Encapsulation of curcumin into layered double hydroxides

2.1 Chemicals

All the chemicals and reagents used were of analytical grade purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. Curcumin was extracted from locally available turmeric, and the crude extract was used without further purification.

2.2 Extraction of crude curcumin from turmeric

2.3 Synthesis of layered double hydroxide (LDH)

Mg-Al-LDH with nitrate anions within the layers was used for comparison purposes. LDH was prepared by a pH controlled co-precipitation method by adding a solution containing Mg²⁺ and Al³⁺ ions (300.00 cm³ of Mg²⁺ and Al³⁺ solution was prepared by dissolving 1 mol dm^-3 Mg(NO₃)₂.6H₂O and 1 mol dm^-3 Al(NO₃)₃.6H₂O in 2:1 ratio) dropwise to a solution containing 300.00 cm³ of 1 mol dm^-3 NaNO₃ under vigorous stirring conditions at 60°C. During the addition period, the pH of the solution was maintained at 9 by adding 1 mol dm^-3 NaOH. Nitrogen gas was purged in order to avoid any contamination by atmospheric CO₂. The slurry was then stirred overnight in a closed container at 60°C. Finally, it was filtered and washed thoroughly with distilled water to remove impurities and was dried at 90°C resulting in a white solid [11, 12].

2.4 Synthesis of curcumin-encapsulated LDH: in-situ method

Curcumin-encapsulated Mg-Al-LDH was prepared by an in-situ co-precipitation method. A solution containing Mg²⁺ and Al³⁺ (Mg²⁺:Al³⁺ ratio 2:1) was added dropwise to a solution containing 200.00 cm³ of curcumin under vigorous stirring conditions at 60°C. During the addition period, the pH of the solution was maintained at 9 by adding 1 mol dm^-3 NaOH. Nitrogen gas was purged in order to avoid any contamination by atmospheric CO₂. The slurry was then stirred overnight in a closed container at 60°C. Finally, it was filtered and washed thoroughly with distilled water to remove impurities and was dried at 90°C [11, 12].

2.5 Characterization techniques

Functional group identification was done using Bruker Vertex 80 FT-IR (Fourier transform-infrared spectroscopy) with OPUS 6.5 software. The desiccated sample was added to standard KBr in 1:100 ratio and mixed thoroughly. The portion of the mixed sample was pelletized, and the transmission spectra of the resulting pellet were obtained in 1000–4000 cm^-1. The composition and purity of LDH and LDH-curcumin were determined by Bruker D8 focus Powder X-ray diffractometer (PXRD) fitted with a copper tube (CuKα radiation, 1.541 Å source), an incident beam monochromator, and a scintillation detector. Diffraction patterns were collected over the range 1°–70° 2θ and a step size of 0.020°. Thermogravimetric analysis was done at the temperature range of 25–900°C on a Thermo SDT Q600 TG-DTA analyzer under N₂ atmosphere at a heating rate of 10°C/min. TGA results may be used to determine the water content and properties such as thermal stability, decomposition, and the composition of the material. UV-Visible spectroscopy was done using Shimadzu UV-3600 (using the solid state attachment, a thin layer of sample pressed onto a substrate of analytical-grade BaSO₄) to evaluate the structural and bonding stability of LDH and LDH-curcumin nanocomposites. The release studies were carried out using the same spectrophotometer but with the solution state attachment. Surface morphological analysis was carried out using a Hitachi SU 6600 SEM (scanning electron microscope), which has an accelerating voltage of 20.0 kV.

2.6 UV degradation of curcumin

Approximately 20 g of crude curcumin was placed uniformly in the bottom of a 1-l glass beaker, and 1000.00 cm³ of pH-adjusted buffer solution was carefully added along the wall of the beaker to avoid agitation. The degradation of curcumin was quantitatively monitored in the presence of a UV source over 2 h of fixed time period. The total volume of the beaker was kept constant, and 5.00 ml of solution was withdrawn every 30-min time interval. UV-Visible absorption spectroscopy was used to quantify the concentration of de-intercalated curcumin in the aqueous phase (total volume of the solution was kept constant by adding the removed solutions back).

2.7 UV degradation of LDH-curcumin

Approximately 50 g of well-dried LDH-curcumin nanocomposite was placed uniformly in the bottom of a 100-cm³ glass beaker, and 100.00 cm³ pH-adjusted buffer solution was carefully added along the wall of the beaker to avoid agitation. Degradation of curcumin being released from the LDH-curcumin nanocomposite was quantitatively monitored in the presence of a UV source over 2 h of fixed time period in acidic medium. The total volume of the beaker was kept constant, and 5.00 ml of solution was withdrawn every 30-min time interval. UV-Visible absorption spectroscopy was used to quantify the concentration of de-intercalated curcumin in the aqueous phase.

2.8 Release studies of LDH-curcumin nanocomposite

Approximately 50 g of LDH-curcumin nanocomposite was placed uniformly in the bottom of a 100-cm³ glass beaker, and 100.00 cm³ of pH-adjusted buffer solution was added carefully along the wall of the beaker to avoid agitation. Under different pH conditions (pH: 2, 5, 8), the release of curcumin was quantitatively monitored over 2 h of fixed time period, withdrawing 5.00 ml of solution every 15-min time interval, and the UV-Visible absorption spectra was recorded to quantify the concentration of de-intercalated curcumin in the aqueous phase (during the period of study, the total volume was kept constant).

3.1 Thin-layer chromatography (TLC)

All the eluted sample fractions in silica gel column chromatography were collected quantitatively in test tubes (approximately 20 cm³) and were subjected to TLC technique using chloroform:methanol (95:5) as the developing solvent system. Curcumin can be easily detected as yellow spots, and fractions were pooled according to their R_f values [7].

According to Figure 1, the curcumin component was comparatively nonpolar compared to demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC). Hence, after the sample introduction, curcumin eluted during the mobile phase, which was having a lower affinity during the stationary phase. The component, therefore, moved along the glass column at a faster rate compared to the other components. Then, the pure curcumin fractions (Figure 2) were collected at fractions 1–6 (R_f value of curcumin is around 0.75).

Figure 1

Structures of curcumin, demethoxycurcumin, and bismethoxycurcumin.

Figure 2

TLC plate of corresponding fractions 1–7. R_f [1]: 7.3/9.9=0.73; R_f [2]: 7/9.9=0.7; R_f [3]: 7.1/9.9=0.71 R_f [4]: 6.9/9.9=0.69; R_f [5]: 7/9.9=0.7; R_f [6]: 7/9.9=0.7; R_f [7]: 7/9.9=0.7, and Rf [7′]: 0.55/9.9=0.55.

In Figure 3, the three main components in turmeric are listed according to their R_f values. Therefore, the seventh and eighth fractions were contaminated with demethoxycurcumin (DMC), which has an R_f value of approximately 0.57. Then, the pure demethoxycurcumin (DMC) component was collected using fractions 9–11 (Figure 4). But further extraction can be ignored as the study was completely focused on curcumin.

Figure 3

TLC plate for turmeric extracts using chloroform:methanol (95:5) as a solvent system.

Figure 4

TLC plate of corresponding fractions 8–11. R_f [8]: 0.6/9.9=0.69 and R_f [8′]:0.56/9.9=0.56; R_f [9]: 0.55/9.9=0.55; R_f [10]: 0.58/9.9=0.58; R_f [11]: 0.53/9.9=0.53.

3.2 Characterization of curcumin-LDH nanocomposites

The nature of the changes in the bonding environment of curcumin after encapsulation into LDH was studied using Fourier transform infrared spectroscopy (FTIR). Figure 5 compares the FTIR spectra of pure LDH, curcumin, and curcumin-intercalated LDH. FTIR spectra of pure LDH and LDH-curcumin show bands in the region of 3300–3400 cm^-1 and indicate the presence of O-H groups. After the intercalation, there is a noticeable shift of the O-H stretching vibrational bands. For pure LDH, pure curcumin and LDH-curcumin the -OH stretching (v_-OH) vibrations were observed at 3385, 3357 and 3380 cm^-1 respectively (Table 1). This is due to the formation of strong hydrogen bonds between curcumin and -OH groups in LDH. The change in broadness of the O-H peak further confirms the organic nature of the interlayer region compared to the parent nitrate.

Figure 5

FT-IR spectra of (a) curcumin, (b) pure LDH, and (c) LDH-curcumin.

For LDH-curcumin, the band position of the stretching vibration of C=0 groups present in the curcumin has shifted from 1383 cm^-1 to 1364 cm^-1 due to the interaction with LDH where this shift agrees with a strong van der Waals bonding network. A band at 1358 cm^-1 for pure LDH is due to the stretching vibration of C=0, which might have formed due to the absorption of atmospheric carbon dioxide gas.

For pure curcumin and LDH-curcumin, C-H stretching bands were observed at 2934 and 2919 cm^-1, respectively. In pure LDH, the typical bands were observed at 942 cm^-1 for Al-O-H stretching and 769 cm^-1 for Mg-O-H stretching. For pure LDH, pure curcumin, and LDH-curcumin, peaks at 1635, 1614, and 1629 cm^-1 correspond to the bending vibration mode of hydrated water molecules and weakly bonded water molecules [13, 14].

Powder X-ray diffraction analysis (PXRD) was used to understand the successful encapsulation of curcumin into the LDH. PXRD pattern of the pure nitrate-LDH and LDH-curcumin are shown in Figure 6. A typical diffraction peak at 2θ=10° was observed for the pure nitrate-LDH corresponding to the basal reflection. This peak position and the interlayer spacing of 8.75 Å agrees with that reported for nitrate-intercalated LDHs [15]. Surprisingly, for curcumin-LDH, the basal reflection has shifted to a higher 2θ value of 11.5°, and the observed interplane spacing for curcumin-LDH is 7.66 Å.

Figure 6

PXRD patterns of (a) pure nitrate-LDH and (b) LDH- curcumin nanocomposite.

The possible encapsulation reaction of curcumin into LDH can be explained by considering the structure of the curcumin. The structure of LDH consists of positively charged cation layers and anions in the interlayer spacing and water molecules. The keto-enol tautomerism of curcumin as given in Figure 7A allows to form a negative charge on the curcumin structure at basic pH values; hence, as a result, curcumin can be intercalated or encapsulated into the interlayer spacing and surface hydroxyl groups during the co-precipitation reaction. Although curcumin molecules demonstrate an overall hydrophobic nature, the presence of hydrophilic hydroxyl groups and the negative charges originated as a result of keto-enol tautomerism drives the curcumin groups into the interlayer spacing of LDHs.

Figure 7

(A) Curcumin exhibits keto-enol tautomerism. (B) Plane of curcumin perpendicular to the cation layer. (C) Plane of curcumin parallel to the cation layer.

As a result of keto-enol tautomerism, curcumin is able to adapt into a flat conformation allowing the plane of the curcumin to be placed within the interlayer spacing either parallel or perpendicular to the cation layer.

The interlayer spacing compared to the smaller nitrate group reduced when curcumin was intercalated.

According to Figure 8, when comparing the width of the curcumin (approximately 6.9 Å) molecule with the basal spacing between the cation layer, it is almost impossible or less feasible to stack in a perpendicular arrangement to the cation plane, hence, to accommodate the charge balance and the most stable form of staking between the interlayer in a parallel conformation. The other reason for the lower interlayer spacing is attributed to the reduction of the degree of hydration between the layers, in the presence of large organic molecules. Additionally, some extra peaks corresponding to un-intercalated crystalline curcumin molecules are observed. These peaks do not disappear upon washing with excessive water or acetone indicating a strong affinity toward the nanolayers. These molecules are expected to be encapsulated onto the layers and layer edges.

Figure 8

Width of curcumin molecule.

Typical TGA curve for pure LDH (Appendix Figure 1) showed weight loss around 100°C due to the evolving of surface water. The second weight loss was at around 200°C corresponding to the removal of the interlayer water in the layered structure [16].

The structure of LDH consists of positively charged cation layers and anions in the interlayer spacing. In addition, water molecules in between the layers are hydrogen bonded to the interlayer anions and to the -OH groups on the surface of the brucite-like structure. Hence, the LDH consists of much higher water content than other respective composites. In TGA analysis of pure LDH, the initial weight loss between room temperature and 200°C was reported around 59.2% which is due to the removal of interlayer and physisorbed water (Appendix Figure 2). The weight loss between 450°C to 1000°C is attributed to the complete dehydroxylation of the Mg-Al hydroxide layers and the temperature treatment beyond 600°C caused a collapse of the layered structure (Table 2).

In LDH-curcumin nanocomposites, the interlayer spacing consists of organic anions, water molecules, and other anions (hydroxide) that may intercalate during the synthesis. As a result of the presence of organic molecules, which are less prone to be hydrated, LDH-curcumin consists of much lower water content compared to pure LDH. In TGA analysis, the weight loss of LDH-curcumin due to the removal of water was reported around 18%. This observation further corroborates the PXRD evidences of reduction in the degree of hydration. After the removal of physisorbed and chemisorbed water, the temperature between 200°C and 450°C LDH-curcumin showed complete dehydroxylation (8.6% weight loss) and completely decomposed after 406°C [17, 18].

The observations suggest that intercalation of curcumin into an LDH thermally stabilizes the curcumin molecule. It shifts the decomposition temperature from 391°C to 406°C after encapsulation. This improved stability allows better processing stability in pharmaceutical applications during industrial applications.

UV-Visible spectra have been used to evaluate the change in λ_max value in the pure compounds and the composites. As observed by UV-visible spectroscopy pure curcumin absorbs at λ_max of 425 nm while the LDH-curcumin nanocomposite absorbs at 459 nm (Table 3).

Curcumin consists of two chromophores (C=0) and exhibits keto-enol tautomerism, which allows the overall structure to become more negatively charged and forming H-bonds with LDH. During these interactions, the level of conjugation has increased when curcumin curcumin is encapsulated within the LDH matrix suggesting an increased stability of curcumin. This observation clearly concludes that the photo stability of the curcumin has improved after intercalation into the layered structures.

Scanning electron micrograph of pure Mg-Al-LDH and LDH-curcumin presented in Figure 9A shows a typical plate-like morphology for the pure LDH. Interestingly, as shown in Figure 9B, the same plate-like morphology is preserved after the synthesis of the nanocomposite, indicating a topotactic reaction mechanism. However, the crystallinity of the composite has reduced once the organic guest molecules are intercalated. This observation is further supported by the PXRD data. However, any crystalline phases of other compounds were not observed in SEM imaging.

Figure 9

SEM images of (A) Pure Mg-Al-LDH, (B) LDH-curcumin nanocomposite.

3.3 UV degradation of curcumin

The main coloring component in turmeric is curcumin, which is relatively unstable against the constant irradiation of UV light at acidic medium.

According to Figure 10, the initial concentration of curcumin is high, but when it is exposed to UV irradiation, it undergoes a rapid loss in curcumin concentration. The possible degradation products of curcumin at pH 2 were determined by HPLC technique and is listed in Figure 11 [19].

Figure 10

The graph of absorbance vs. time of UV degradation of curcumin.

Figure 11

Degradation products of curcumin.

The degradation products of curcumin under various pH conditions are mainly due to the autoxidation process where the radical chain reaction leads to the incorporation of oxygen into curcumin, resulting in the major products such as dioxygenated bicyclopentadione products and ferulic acid, feruloylmethane, vanillin as minor products [20].

3.4 UV degradation of LDH-curcumin

This evaluation is mainly focusing on the releasing stability of LDH-curcumin nanocomposite in the presence of UV light at acidic pH. Figure 12 shows that the LDH-curcumin nanocomposite releases curcumin in a consistent level despite the fact that curcumin, itself, degrades against the UV light in considerable rate according to Figure 10, which means the UV irradiation directly affects curcumin to degrade but showed a minimal effect on the releasing ability of LDH-curcumin nanocomposite due to the layered structure of LDH, which provides stability to curcumin to remain stable slightly longer under UV irradiation and to maintain the constant release.

Figure 12

The graph of absorbance vs. time of UV degradation of LDH-curcumin.

In other words, with time, curcumin molecules are released to the medium in a sustained manner. In the process, the released amount of free molecules is higher than the rate of degradation; therefore, increase in the concentration is observed.

3.5 Release behavior of LDH-curcumin

The releasing ability of LDH-curcumin has been tested in different pH conditions (pH: 2, 5, 8) under selected kinetic models. Curcumin has a possibility of exhibiting keto-enol tautomerism at pH 8. Owing to the presence of a high concentration of OH^- ions in the medium, curcumin stabilizes between the LDH nanolayers. In other words, at pH>7, the curcumin molecules exist in keto-enol tautomerism, and it drives the nanocomposite formation with the double hydroxide layers. As a result, the curcumin molecules are well protected within the layers preventing any degradation or release at higher pH values. Therefore, the LDH-curcumin nanocomposite showed insignificant release at basic pHs.

One the other hand, the medium of pH 2 consists of more H⁺ ions than OH^- ions compared to that with pH 5, which does not allow curcumin to undergo keto-enol tautomerism, which enables curcumin to release gradually due to the destabilization of interaction between LDH and curcumin. The release pattern demonstrates a slow releasing behavior at acidic pHs under zero-order kinetics when curcumin intercalated to LDH, but the release patterns of pure curcumin cannot be followed due to the instant dissolution of curcumin under the low pH values (Figure 13).

Figure 13

(A_t/A₀) vs. time for release of curcumin at pH 2 (zero order).