Concise behavior of Curcumin in water-ethanol: Critical Water Aggregation Percentage and multivariate analysis of protolytic equilibria

Highlights

• Critical Water Aggregation Percentage of CUR was determined in water-ethanol.

• Multivariate Analysis was employed to evaluate the protolytic equilibria of CUR.

• CUR presents three pKa values being the most acidic related to enol/enolate.

• pK_a2 and pK_a3 were related to the phenol/phenolate equilibria of CUR.

• The K-matrix leads to the pure spectra and the molar absorptivity of the species.

Abstract

Curcumin (CUR) is a promising natural product in the field of biomedical applications, having a range of pharmacological effects. Despite this, clinical trials have reported poor absorption, low bioavailability, and rapid metabolism, which reduce its effectiveness. In general, these problems are related to the highly hydrophobicity of CUR, a limiting factor not only in its applications but also in its characterization. In fact, the spectroscopic description of CUR in homogeneous/microheterogenous media has a range of controversies, mainly about its self-aggregation process and its pK_a determinations. To solve these drawbacks, the present work describes the concise spectroscopic analysis of CUR in different water-ethanol mixtures. The Critical Water Aggregation Percentage (CWAP), i.e., the limit percentage in which monomers are predominant is 50% of water. The Multivariate Analysis that employs the chemometric tools (MASDA) by Q-mode method was efficient in demonstrating that above 75% (water: ethanol v/v) CUR exists predominantly in self-aggregated form. In addition, the molecular modeling conducted in the ORCA 4.2 using the Hartree-Fock method associated with MASDA demonstrated that even at 30% (water: ethanol v/v) CUR dimers are present (∼6%). From these conditions, the complex protolytic equilibria of CUR was evaluated in water-ethanol (30:70 v/v) with or without ionic strength. The pKa determination was also conducted by Multivariate Analysis. The obtained pKa values in the presence or absence of ionic strength control do not differ significantly. The protolytic equilibria can be represented in terms of the principal species: CUR^{0 ⇌} CUR⁻¹ (enol/enolate), CUR^{−1 ⇌} CUR⁻² (phenol/phenolate) and CUR^{−2 ⇌} CUR⁻³ phenol/phenolate). The chemometric also allows to obtain the spectra of the pure species and the molar absorptivity for each one. This set of results is especially important to complement the literature about the basic spectroscopy and physicochemical properties of CUR.

Introduction

Curcumin (CUR) or according to IUPAC (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione is a polyphenolic compound of the ginger family, native of the tropical Asia region [[1], [2], [3]]. CUR presents in its structure two aryl rings with phenolic, –OH groups and a symmetrically linked ortho-methoxy groups. CUR also presents a diketone portion, which gives it a keto-enol tautomerism equilibria in neutral form, according to Fig. 1 [[4], [5], [6]].

Curcumin has been widely employed as food dye due to its strong absorption and fluorescence emission in the visible region, mainly in its monomeric form [3,7]. In recent years, CUR has been extensively studied in the field of biomedical research [1,2,[8], [9], [10], [11]]. This is due to its wide range of pharmacological effects such as antioxidant, microbial activity, anti-inflammatory, immunoregulatory, anti-parasite and others [8]. Recently, the benefits of CUR against COVID-19 were reported [12,13]. Moreover, current studies confirm the efficacy of CUR in the prevention and treatment of several types of cancer [[14], [15], [16]]. Despite this, CUR is highly hydrophobic, and during clinical trials it has showed poor absorption, low bioavailability, and rapid metabolism, factors that limit its clinical effectiveness [1,17]. The high hydrophobicity of CUR is one of the main factors related to these problems [1,17]. In this way, several studies have been carried out in order to evaluate its self-aggregation process in homogeneous/microheterogenous media [6,18,19]. In this sense, its spectroscopic properties are especially useful for the analysis of its behavior.

The spectroscopic properties of CUR are extremely dependent on the solvent and the pH conditions [18,20]. Thus, in acidic or neutral aqueous solution, CUR has strong absorption with the maximum wavelength (λ_max) ranging from 410 to 430 nm and a weak absorption between 260 and 280 nm, due to π → π* transitions [7,18,21]. Besides that, CUR presents a slightly transition n - π*, nearby 375 nm [22]. As demonstrated in Fig. 1, under these conditions, the diketo group exhibits keto-enol tautomerism [4,18,23]. The enolic species is more stable than the diketo form [6,23], given its planar structure [4]. Furthermore, experimental and computational studies have shown that the amount of enolic form is predominant and the main responsible for spectral absorption of the CUR [19].

In alkaline conditions, CUR presents unstable behavior, suffering chemically from decomposition. The main degradation products are: trans-6-(4-hydroxy3-methoxyephenyl)-2,4-dioxo-5-hexenal, dehydrozingerone (half Curcumin), ferulic acid and vanillin [3,[24], [25], [26]].

In this sense, the behavior of CUR is highly dependent on the pH of the medium and can exist in different protolytic species: neutral (Keto or Enol - CUR⁰), monoanionic (CUR⁻¹), dianionic (CUR⁻²) and trianionic form (CUR⁻³). And depending on the pH, two or more species can coexist [3]. Thus, the understanding about the protolytic and tautomeric species in each pH condition is crucial, especially for biological applications. Therefore, depending on the type of administration, the drug may be exposed to different pH (gastric, intestinal, physiologic, e.g.). Despite this, there is a countless controversy in the literature about the order of deprotonation and assignment of pKa values in aqueous solution or in homogeneous/microheterogenous aqueous mixtures [21,[27], [28], [29], [30], [31]]. The problems begin with the high spectral overlap existing between the different protolytic and tautomeric species [32]. In addition, the high hydrophobicity of CUR is a factor that limits the pKa determination in pure water [21,28,29,33].

To overcome these problems, in the present study we propose the pKa determination of CUR in water-ethanol microheterogenous media in conditions above the Critical Water Aggregation Percentage (CWAP) [34,35]. Supplementarily, we also propose the utilization of a chemometric tool that is a promising strategy to accurately determine the protolytic equilibria of the CUR by spectrophotometric titration [32,36,37]. The advantage of chemometric methods is the multivariate analysis, i.e., the search for the contribution of each species in each wavelength of the absorption spectrum [36,38]. In the traditional univariate analysis, only one specific wavelength is analyzed, and much information is overlooked, leading to serious mistakes.

According to the literature, chemometric tools are very efficient in cases in which there is an intense spectral overlap, poor analytical signal quality and when the pKa values are close to each other, which causes an interference between the chemical equilibria [32,36,[39], [40], [41]]. Regardless, various chemometric methods have been applied [[42], [43], [44], [45]], being the R-type mode the most commonly employed. The proposed methodology presented here for water-ethanol microheterogenous media is based on the Q-mode method, in which the focus is the relation between objects. The method evaluates the interrelationship among the samples, in order to search for similarities to determine the relative concentrations [[46], [47], [48], [49]]. Some recent papers already detailed the application of this methodology for aqueous systems [36,50]. The K-matrix method, where Beer's law is applied in matrix form, allows to retrieve the spectra of pure species and molar absorptivity of each species.

Summarily, the present study provides a concise determination of the behavior of CUR in aqueous medium and in different water-ethanol mixtures. The study basically makes use of the Critical Water Aggregation Percentage (CWAP) to delimit the monomeric form of CUR. From the established CWAP, the pKa values of CUR in microheterogenous water-ethanol will be determined in its monomeric form.