Synthesis of Curcumin Nanoparticles from Raw Turmeric Rhizome.

Turmeric (Curcuma longa L.) has been used as a spice and a medicinal herb since ancient times. The main active ingredient of turmeric is curcumin, a polyphenol that helps prevent and control neurological, respiratory, cardiovascular, metabolic, inflammatory, and autoimmune diseases and some cancers. However, curcumin has drawbacks such as low water-solubility, poor absorption, fast metabolism, quick systemic elimination, low bioavailability, poor pharmacokinetics, low stability, and low penetration targeting efficacy. To overcome these drawbacks, a common method used is encapsulating curcumin in nanocarriers for targeted delivery. However, the degraded products of nanocarriers have raised concerns. In this research, we synthesized nanoparticles of curcumin, nanocurcumin without using nanocarriers. To do so, curcumin was soxhlet extracted from raw turmeric rhizome. The stock solutions of different curcumin concentrations prepared in dichloromethane were added to boiling water at different flow rates and sonicated for different time intervals. An average particle size of 82 ± 04 nm was obtained with 5.00 mg/mL stock solution concentration, at 0.10 mL/min flow rate and 30 min sonication time. The particle size tends to increase with the flow rate and the concentration of curcumin in the stock solution but decreases with the sonication time. X-ray diffraction shows sharp and intense diffraction peaks for curcumin, indicating its identity and high crystallinity, but nanocurcumins are amorphous. Fourier-transform infrared spectroscopy spectra confirm the presence of all the functional groups of curcumin in nanocurcumin. Transmission electron microscopy and scanning electron microscopy images show the perfectly spherical morphology of nanocurcumin. Although curcumin is not water-soluble, nano-curcumin formulations are freely dispersible in water.

Introduction

Nanotechnology is a field of modern applied science that aims to develop materials and devices with unique and inherent properties, in their size of 1–100 nm, at least in one dimension.[1] Nanomedicine is a branch dealing with the applications of nanotechnology in the treatment, diagnosis, monitoring, and control of biological systems.[2] The use of nanomaterials in targeted drug delivery (TDD) systems is gaining increased attention in cancer chemotherapy,[3] overcoming microbial resistance to antibiotics,[4] and in the safe and efficient delivery of vaccines, including newly developed SARS-CoV-2 vaccines.[5] The use of nanoparticle carriers in TDD systems is found to result in enhanced solubility, stability, bioavailability, and pharmacological activity while avoiding physical and chemical degradation, cytotoxicities to healthy cells, and higher dosage requirements.[6] Among these, the drugs and formulations derived from natural sources, such as plants and minerals, are gaining upsurged worldwide attention. In fact, half of the approved anticancer drugs have plant-based origin, which include vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine), taxanes (paclitaxel and docetaxel), podophyllotoxin and its derivatives (etoposide and teniposide), camptothecin and its derivatives (topotecan and irinotecan), anthracyclines (doxorubicin, daunorubicin, epirubicin, and idarubicin), and many others.[7] Their targeting toward diseased cells, tissues, and organs is currently under intense investigation. This has been achieved by developing nanodosage formulations in carriers such as polymeric nanoparticles (nanospheres and nanocapsules), liposomes, proliposomes, solid lipid nanoparticles, nanoemulsions, inorganic nanoparticles, and biological nanomaterials such as red blood cells, DNA, and so forth as drug and vaccine carriers.[8−14] The encapsulation of the active ingredients isolated from plant products, such as curcumin from turmeric, in such nanocarriers for TDD, has been recently reported.[15−17] While encapsulated drugs in nanocarriers have many benefits, the toxicity issues of the degraded products of nanocarriers, such as liposomes, have become a concern. Such concerns can be avoided if the nanoparticles could be made directly from the drug molecules without having to use separate nanocarriers. With this idea in mind, we investigated the synthesis of nanoparticles of curcumin, which we call nanocurcumin, from curcumin extracted from turmeric by developing a novel and inventive methodology based on mechano-chemical synthesis.

The reason for choosing curcumin is because it has been used as a traditional medicine for the diseases such as biliary disorders, anorexia, cough, diabetic wounds, hepatic disorders, rheumatism, and sinusitis.[18,19] Recent studies have revealed that turmeric has anticancer activity as well.[20−22] However, the yield and the quality of curcumin obtained depend on the methods of turmeric processing and curcumin extraction. Different methodologies are used to process turmeric though it consists mainly of four basic steps: washing, curing, drying, and polishing.[23] Color, aroma, and other chemical properties of processed turmeric directly depend on the processing procedure.[24] Therefore, specific optimum parameters should be used to process the turmeric rhizomes to obtain a maximum yield of volatile oil and oleoresin, safeguarding the chemical constituents in turmeric. There are specifications given by organizations such as the American Spice Trade Association (ASTA), the Food and Drug Administration (FDA), and the Sustainable Livelihood Security Index (SLSI) regarding the maximum and minimum levels of particular compounds in turmeric powder.[23] Turmeric powder contains about 60–70% carbohydrates, 6–13% water, 6–8% protein, 5–10% fat, 3–7% dietary minerals, 3–7% volatile oil, 2–7% dietary fiber, and 1–9% curcuminoids.[23] About 235 compounds have been identified in turmeric, they are categorized under phenolic compounds and terpenoids.[25] The identified compounds consist of 22 diarylheptanoids, 8 phenylpropene, other phenolic compounds, 68 monoterpenes, 109 sesquiterpenes, 5 diterpenes, 3 triterpenoids, 4 sterols, 2 alkaloids, and 14 other compounds.[25] Curcuminoids are comprised of curcumin, demethoxycurcumin, and bisdemethoxycurcumin, which all belong to the diferuloylmethane group of phenolic compounds.[26,27] The percentage composition of curcuminoids depends on geographical conditions; it roughly contains 80% curcumin, 17% demethoxycurcumin, and 3% bisdemethoxycurcumin.[28] The most important active ingredient is curcumin, and its chemical structure is shown in Figure .

Figure 1

Chemical structure of curcumin.[29]

Widely used methods to extract curcumin from the turmeric rhizome are soxhlet extraction, microwave-assisted extraction, supercritical fluid extraction, enzyme-assisted extraction, and ultrasound-assisted extraction.[28,30] Soxhlet extraction is one of the foremost and most common extraction techniques that allow the sample to repeatedly contact with the solvent, thus enhancing the extraction yield of the target compound.[28] Further, since curcumin is a liposoluble compound, organic solvents such as hexane, acetone, methanol, isopropanol, ethyl acetate, and ethanol have been used for extractions in turmeric.[31,32] In most extraction experiments, ethanol has been used as the extraction solvent due to its high solubilization capacity.[28] As such, in this study, we used the soxhlet extraction to isolate curcumin and purposely developed a mechano-chemical method to synthesize nanocurcumin.

Results and Discussion

The moisture percentage, volatile oil percentage, oleoresin percentage, and the curcumin percentage of the obtained turmeric powder are 10.00 ± 0.01, 5.85 ± 0.05, 17.55 ± 0.05, and 6.47 ± 0.01, respectively (see Supporting Information: Figure S1). Accordingly, the quality parameters of the turmeric powder complied with the standards; thus, it can be confirmed that good quality turmeric powder was obtained. Curcumin was successfully extracted from turmeric powder. 2.60 ± 02 g of crude extract was obtained from 15.00 g of ground turmeric powder. 0.40 ± 0.02 g of crude curcuminoid powder was extracted from 1.00 g of the crude extract. 0.30 ± 0.01 g of crystalline curcumin was obtained from 1.00 g of crude curcuminoid powder. Accordingly, the percentage extractability of crude extract, crude curcuminoid powder, and crystalline curcumin, with respect to the turmeric powder, is 17.49 ± 0.14, 6.95 ± 0.31, and 5.16 ± 0.44%, respectively (see Supporting Information: Figure S2). The particle size of the synthesized nanocurcumin was studied using dynamic light scattering (DLS), and the distributions of the hydrodynamic diameter of the synthesized particles are shown in Figure . Since there is a single peak in the distribution of the hydrodynamic diameter, it can be assumed that spherical particles have been synthesized. The average hydrodynamic diameter of each treatment was statistically analyzed. The results are depicted in Table S2. Mean values were comparatively analyzed according to the analysis of variance (ANOVA) procedure. Since the P-value is lower than the alpha P value (P = 0.0001, therefore P < 0.05), the null hypothesis is rejected. There was a significant difference between the average hydrodynamic diameters of the synthesized nanocurcumin particles at a 0.05 significance level. Duncan comparison analysis was carried out between the treatments to find out which means are different. There are significant differences between some treatments, while some treatments did not have significant differences. The smallest average hydrodynamic diameter is 82 ± 04 nm, which is from treatment 01. The highest average hydrodynamic diameter is 316 ± 15 nm, which is from treatment 12 (see Figure ).

Figure 2

Particle size distribution of synthesized nanocurcumin.

According to the results, the particle size is proportional to the flow rate and the curcumin concentration of the stock solution, while it is inversely proportional to the sonication time.[33] The absorption spectrum of curcumin results in an absorbance peak at 415 nm, and the absorption spectrum of nanocurcumin results in an absorbance peak of 411 nm (see Supporting Information: Figure S3). Since the characteristic feature of curcumin and nanocurcumin is obtained, it can be confirmed that curcumin and nanocurcumin are synthesized successfully.[34,35]

The crystallinity and purity nature of curcumin and nanocurcumin were analyzed using XRD, a strong analytical tool used to assess the purity nature and crystalline phases of sample particles (Figure ). Accordingly, it shows that the synthesized curcumin is pure and crystalline, whereas the synthesized nanocurcumin is amorphous.[34] Curcumin demonstrated sharp, intense diffraction peaks at 2θ values of 9.04, 12.35, 14.68, 17.42, 18.30, 23.46, 24.75, 25.75, and 27.53°, indicating its purity and high crystalline nature.

Figure 3

XRD pattern of curcumin and nanocurcumin.

The Fourier transform infrared spectroscopy (FTIR) spectra of curcumin and nanocurcumin were scanned at the mid-infrared region (4000–400 cm–1), and the spectra are depicted in Figure . The bands obtained correspond to the respective vibrations of the functional groups present in curcumin and nanocurcumin, as detailed below (Table ).

Figure 4

FTIR spectra of curcumin and nanocurcumin.

Table 1

Functional Groups Responsible for IR Absorption of Curcumin and Nanocurcumin

wavenumbers (1/λ) (cm–1)
curcumin	nano-curcumin	functional groups, along with the mode of vibration
3501	3350	stretching vibration of hydrogen-bonded O–H
2916	2922	asymmetric stretching vibrations of Csp2–H
2847	2850	asymmetric stretching vibrations of Csp3–H
1625	1623	C–H bending frequency of the aromatic overtone
1600	1581	stretching vibration of double-bonded C=C
1501	1503	stretching vibration of conjugated carbonyl (C=O) group
1454, 1423	1447, 1424	aromatic stretching vibrations of the benzene ring
1266	1262	stretching vibration of Csp2–O bond
1154	1118	stretching vibration of Csp3–O bond

The intense band at wavenumber 3501 cm–1 corresponds to the stretching vibration of hydrogen-bonded O–H present in curcumin. Bands at 2916 and 2847 cm–1 originate from asymmetric stretching vibrations of Csp–H and Csp–H bonds, respectively. C–H frequency of the aromatic overtone is obtained at 1625 cm–1. Bands at 1454 and 1423 cm–1 correspond to the aromatic stretching vibrations of the benzene ring. The intense characteristic band centered at 1501 cm–1 corresponds to the stretching vibration of the conjugated carbonyl (C=O). The band centered at 1600 cm–1 originates from double-bonded carbon stretching vibration, while bands centered at 1266 cm–1 and centered at 1154 cm–1 originate from the stretching vibrations of Csp–O and Csp–O bonds, respectively.[32,36]

Considering nanocurcumin, the broad, intense band at wavenumber 3350 cm–1 corresponds to the stretching vibration of hydrogen-bonded O–H present in nanocurcumin. Asymmetric stretching vibrations of Csp–H and Csp–H are shown at 2923 and 2850 cm–1, respectively. C–H frequency of the aromatic overtone is obtained at 1623 cm–1. Bands at 1447 and 1424 cm–1 correspond to the aromatic stretching vibrations of the benzene ring. The intense characteristic band centered at 1503 cm–1 corresponds to the stretching vibration of the conjugated carbonyl (C=O). Stretching vibrations of double-bonded carbon, Csp–O, and Csp–O bonds are shown at 1581, 1262, and 1118 cm–1, respectively.

The shape, size, morphology, and surface texture of the curcumin nanoparticles could be detected using numerous sophisticated analytical techniques such as transmission electron microscopy (TEM) or scanning electron microscopy (SEM).

Based on the TEM image shown in Figure a, the nanocurcumin shows perfectly spherical polydisperse particles with an average diameter of around 100–200 nm. Most of the visible nanoparticles showed a round and smooth surface in TEM. The SEM of nanoparticles further proved their smooth surface texture (Figure c,d). On the other hand, higher sonication leads to higher fluidization by which nanocurcumin is distributed more uniformly to produce a homogeneous dispersion and triggers more efficient loading. Besides, frequent collisions among curcumin nanoparticles lead to less accumulation. When curcumin and nanocurcumin were dispersed in water to study and compare the behavior of particles in water, curcumin is not soluble in water, while nanocurcumin is freely dispersible in water (see Supporting Information: Figure S4).[37]

The extracted curcumin is highly crystalline. During the formation of nanocurcumin, there was a sudden temperature change when adding into hot water, which was under sonication condition. This may have changed the crystalline nature of curcumin resulting in an amorphous nature in the synthesized nanocurcumin.[38,39] Dichloromethane is a solvent that dissolves curcumin.[40] However, once we dissolve them in dichloromethane, curcumin molecules get separated. When boiling water is added, the solvent dichloromethane evaporates since its boiling point is below the boiling water temperature. This evaporation of solvent helps the curcumin molecules to self-assemble in all directions. Since molecules are assembling from all directions, the resultant nanoparticles assume a perfectly spherical morphology, as witnessed by their TEM images (see Figure a,b).

Figure 5

TEM images of the spherical nanocurcumin synthesized from treatment 01 are detailed in Table S1 (a) 100k magnification and (b) 200k magnification. SEM images of spherical nanocurcumin (c) 500 nm resolution (d)100 nm resolution.

Nanoparticle formation depends on two factors: nucleation and growth.[41] The relative kinetics of these two processes determines the size and shape of the nanoparticles formed.[42] Since nucleation happens throughout the solution and each nucleus formed undergo subsequent growth, depending on the concentration of the initial molecules and rates of these two processes, the particle formed assumes the said size and the shape.[43,44] The flow rate should be very low to form nanocurcumin in the nanorange. Using a higher flow rate or turbulent addition leads to particle aggregation, thus leading to the increase of particle size. When we add a drop of curcumin dissolved in dichloromethane, at a very low flow rate, the solvent evaporates suddenly, and the available curcumin remains in the water. Still, it does not tend to agglomerate due to the sonication condition, which results in nanocurcumin. Although curcumin does not dissolve in water and appears as clumps in water, it does not tend to become nanocurcumin under normal sonication conditions.

Conclusions

Raw turmeric rhizomes are processed to obtain turmeric powder with 6.47 ± 0.01% curcumin percentage. Curcumin is successfully extracted from turmeric powder using the soxhlet extraction method, and the extractability of curcumin is 5.16 ± 0.44%. The mechano-chemical fabrication method is used to synthesize nanocurcumin from the extracted curcumin. When curcumin and nanocurcumin are dispersed in water, curcumin is not soluble in water, while nanocurcumin is freely dispersed. However, there is no change between the chemical structures of curcumin and nanocurcumin. According to the results, the particle size of nanocurcumin increases with the flow rate and the curcumin concentration of the stock solution, while it decreases with the sonication time. TEM and SEM images provide evidence of spherical and smooth surface morphology and the size range between 100 and 200 nm. In contrast, the other analytical techniques such as UV–visible spectroscopy, FTIR, and XRD further confirm the characteristic features of nanocurcumin synthesis. The present study is the first evidence to synthesize nanocurcumin, using the natural turmeric rhizome as the raw material, bringing a new insight on natural substances such as turmeric.

Materials and Method

Materials

Toluene, ethanol, hexane, isopropyl alcohol, and dichloromethane of analytical grade were purchased from Sigma-Aldrich. The raw turmeric rhizomes were obtained from the locally grown turmeric plants in the Ampara district in the Eastern Province of Sri Lanka.

Methods

Turmeric rhizomes were cleaned well; the fingers and the mother rhizomes were separated and blanched in a closed pot filled with 3/4 of water for 30 min and 45 min, respectively. The rhizomes were dried under sunlight until the moisture percentage was reduced to 10%. Then, the removal of the skin in the rhizomes was done by the turmeric polisher machine, and it was ground using the grinder (I.K.A. Laboratechnik 6000 min–1, France) to obtain turmeric powder.

The moisture percentage of the turmeric powder was determined by the Dean and Stark method (AOAC 17th edition 2000 Official Method 986.21) which was used to determine the volatile oil percentage of turmeric powder. The soxhlet extraction method was used to determine the oleoresin percentage using ethanol as the solvent. ASTA 18.0 method was used to determine the curcumin content of the turmeric powder.[45]

Curcumin was extracted using the soxhlet extraction method. To do so, ground turmeric powder was weighed. A sample of 15.00 g was embedded in a thimble and put in the soxhlet apparatus, which was gradually filled with ethanol as the extraction solvent. The extraction was carried out at 60 °C for 8 h by then; all the colored compounds are extracted to the solvent. Upon completing the extraction, the ethanol was separated from the extract using the rotary evaporator (Heidolph, Germany). 1.00 g of the crude extract was mixed with 25.0 mL of hexane, stirred gently, and was kept for 12 h. The solution was then stirred using the magnetic stirrer (Velp Scientifica, Europe) at 600 rpm for 3 h to obtain the powder. The solution was centrifuged. The powder obtained was separated and dried at 40 °C in the oven for 2 h. 1.00 g of the crude curcuminoid powder was mixed with 10.0 mL of a hot solvent mixture of isopropyl alcohol: hexane in 1:1.5 molar ratio.[32] The solution was then cooled at room temperature to obtain pure crystalline curcumin, and the extracted curcumin was separated by filtration.

Nanocurcumin was synthesized by a physicochemical fabrication method. The stock of curcumin solution (5.00 mg/mL) was prepared by dissolving the extracted curcumin powder in 20.0 mL of dichloromethane. 1.00 mL of stock solution was added to 50.0 mL of boiling water, in a dropwise manner at 0.1 mL/min flow rate, under ultrasonication conditions (Velp Scientifica, Europe). The solution was sonicated for 30 min. Then, the mixture was stirred at 800 rpm for 20 min till an orange-colored precipitate was obtained. The supernatant was discarded, and the synthesized nanocurcumin was obtained for further studies. The effect of the curcumin concentration of the stock solution, flow rate, and the sonication time was examined to achieve an optimal formulation of nanocurcumin. Accordingly, twelve treatments were used to synthesize nanocurcumin by changing the concentration of the stock solution, flow rate, and the sonication time, as shown in Table S1.

The absorbance spectra were scanned using UV–visible spectrophotometry (Agilent Cary 60, Australia) in the range of 200–800 nm wavelengths. The diffraction pattern was recorded by a Bruker D8 ADVANCE Eco powder X-ray diffractometer (XRD) with Cu Kα radiation of wavelength λ = 0.154 nm, incident at an angle of 1° and 2θ intervals from 20° up to 80° with the step size 0.005°. The dried powder sample was placed on a Smart iTR attenuated total reflectance (ATR) accessory composed of diamond crystal as the sample holding technique at a controlled ambient temperature (25 °C). The sample was scanned using a Jasco FTIR 6700 spectrophotometer from wavenumbers 4000–400 cm–1 to identify the chemical composition and bonding present.

The mean particle diameter of nanocurcumin was investigated using DLS, CILAS particle size analyzer—NANO DS. 5.00 mg of nanocurcumin was dispersed in 30.0 mL of distilled water and sonicated for 15 min. The colloidal solution was then diluted with distilled water in a 1:1 ratio and sonicated again, and the colloidal solution was used for size distribution analysis. Each experiment was performed in triplicates. Data were analyzed using ANOVA using the SAS 9.0 statistical package at 5% level of significance. Means were compared with Duncan’s Multiple Range Test. The morphological analysis of nanocurcumin was conducted by TEM (JEOL JEM-3010) operating at 300 kV. SEM images were obtained using a FEI Quanta 450 FEG-SEM microscope operating at 30 kV. 5.00 mg of nanocurcumin was dispersed in 30.0 mL of ethanol. The particle dispersion prepared was evaporated to dryness and was analyzed by coating on the surface of carbon and sputtering with gold.