopreventive properties in preclinical models. Their clinical pharmacology is only poorly understood. In this pilot study, anthocyanins and their metabolites were analysed in the urine of two patients with colorectal liver metastases. They received a single dose of 1.88 g standardized bilberry extract (mirtoselect) via either nasogastric or nasojejunal tube intra‐operatively during liver resection. HPLC‐MS/MS and HPLC‐UV analysis showed there were more anthocyanins and metabolites in the urine of the patient who received mirtoselect via the stomach than via the jejunum. This result is consistent with information obtained in rodents which suggests the stomach is the predominant site for anthocyanin absorption. Copyright © 2010 John Wiley & Sons, Ltd.     

Anthocyanins HPLC-DAD and MS Characterization,Total Phenolics,and Antioxidant Activity of Some Berries Extracts

Extracts of indigenous wild blackberries, mulberries, bilberries, and blackthorns were analyzed for anthocyanin composition, anthocyanin content, total phenolics, and antioxidant capacity. Anthocyanins extraction with acidified methanol in ultrasonic condition (59 kHz, 60 min., 25°C) was carried out. The extracts were analyzed by high-performance liquid chromatography (HPLC) using a Dionex Ultimate 3000 apparatus equipped with photodiode array detector for qualitative characterization of the anthocyanins. The chromatograms revealed the presence of a large number of anthocyanins in fruits extracts: blackberries, 4 compounds; mulberries, 3 compounds; bilberries, 18 compounds; and blackthorns, 5 compounds. The most abundant anthocyanins were cyanidin-3-glucoside in blackberry, mulberry, and bilberry, and cyanidin-3-rutinoside in blackthorn extract. Structural information about anthocyanins was obtained by using a mass spectrometric method based on fully automated chip-nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT). Anthocyanin content was quantified by the pH differential method and total phenolics were determined by Folin-Ciocalteu method. A Jasco V 530 UV-VIS spectrophotometer was used for absorbance measurements. The free radical scavenging activity of the berries extracts was performed by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The reduction of DPPH was followed by a spectrophotometric method. Also, a correlation of the antioxidant capacities of the extracts with their anthocyanin content and total phenolics was attempted.     

Analysis of anthocyanin pigments in Lonicera (Caerulea) extracts using chromatographic fractionation followed by microcolumn liquid chromatography-mass spectrometry

Anthocyanins from the fruit Lonicera caerulea L. var. kamtschatica (blueberry honeysuckle, Caprifoliaceae) were studied via (semi)preparative chromatographic fractionation followed by MS and μLC/MS analysis. The extraction procedure was optimized with respect to analytical purposes as well as its potential use for the preparation of nutraceuticals. The highest yield of anthocyanins was obtained using acidified methanol as the extraction medium. A comparable total anthocyanin content was obtained using a mixture of methanol and acetone. However, when Lonicera anthocyanins were in contact with acetone, a condensation reaction occurred to a large extent and related 5-methylpyranoanthocyanins were found. The effect of other extraction media, including ethanol as a "green" solvent, is also discussed. The potential of two fractionation procedures for extract purification differing in their chromatographic selectivity and scale was studied (i.e. using a Sephadex LH-20 gel column and a reversed phase). Fractions obtained by both procedures were used for a detailed analysis. MS and μLC/MS(2) methods were used for monitoring anthocyanin and 5-methylpyranoderivatives content as well as identifying less common and more complex dyes (dimer of cyanidin-3-hexoside, cyanidin-ethyl-catechin-hexosides, etc.). These more complex dyes are most likely formed during fruit treatment.     

Functional Characterization of Flavanone 3-Hydroxylase (F3H) and Its Role in Anthocyanin and Flavonoid Biosynthesis in Mulberry

Mulberry (Morus spp., Moraceae) is an important economic crop plant and is rich in flavonoids and anthocyanidins in ripe fruits. Anthocyanins are glycosides of anthocyanidins. Flavanone 3-hydroxylase (F3H) catalyzes the conversion of naringenin into dihydroflavonols and is responsible for the biosynthesis of flavonols and anthocyanidins. In this study, MazsF3H was cloned and characterized from Morus atropurpurea var. Zhongshen 1. Conserved motif analysis based on alignment and phylogenetic analysis indicated that MazsF3H belonged to 2-oxoglutarate-dependent dioxygenase and MazsF3H clustered with F3Hs from other plants. MazsF3H was located in both nucleus and cytosol. MazsF3H was expressed in stems, leaves, stigmas and ovaries, except buds. F3H expression levels showed a positive and close relationship with anthocyanin content during the anthocyanin-rich fruit ripening process, while it showed a negative correlation with anthocyanin content in LvShenZi, whose fruits are white and would not experience anthocyanin accumulation during fruit ripening. Significantly different F3H expression levels were also found in different mulberry varieties that have quite different anthocyanin contents in ripe fruits. Overexpression MazsF3H in tobacco showed unexpected results, including decreased anthocyanin content. Down-regulation of F3H expression levels resulted in co-expression of the genes involved in anthocyanin biosynthesis and a significant decrease in anthocyanin content, but the change in total flavonoid content was subtle. Our results indicated that F3H may play quite different roles in different varieties that have quite different fruit colors. In addition, possible complex regulation of flavonoid biosynthesis should be further explored in some of the featured plant species.     

CZE separation of strawberry anthocyanins with acidic buffer and comparison with HPLC

Anthocyanins, the major colourants of strawberries, are polar pigments that are positively charged at low pH. Herein, we have assessed a new analytical method for the separation of anthocyanins using CZE. Acidic buffer solutions (pH <2) were employed in order to maintain pigments in the cation flavylium form and achieve high molar absorptivity at 510 nm. These spectral properties enabled us to identify strawberry anthocyanins in a preliminary stage by detection in the visible range, although the method was optimised at 280 nm to obtain the best S/N. The effects of buffer composition highlighted the necessity of adding an organic modifier to the running buffer to obtain a suitable separation. The electrophoretic method permitted the separation of the three main anthocyanins of strawberry extracts, namely pelargonidin 3-glucoside (Pg-glu), pelargonidin 3-rutinoside and cyanidin 3-glucoside. The electrophoretic results, expressed as retention time and separation efficiency of the major anthocyanin (Pg-glu), were compared to those achieved in HPLC, the analytical technique traditionally used for the investigation of anthocyanins in vegetable matrix. The content of Pg-glu in strawberries (cv. Camarosa), calculated with HPCE and HPLC methods, resulted respectively in 11.41 mg/L and 11.37 mg/L.     

Thin-layer chromatographic investigation of plant pigments in selected juices and infusions of cosmetological importance and their antioxidant potential

ABSTRACT

Fruit preparations (e.g., fruit juices and nectars) are rich in plant pigments and of great demand by the alimentary and pharmaceutical industry, basically not only due to their health-enhancing properties, but also due to their attractive colors and an overall high esthetic valor. Anthocyanins and anthocyanidins are an interesting group of plant pigments, and in this study, a thin-layer chromatographic detection was carried out of two anthocyanins (cyanin and keracyanin) and two anthocyanidins (pelargonidin and delphinidin) in a selection of the commercial and homemade fruit juices and the infusions prepared of dried plants. Moreover, the same preparations were evaluated for their antioxidant properties by means of two spectrophotometric methods (based on the 2,2′-azino-di-[3-ethylbenzthiazoline] sulfonate (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) tests, respectively) and by the dot-blot test (based on DPPH). Finally, an effort was undertaken to correlate the chromatographically established occurrence of plant pigments in the investigated fruit preparations with their antioxidant properties. However, this correlation was not straightforward, and for at least two reasons. First, the four plant pigments considered are not the only secondary plant metabolites which exert an antioxidant activity. Second, different chemistries stand behind the two spectrophotometric antioxidant activity tests (ABTS vs. DPPH), and the different measuring techniques (spectrophotometry vs. dot blot) are assumed. Nevertheless, it was established that the juices and infusions with the highest detected numbers of plant pigments characterize with the high antioxidant activities also (with blueberries, chokeberries, and hibiscus flower on the top positions). In that way, confirmation of the antioxidant potential of the plant pigments was obtained. Moreover, the presence of certain plant pigments in fruit juices and plant infusions was reported for the first time.