**1. Introduction**

Curcumin (abbreviated CUR), known as diferuloyl methane, is an intense orange-yellow solid and a natural ingredient of the plant rhizome of *Curcuma Longa* L. Two derivatives of CUR, demethoxycurcumin (abbreviated DMC) and bis(demethoxy)curcumin (abbreviated BDMC), can be found in the plant as well. Altogether they are known as curcuminoids (abbreviated CURD). Depending on the soil condition, the total content of CURDs in the plant rhizome varies between 2 and 9%. With approximately 70% of the total CURD content CUR represents the major component in turmeric [1–3]. As highlighted in Figure 1, the presence or absence of a methoxy functional group on o-position to a phenolic group represents the only difference in the chemical structure of the three CURDs. The molecular structure of CUR comprising two equally substituted aromatic rings linked together by a diketo group, which exhibits keto-enol tautomerism, plays a crucial role in the reactivity of CUR [4,5].

**Figure 1.** Curcuminoids extracted from the rhizome of Turmeric (*Curcuma longa* L.) and molecular structures of the three major constituents (curcumin (CUR), demethoxycurcumin (DMC) and bis(demethoxy)curcumin (BDMC)).

Studies show that CUR can be potentially used to treat over 25 diseases due to its anti-oxidative, immunosuppressive, wound-healing, anti-inflammatory and phototoxic effects [6–8]. These include, in particular, neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, diabetes, heart sickness, bacterial, viral and fungal diseases, AIDS and over 20 different cancers [9–12]. In addition to CUR, also the potential use of DMC and BDMC in the prevention of cancer was emphasized [13–15]. It was reported that DMC has the stronger effect on the inhibition of human breast tumor cells, followed by CUR and BDMC [16]. Ruby et al. described the higher bioavailability and cytotoxic activity of BDMC in animal cells [17].

Due to the higher reactivity of CUR associated with the stronger pharmacological activity on the human body comparable to the two other derivatives, CUR currently remains the targeted turmeric compound [18]. Despite the diverse pharmacological effects, the practical insolubility of CUR in water results in a very low bioavailability of the molecule and therewith leads to a limited usage as a drug [19]. To improve the bioavailability, formulation of curcumin nanoparticles or metal complexes were successfully implemented [20,21]. In addition, the application of CUR together with artemisinin in a CUR-artemisinin combination therapy against malaria was reported to decrease the drug resistance [22]. Moreover, the formulation of a CUR-artemisinin co-amorphous solid showed a higher therapeutic effect in the treatment of cancer than the single drug formulation [23]. For each of the application, CUR has to be available in chemically pure form and in sufficient amount.

H.J.J. Pabon described the preparation of synthetic CUR and related compounds [24]. Kim et al. recently published a process for production of CURDs in engineered Escherichia coli [25]. Nevertheless, the separation of CUR by means of solvent extraction from the plant rhizome still represents the most economical way of CUR production. In addition to plant proteins, oils and fats, the final extract contains 80% of the ternary CURD mixture [26]. In this mixture CUR is the major component with approximately 64% share of the total CURD content, together with 21% DMC and 15% BDMC [27]. Commercially available mixture usually contains 77% CUR, 17% DMC and 6% BDMC [28]. Consequently, CUR has to be purified from the ternary mixture.

There are two methods for separation of CUR from the mixture of CURDs described in the literature: by means of column or thin layer chromatography and by crystallization from solution.

For the chromatographic separation of CUR, silica gel (untreated or impregnated with sodium hydrogen phosphate) is commonly used as a stationary phase and various binary solvent mixtures of dichloromethane, chloroform, methanol, acetic acid, ethyl acetate and hexane as the mobile phase [29]. At the end of the process, three chromatographic fractions are enriched with the three CURDs, respectively [30,31]. Usually crystallization is applied as the final formulation step providing the solid product with desired specifications.

In the last decade, crystallization as a single separation technique was studied to purify CUR from the ternary mixture of curcuminoids [32–34]. Processes were described exploiting anti-solvent addition or system cooling, using methanol, ethanol and 2-propanol as process solvents and water as anti-solvent (Table 1).


**Table 1.** Overview of the results of published studies on CUR purification via crystallization: References 1–3 relate to [32–34], respectively.

<sup>1</sup> not specified; <sup>2</sup> optimized crystallization conditions.

As summarized in Table 1, from initial CURD mixtures crystalline CUR with purities of 92.2%, 96.0% and 99.1% at overall yields between 40 and 50% were obtained. The used separation methods were implemented as multi-step processes consisting of at least two successive sub-steps. It is reported that the main part of BDMC could be depleted after the first separation step, full removal was achieved after the second crystallization step [33,34]. DMC was always present in the final product. Ukrainczyk et al. observed an exponential decrease of the removal efficiency of DMC with increasing number of successive crystallization steps [34].

In order to reach the desired product purity and also to improve the overall process yield, a combination of the two separation techniques, chromatography and crystallization, was recently studied. Horvath et al. successfully implemented this integrated process for recovery of 99.1% pure artemisinin from an effluent of a photocatalytic reactor with 61.5% yield [35]. Heffernan et al. demonstrated the purification of single CURDs from the crude curcumin extract. There, the firstly performed crystallization process comprised three crystallization cycles, which provided 99.1% pure CUR in the final crystalline product. In the second process step, the remaining mother liquor was processed by column chromatography to isolate DMC and BDMC with purities of 98.3% and 98.6% and yields of 79.7% and 68.8%, respectively [36].

As has been demonstrated for other natural product mixtures, crystallization is a powerful technique to isolate a target compound from a multicomponent mixture within a single crystallization step [37,38]. Due to the fact that a 98% minimum purity of CUR is already sufficient for further drug application in pharmaceutical preparations [22], this study is directed to develop a separation process for isolation of pure crystalline CUR from the ternary mixture of CURDs within a single crystallization step.

To separate a target compound from a multi-component mixture, seeded cooling crystallization is preferably applied. Anti-solvent is usually added either at the beginning of the cooling step to generate the supersaturation in the solution or at the end of the process to increase the overall crystallization yield [39].

To purify CUR from the crude CURD mixture, seeded cooling crystallization processes were designed on the basis of solubility and nucleation measurements of pure CUR and CUR in presence of the CURDs mixture components in acetone, acetonitrile, ethanol, methanol, 2-propanol and selected binary mixtures thereof. Finally, with respect to the solubility results, 2-propanol, acetonitrile and water were considered as anti-solvents to improve the overall process yield.
