This study focuses on the encapsulation of natural curcumin present in turmeric (Curcuma longa L.) into a layered double hydroxide (LDH), which demonstrates slow release properties with future potential in therapeutic applications such as slow release wound dressings. Turmeric has been used in traditional medicinal applications since ancient times. The main active substances in turmeric are curcumin together with two related compounds, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC), which have been extensively studied as antibacterial compounds. However, these molecules are unstable and, therefore, demonstrate limited biological activity and practical applications. In this study, attempts were made to synthesize curcumin in-situ encapsulated layered LDHs in order to stabilize the curcumin molecules within the nanolayers of the LDHs. The curcumin intercalated LDHs were synthesized by a simple in-situ co-precipitation method. The release characteristics of curcumin from the nanocomposites were quantitatively monitored under different pH conditions using UV-Visible spectroscopic methods, and the results indicate that the nanocomposite has the future potential in slow release therapeutic applications.
Layered double hydroxide (LDH) is a class of anionic clay minerals, which is represented by the general formula [MII1-xMIIIx(OH)2]x+(An-)x/n·yH2O. Here, MII is a divalent metal ion, MIII is a trivalent metal ion, and An- is an anion . LDHs have attracted immense industrial and scientific significance due to the diversity of potential compositions and wide range of physical and chemical properties, which include features such as anion exchange and ease of reconstruction following mild experimental conditions. Structural properties known for their applications are as catalysts, adsorbent materials, and anion exchangers, while recent studies have emphasized other applications including the controlled release of pharmaceutically active drug components . The most decisive properties that make LDH useful in these industries are their low toxicity, biocompatibility, high specific surface area, and buffering effect . One of the major challenges in the pharmacology is to formulate active and more stable drugs that can be released at a controlled rate in the human body. In this context, LDHs have proven to be beneficial as a matrix for encapsulation of a wide spectrum of pharmaceutical and biologically active agents for these purposes .
Turmeric (Curcuma longa L.) is a well-known medicinal plant found in South Asian countries such as Sri Lanka and extensively used in Ayurveda, Unani, and Siddha medicinal treatments as remedy for various diseases . Current Indian traditional medicine uses it for biliary disorders, anorexia, cough, diabetic wounds, hepatic disorders, rheumatism, and sinusitis . Curcumin is the main coloring substance, which is responsible for the characteristic yellow color in turmeric and finds many applications in food and dye industries and agriculture . More importantly, it demonstrates antibacterial properties.
The wound healing ability of curcumin involving the mechanisms of reduced inflammation, granulation, and remodeling of the tissue has been intensively examined in rats and guinea pigs. The recent studies have highlighted that punch wounds in curcumin-treated animals heal much faster than curcumin-untreated animals . However, the instability of curcumin under normal environmental conditions has limited its practical applications.
In this study, attempts were made to stabilize the unstable curcumin molecules within the nanolayers present in LDHs. The high aspect ratio in LDHs enables to take the advantage of “more for less” as expected in nanotechnology applications [8, 9].
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.
Turmeric extract was prepared by using 20 g of ground turmeric rhizome dissolved in 100.00 cm3 acetone and left for 7 days at room temperature. After 7 days, turmeric extract was filtered, evaporated, and concentrated.
The acetone extract was filtered and concentrated in a rotary evaporator, then re-precipitated with petroleum ether and vacuum dried. The crude curcuminoid mixture contained curcumin, DMC, and BDMC. This mixture was passed through a silica gel (60–120 mesh) glass column. Approximately 4 g of crude curcuminoids were mixed with 8 g of silica gel by using dry column packing method. Then, the sample was injected on top of the column and eluted with chloroform followed by chloroform:methanol ratio with increasing polarity. The collected fractions were subjected to TLC test using chloroform:methanol (95:5) as a solvent system, and spots were detected by its characteristic yellow color. Fractions with similar retention time, Rf value, were pooled .
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].
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].
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.
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).
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.
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).
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 Rf values .
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 (Rf value of curcumin is around 0.75).
In Figure 3, the three main components in turmeric are listed according to their Rf values. Therefore, the seventh and eighth fractions were contaminated with demethoxycurcumin (DMC), which has an Rf 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.
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.
|Type of bond||Pure LDH cm-1||Pure curcumin cm-1||LDH- curcumin cm-1|
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 . 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.
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.
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.
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 .
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).
|Sample||Room temperature to 200°C weight loss/%||200–400°C Weight loss/%||400–1000°C Weight loss/%|
|Pure LDH||55.8 (94°C) (removal of surface water)||4.8 (305°C) (complete dehydroxylation)||3.6 (482°C) (complete decomposition)|
|3.4 (187°C) (removal of interlayer water)|
|Curcumin||3.3 (174°C) (dehydroxylation of OH groups)||45.9 (391°C) (complete decomposition)||–|
|LDH-curcumin||17.6 (72°C) (removal of water)||8.6 (235°C) (complete dehydroxylation)||7.6 (406°C) (complete decomposition)|
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 λmax value in the pure compounds and the composites. As observed by UV-visible spectroscopy pure curcumin absorbs at λmax 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.
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 .
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 .
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.
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.
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).
Curcumin derived from natural turmeric was successfully encapsulated into Mg-Al-NO3-LDH by co-precipitation method. The FT-IR spectra confirmed the presence of curcumin in the LDH. The change in the interlayer spacing in nitrate LDH obtained by PXRD characterization further suggests the successful encapsulation reactions between LDH nanolayers and curcumin. Higher thermal stability was observed from TGA characterization. Comparing the UV stability of curcumin and LDH-curcumin, a better UV stability in encapsulated products were observed. Slow release characteristics were observed at pH 2. The product therefore, demonstrates future potential as wound dressings.
The authors acknowledge all the academic and non-academic members of SLINTEC for their valuable guidance and the discussions and for providing necessary facilities, and Institute of Chemistry, Ceylon, for the lab facilitation. The authors wish to extend their gratitude to Prof. B.M.R. Bandara, Department of Chemistry, University of Peradeniya, Sri Lanka, for the valuable discussions and guidance.
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