**4. Application of DES in anthocyanins extraction**

The tendency of use and research about anthocyanin extraction using DES or NADES is growing since 2015, as can be observed in **Figure 3**. Although, their use in the extractions of other nutraceutical compounds (phenolics, flavonoids, alkaloids, terpenoids) began some years before [21, 34, 42]. Now, DES have been studied in the extraction of anthocyanins of different parts of plants such as fruits, flowers and vegetables (**Table 1**). All examples cited in this work mention anthocyanins as primary compounds in the extraction), especially in the recovery of pigments from agroindustrial residues like the ones coming from the winemaking industry [69–72, 76] or juices industry [73].

Zuo et al. [21] have mentioned that due to the variety in molecule structure of the extracted compounds it has to take into account some considerations to make the extractions. For example, hydrophobicity or hydrophilicity, hydrogen-bonding capacity, polarity, viscosity, and the acidity tolerance range. One of the main advantages of DES is the feasibility of tuning their physical and chemical characteristics, in order to secure the desired extraction. In terms of anthocyanin extraction, a wide variety of DES has been used. The common composition of DES used is based on choline chloride (ChCl) as the hydrogen bond acceptor and polyalcohols, organic acids, sugars, or amides as hydrogen bond donors.

### **4.1 Influence of properties of DES due to composition**

Considering that, there are multiples HBA and HBD combination to synthesize a DES and that each one has its own properties, it is difficult to know how it will behave during extraction. Therefore, some authors focused on screening different DES in the extraction of anthocyanins. Alañón et al. [43] evaluate eight different DES based

**Figure 3.** *Number of articles published on DES (NADES) for anthocyanin extraction.*


*Deep Eutectic Solvents: A Promising Technique to Anthocyanin Extraction for Food Coloring… DOI: http://dx.doi.org/10.5772/intechopen.105162*


*ChCl: Choline chloride; Ox: Oxalic acid; CA: citric acid; EtGly: ethylene glycol; SodAce: Sodium acetate; FoA: Formic acid; LA: Lactic acid; Glu: Glucose; Suc: Sucrose; Fru: Fructose; 1,2-pro: 1,2-Propanediol; Glyc: Glycine; Gly: Glycerol; MaA: Malic acid; Pro: Proline; Sor: Sorbose; TA: Tartaric acid; 1,4-But: 1,4-Butanediol; Mal: Maltose; SodBen: Sodium benzoate; PrGly: Propyleneglycol; GlyA: Glycolic acid; MAE: microwave assisted extraction; UAE: Ultrasound assisted extraction; MA: Maceration; NPCE: Negative pressure cavitation extraction; HSH-CBE: high speed homogenization and cavitation burst extraction; and PHWE: Pressurized hot water extraction.*

#### **Table 1.**

*Source of anthocyanin extracted with DES reported in the literature.*

in choline chloride for the extraction of anthocyanins of *Hibiscus sabdariffa* calyces. All DES tested were able to extract delphinidin-3-sambubioside (hibiscin) and cyanidin-3-sambubioside (gossypicyanin). Although DES that contains an organic acid (lactic and oxalic acid) reached the highest yields. Another example was reported by Bi et al. [55]; they evaluated the effect of ten DES based in choline chloride or choline bitartrate or choline acetate and organic acids, polyalcohols, and amide in the anthocyanin extraction of mulberry puree. The DES with urea (amide) were unable to extract anthocyanin, due to the basicity of the solvent. The organic acid-DES combinations were the ones with high extraction values, especially ChCl-lactic acid. In addition, these authors mentioned that there is not a clear relation between viscosity and extractability as other authors previously said. The organic acid relation with extractability was confirmed by Guo et al. [56] in mulberry, Kou et al. [67] in blue honeysuckle fruit (*Lonicera caerulea* L.), and Kou et al. [59] in *Ribes nigrum* L. On the contrary Dai et al. [48] and Alrugaibah et al. [73] mentioned that pH did not impact the extraction efficiency. However, the pH of DES is not the only parameter to consider. Other physicochemical properties such as solubility, viscosity, surface tension, and polarity are also very important [59].

The interaction of anthocyanins, which are polar molecules (depending on the number of hydroxyl groups, degree of methylation, and acylation pattern), with polar DES could increase the extractability. Anthocyanin compounds could be dissolved into a solvent with similar polarity by complying with the principle of similar compatibility [59]. This agrees with results reported by Loarce et al. [72] in grape pomace, Kou et al. [59] in *R. nigrum* L., Cvjetko Bubalo et al. [53] in grape skin, and Xue et al. [76]. In the former research, ten DES of different composition (based in ChCl with polyalcohol and sugars) and molar ratios were screened, finding that DES with similar polarities to that of anthocyanins yielded the higher values. These results differ with Dai et al. 2016 [48] and Sang et al. [60]. In the last-mentioned work, authors evaluate different ChCl and lactic acid, 1,2-Propanediol, 1,4-Butanediol. Organic acids had the lowest extraction yield compared to polyalcohol-based DES which are the less polar DES [81]. Authors also evaluated different ratios of ChCl, suggesting that anthocyanin extraction could be enhanced by making more hydrogen–hydrogen bonds between DES and anthocyanins. However, an excessive hydrogen-bound increase viscosity of the DES affecting the mass transfer parameters.

The high viscosity of the DES could affect the penetration of DES into the pores; therefore, a decrease in viscosity could increase diffusivity, and reduce surface tension [67]. Also, as it was mentioned above high viscosity affects the hydrogenboding between DES and anthocyanin compounds by making it more difficult to form [48, 50, 53, 60, 69]. Viscosity could be changed by adding water into the DES mixture. But the increase in water content could affect the polarity properties by weakening the interactions between DES and anthocyanin [56]. Cvjetko Bubalo et al. [53] evaluated the extraction of anthocyanin from grape skin. They observe that the eight anthocyanins founded in grape skin were better extracted when the water content was between 10 to 50% as well as the content/yield of the specific anthocyanins, increasing the polarity effect. For example, high polar anthocyanins (anthocyanin-3-*O*-monoglucosides) were better extracted when the water content was 50%. Meanwhile, less polar anthocyanins (malvidin-3-*O*-acetylmonoglucosides) increase when DES contain 25% of water. Those observations are in agreement with Bosiljkov et al. [69]; they found that certain levels of water into the eutectic mixture improve the extraction due to the polarity (by approaching the polarity of DES close to the water polarity, decreasing it in high polar DES like the organic acid–base or increase in polyalcohol or sugar DES with low polarity), favoring the mass transfer properties due to low viscosity.

### **4.2 Influence of DES due to process properties**

Usually, the most critical factors that affect the extraction are the inherent properties of the solvents. However, extraction variables such as temperature and time also play an important role during the extraction. Furthermore, the combination of the solvent with other technologies (microwave-assisted extraction (MAE) or ultrasound-assisted extraction (USE), among others), and the variables of the process could improve the extractability.

The temperature is related to the change in physical characteristics of the DES, particularly, the reduction of viscosity and surface tension (similar to an increase in water content in DES) when temperature increase. Changes due to temperature could be attributed to the intermolecular forces by decreasing the strength of van der Waals forces and hydrogen bonding interaction, improving the flowability characteristics of DES [20, 34, 35]. Additionally, as previously mentioned, an improvement in mass

### *Deep Eutectic Solvents: A Promising Technique to Anthocyanin Extraction for Food Coloring… DOI: http://dx.doi.org/10.5772/intechopen.105162*

transfer parameters facilitates the penetration of DES in cells and the interaction with target compounds. Xue et al. [76] observed this behavior, but an excessive increase in temperature causes degradation of anthocyanins. Similar behavior was observed by Sang et al. [61] and Bozinou et al. [80]. Xue et al. [76] and Bozinou et al. [76, 80] focused their research in optimizing the extraction time [61, 76, 80], showing that shortest times do not allow the complete extraction, on the contrary, at longer time expose to the environmental conditions the quantity decrease [21]. Bozinou et al. [80] observed a fast extraction during the first minutes, reaching the equilibrium condition after 400 min, and a slight reduction after 1400 min. In another study Xue et al. [76] found that the reduction in anthocyanin content was notable after 30 min.

Both parameters, temperature and processing time are still involved when the extraction is assisted by some alternative technology. Microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE) are the most used methods. The use of UAE and MAE reduces the temperature and time process by causing a disruption of cell structure (by cavitation or because of a rapid heath inside the cell, respectively) and releasing the internal compounds. The use of MAE and UAE reduce processing temperature and time, and the effect of both parameters is similar to the simple maceration method. It has been reported that increase the temperature and time at a certain point increases extraction yield and an excess of both can decrease the anthocyanin content. This effect was evaluated by Cvjetko Bubalo et al. [53], Fu et al. [75], Kou et al. [59], MacLean et al. [68], Grillo et al. [58], among others. Some of them optimize both parameters, among other parameters such as water content in DES (%), solid–liquid solvent ratio, power (W). However, the optimization parameters will depend on DES type, molar ratio, water content; type of material, state, and metabolite of interest.

## **4.3 Stability**

In addition to the high extraction performance of DES and the physicochemical characteristics of the extraction method, some researchers have focused their studies in evaluating the stability of anthocyanins after being extracted with DES [41, 48, 55, 56, 67]. All observed that the stability of anthocyanins extracted by DES was higher compared to that obtained with a conventional solvent like ethanol. The stability could be attributed i) to the anthocyanin molecule interaction with DES mainly hydrogen bonding with the carboxyl and hydroxyl of cyanidin [48], ii) protection of the chromophore against nucleophilic attack [67], iii) substitution of core flavylium cation of anthocyanins [56], and iv) the reduction of the interaction of solutes with oxidative factors by reducing molecular movement, avoiding oxidative degradation [48, 55, 56].
