**1. Introduction**

Turmeric (*Curcuma longa*) is a notorious spice, highly esteemed not only by the scientific world but also by gastronomes as it is the primary source of curcumin (CR) or *(1E,6E)*-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione, a renowned natural antioxidant polyphenol that can scavenge free radicals undergoing electron transfer or abstract H-atoms either from the phenolic OH groups or the CH2 group of the β-diketone moiety [1–4]. Depending on the chemical environment, CR, an <sup>α</sup>,β-unsaturated β-diketone can adopt two different conformations, either the diketonic form or the enolic one (Figure 1) [2].

**Figure 1.** Keto-enolic tautomerization of curcumin.

The antioxidant activity of this natural polyphenol is a controversy for the scientific world. Some consider that the enol tautomer characterized by a better conjugation between the two aromatic rings containing the phenolic OH groups holds the main responsibility for the presence of the antioxidant activity highlighted as an inhibition of superoxide radicals, hydrogen peroxide and nitric oxide radical [3,5], while others acknowledge better the keto form due to its existence in slightly acidic media [6,7]. It was also suggested that the presence of CR accelerates the processes catalyzed by several antioxidant enzymes such as catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx) and heme oxygenase-1 (OH-1) [5]. Since this compound has low solubility in water at neutral pH, its absorption and bioavailability are poor. In addition to that, it has also reduced stability towards oxidation, light, alkalinity, enzymes and heat. Therefore in order to increase its pharmacological effectiveness, the latest researches were focused on improving the bioavailability, chemical and photochemical stability of CR using different methods such as: conjugation with cyclodextrin, β-diglucoside, αS1-Casein, β-Lactoglobulin [8–11], encapsulation in nanoparticles of biocompatible polymers, exosomes, lipid nanoparticles, dextrin nanogels, dendrimers, metal oxide nanoparticles [12–18], complexation with multivalent metal cations [19,20].

Layered double hydroxides are substances belonging to the class of anionic clays, having the general formula [M<sup>2</sup>+1-xM3<sup>+</sup>x(OH)2]x<sup>+</sup>[An<sup>−</sup>x/n]x−·mH2O (*M2*<sup>+</sup> and *M3*<sup>+</sup> are metal cations that can adopt an octahedral arrangemen<sup>t</sup> similar to the one adopted by Mg<sup>2</sup>+ in brucite, *An*− is a compensation anion, *x* is a value in the range of 0.2–0.33 and *m* is the number of water molecules) [21]. The presence of the trivalent cations leads to an excess of positive charge in the brucite-type layer which is compensated by anions *An*<sup>−</sup>, which are located in the interlayer region along with the crystallization water molecules. Even though the hydrotalcite (Mg6Al2(OH)16CO3·4H2O is the most renowned representative of this type of materials, there are also other natural occurring layered double hydroxide (LDH) compounds such as meixnerite (Mg6Al2(OH)18·4H2O), zaccagnaite (Zn4Al2(OH)12[CO3]·3H2O) and pyroaurite (Mg6Fe2(OH)16CO3·4.5H2O). Structurally, these solids consist of positively charged brucite-type layers with balancing anions and water molecules in the interlayer space [21]. Despite the fact that there is a scarce spreading of LDHs in the earth crust, several laboratory methods for their obtaining were developed: co-precipitation at a variable or constant pH, under low or high supersaturation conditions, sol-gel, hydrothermal and mechanochemical synthesis [21–24]. An interesting feature of the LDHs is the so-called "memory effect" which allows the reconstruction of the layered structure when the mixed oxide obtained by thermal decomposition of an LDH precursor at temperatures lower than 550 ◦C is immersed in an aqueous solution containing the desired compensation anion which can be either inorganic or organic [21,22]. LDH compounds are considered to have low toxicity, good biocompatibility and a buffering action when immersed in aqueous solutions, therefore they were also utilized in medical formulations (such as the antiacid TALCID®), for ibuprofen slow release, in colon-targeted drug-delivery, for anticancer methotrexate delivery [25–28] and as matrices for bioinorganic hybrid materials [29–31]. Due to their structure LDHs compounds have anion exchange properties and are frequently utilized as anion exchangers and adsorbents. The insertion of organic anions in the LDH can be performed by ionic exchange, co-precipitation or reconstruction. However, the process is more difficult due to the poorer aqueous solubility of the organic species [22,27,32]. Recently, Kottegoda and coworkers showed that the inhibitory activity against several microbial species (e.g., *Candida albicans*, *Candida dubliniensis*, *Pseudomonas aeruginosa*, *Escherichia coli* and *Staphylococcus aureus*) can be enhanced by CR encapsulation into an inorganic host, namely a layered double hydroxide (LDH) containing Mg and Al as metal cations [33–35].

Considering this state of the art, the present contribution aims to extend the studies towards the incorporation of CR in another type of LDH-matrix, namely Zn3Al-LDH that is less basic than MgxAl-LDH and could bring its own contribution in increasing the antioxidant activity due to the presence of Zn which is known for its antiseptic properties [36]. The target of this study was to choose the modality for obtaining the solid with the best capacity to incorporate CR during synthesis and the best ability to release it in vitro under controlled bu ffer conditions. Therefore, for the synthesis of CR-containing Zn3Al-LDH, two di fferent methods were applied: (*i*) direct co-precipitation (P) and (*ii*) reconstruction (R) of the LDH in the presence of CR, which was added either as an aqueous alkaline solution (Aq) or as an ethanolic solution (Et). Depending on the applied preparation protocol, the names of the synthesized solids were abbreviated as PZn3Al-CR(Aq), PZn3Al-CR(Et), RZn3Al-CR(Aq) and RZn3Al-CR(Et). The structural characterization of the samples was performed using X-ray di ffraction (XRD), attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR) and di ffuse reflectance UV–Vis spectroscopy (DR-UV–Vis). The release of CR from the solids was investigated in bu ffer solutions at di fferent pH values (1, 2, 5, 7 and 8) and the spent solid samples recovered after 24 h were also characterized by ATR-FTIR and DR-UV–Vis. Based on the obtained results, the sample allowing the highest CR-release was selected for further antimicrobial activity tests which are going to be the subject of a future publication.

### **2. Materials and Methods**

Curcumin (CR) from Sigma-Aldrich, Zn(NO3)2•2H2O, Al(NO3)3•9H2O, NaOH and Na2CO3 from Merck were utilized as raw materials for the synthesis of the CR-functionalized LDHs. Absolute ethanol from Fluka and deionized water were used as solvents. Certified SUPELCO bu ffer solutions of pH 1 (glycine, sodium chloride, hydrochloric acid), pH 2 (citric acid, sodium hydroxide, hydrochloric acid), pH 5 (citric acid, sodium hydroxide) and pH 7 (potassium dihydrogen phosphate/di-sodium hydrogen phosphate) were purchased from Merck, while the certified Fischer Chemical bu ffer solution of pH 8 (potassium dihydrogen phosphate/sodium hydroxide) was purchased from Fischer Scientific.

A pristine Zn3Al-LDH with interlayer carbonate anion was prepared by co-precipitation at pH 9 using 160 mL of a metal nitrates Zn(NO3)2·6H2O, Al(NO3)3·9H2O aqueous solution (1.5 M, molar ratio Zn/Al = 3/1) and 160 mL of an aqueous solution containing Na2CO3 and NaOH 160 mL (1 M concentration of Na2CO3, 2.5 M concentration of NaOH) for pH adjustment. The resulting gel was aged 18 h at 50 ◦C. The solid recovered by filtration was washed with deionized water until the conductivity of the wastewater was lower than 100 μS/cm and dried at 90 ◦C for 24 h and the reference material Zn3Al-LDH was finally obtained.

The preparation of CR-containing Zn3Al-LDH was performed using always an amount of CR corresponding to a molar ratio CR/Al = 1/10. For the synthesis by co-precipitation at pH 9 the above-mentioned amount of metal nitrates in aqueous solution were utilized and the pH was adjusted with an aqueous solution of NaOH (2.5 M). Two samples, e.g., PZn3Al-CR(Aq) and PZn3Al-CR(Et), were obtained following this procedure since CR was added either as an aqueous alkaline solution (Aq) or as an ethanolic solution (Et). Then, 100 mL of deionized water were poured in the reactor before starting the precipitation of PZn3Al-CR(Aq) by concomitantly adding the metal nitrates solution and the alkaline solution containing CR under vigorous stirring (350 rot/min). For the obtaining of PZn3Al-CR(Et), 100 mL of CR ethanolic solution were first added in the reactor and then the precipitation took place by concomitantly adding the solutions containing the metal nitrates and the NaOH under similar conditions of stirring. The flowchart for these preparations is presented in Figure 2. The aging of the precipitates was performed under an inert atmosphere (He flow 10 mL/min).

**Figure 2.** Flowchart for the preparation of curcumin (CR)-containing Zn3Al-layered double hydroxide (LDH) by co-precipitation.

For the preparations performed by reconstruction a mixed oxide called CZn3Al obtained by the thermal decomposition of the pristine Zn3Al-LDH at 460 ◦C during 18 h was utilized as raw material. The reconstructions were performed in brown glass vessels at 25 ◦C by contacting CZn3Al powder with a CR-containing solution under magnetic stirring and inert atmosphere (He 1atm) during 24 h. Two CR-containing solutions were prepared, an alkaline aqueous solution containing CR and NaOH at a concentration of 4 × 10−<sup>3</sup> M, and an ethanolic solution containing 4 × 10−<sup>3</sup> M CR. Depending on the type of CR-solution utilized for reconstruction, two solid samples were obtained, e.g., RZn3Al-CR(Aq) and RZn3Al-CR(Et), respectively. The washing of the recovered solids was performed with deionized water for RZn3Al-CR(Aq), and with ethanol for RZn3Al-CR(Et). The flowchart for these preparations is presented in Figure 3.

**Figure 3.** Flowchart for the preparation of CR-containing Zn3Al-LDH by reconstruction.

The content of Zn and Al in the solids was determined by atomic absorption spectrometry (AAS) using a Thermoelemental Solar AAS apparatus, while the content of CR in the solid samples was calculated based on the determination of total organic carbon (TOC) content using HiPerTOC–Thermo carbon analyzer according to a previously described protocol [37]. The value of TOC was calculated as the difference between total carbon (TC content obtained by UV-persulfate oxidation of the samples) and total inorganic carbon (TIC content obtained by mineralization of the samples with HNO3 to convert the bicarbonate and carbonate ions to CO2).

The XRD patterns of the samples were recorded on PANalytical MPD system using Ni-filtered CuKα radiation (λ = 1.5418 Å), with a scan step of 0.02◦ and a counting time of 20 s per step, for 2θ ranging between 5 and 70◦. The average crystallite size (*D*) of the different phases in the samples was determined using the Scherrer formula applied to particular reflections/crystallographic directions.

The characterization of the samples by infrared spectroscopy was performed using a JASCO 4700 FT-IR spectrophotometer equipped with ATR PRO ONE Single-reflection ATR accessory and monolithic diamond crystal on the 4000–400 cm<sup>−</sup><sup>1</sup> domain at 128 scans and a resolution of 4 cm<sup>−</sup>1.

Shimadzu 3600 UV–Vis NIR spectrometer equipped with an integration sphere was utilized for recording the DR-UV–Vis spectra of the solids in the range of 200–800 nm, using BaSO4 as white reference.

In vitro CR release studies were performed in dark brown bottles where 0.5 g of solid sample were contacted with 50 mL of the adequate buffer solution (pH 1, pH 2, pH 5, pH 7 and pH 8) at 25 ◦C during 24 h under mild stirring (100 rot/min). The amount of released CR was determined by UV–Vis spectrometry using a JASCO V650 UV–Vis double-beam spectrophotometer with a photomultiplier tube detector. Liquid samples were withdrawn from the bottles hourly in the first four hours and finally after 24 h and their absorption spectra were recorded in the range 350–550 nm against the corresponding buffer solution as blank. The concentration of CR in the solution was calculated with Equations (1)–(3):

> CCRsolution= A423nm/8153.5 [mol/L] = A423nm/22.13 [g/L] (1)

$$\text{TRCR in 50 mL solution} = \text{C}\_{\text{CR solution}} / 20 \text{ [g]} \tag{2}$$

%CR released= mCR in 50 mL solution × 100/(msolid × CCR in the solid (see Table 1)) (3)


**Table 1.** Chemical composition of the solids.

1 Calculated from TIC (wt. %) values CO3 (wt. %) = TIC/0.2; 2 Calculated from TOC (wt. %) values: CR (wt. %) = TOC (wt. %)/0.684; 3 H2O calculated considering the loss of weight in the temperature range 105-200 ◦C [21].
