Curcumin Increasing its Water Solubility by Ultrasound and Peg
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Sigma Journal of Engineering and Natural Sciences 2017, Vol. 35, Issue 1, pp. 47-58; doi.org/10.62051/ytu.sigma-journal-of-engineering-and-natural-sciences-curcumin-increasing-its-water-solubility-by-ultrasound-and-peg
Abstract
Keywords: Curcumin; solubility; ultrasound; complex formation; polyethylene glycol.
1. Introduction
Curcumin (diferuloylmethane) is a major component of the yellow spice turmeric, derived from the rhizomes of curcuma longa and is commonly used as a flavoring and coloring agent in foods. Curcumin has also been reported to show potential in terms of antioxidant, antiinflammatory, antimicrobial and anticarcinogenic activities [1-6]. In rodent experiments, enzymatic effects in anti-inflammatory and anticancer activities of curcumin were shown by Piper [1]. Chemopreventive effects in skin, stomach and colon carcinogenesis were investigated by Rao and Kawamori [2-3]. Oral toxicity and the effect against human cancers has been investigated in clinical pilot studies [4]. The effects in preventing and treating human cancers were shown by Aggarwal [5]. Mode of action of curcumin on carcinogenesis, gene expression mechanisms and drug metabolism of curcumin and its properties has been investigated by Duvoix [6]. Curcumin is highly hydrophobic. Its insolubility in water is the main obstacle which hinders its usage for production of useful drugs. In order to increase its applicability in food and drug preparations its solubility in water has to be increased. Methods used to increase its solubility include; solid dispersion techniques in polyethylene glycol (PEG) 4000 and 6000 [7], in poly vinylpyrrolidone [8], and cellulose matrices [9]. In the work by Song group curcumin was solubilized by food emulsifiers and deposited in alginate beads [10]. Conjugation onto alginate beads requires chemical synthesis [11]. Increasing the solubility by using cyclodextrin was preferred by many groups [12-19]. Other techniques for improving its solubility include using rubusoside [20], betacasein [21], soyprotein [22], hyaluronic acid [23], and micellization of curcumin in cationic, anionic and non-ionic surfactant solutions [24] and solubilization by biocompatible natural polymers like alginate [25]. Chemical conjugation of curcumin on water soluble polymers is another method to improve the water solubility [26]. The non-toxicity, biodegradability and biocompatibility of PEG makes it suitable for various biomedical applications [27]. All valuable effects of curcumin depend on its solubility in blood stream, in other words its water solubility In this work, the water solubility of curcumin is increased. Highly water soluble PEG is used to provide a curcumin-soluble organic medium and ultrasound (US) is used for efficient mixing for PEG-curcumin complex formation. Curcumin-PEG complexes were also prepared without using US for comparison. Curcumin existence and concentrations in complexes were determined by IR and UV measurements. Zeta potential measurements and size measurements were performed for the complexes. Critical aggregate concentrations (CAC)’s were determined from UV and zeta potential measurements. Antioxidant capacity of PEG-curcumin complexes were determined by the DPPH assay and the ferric-reducing antioxidant powers (FRAP) procedure.
2.1. Materials and Methods
PEG200, PEG600, PEG2000 and Curcumin are Merck products. 2,2 diphenyl-1picrylhydrazyl, 2,4,6-tripyridyl-s-triazine and FeCl3.6H2O were from Sigma Chemical Co (Steinheim, Germany). Sodium acetate trihydrate, methanol, acetic acid, hydrochloric acid and FeSO4. 7H2O were from Merck (Darmstadt, Germany).
2.2. Complex Preparation
Different concentrations of 10 g, PEG solutions were prepared, (PEG concentrations (weight percentages, g/g in water) are given in Table1) and 20 mg of curcumin was added to each of
Curcumin, Increasing its Water Solubility by … / Sigma J Eng & Nat Sci 35 (1), 47-58, 2017
them. One group of samples was prepared by sonicating in an ultrasound bath for 2 hours at 25 oC (Bandelin Sonorex RK, 80W, 35kHz). At this power and frequency, chain scission limit is approximately 100000 ±20000 [28] and significant molecular weight reduction is not expected for PEG chains, however sonication is expected to result in more homogenous size and charge distributions. The other group was prepared by magnetic stirring for 2 hours on a magnetic stirrer. At the end of 2 hours each group was centrifuged for 1 hour at 6000rpm and the dissolved part was decanted and the amount of dissolved curcumin determined by UV spectroscopy. Neither alcohol nor any other organic chemical were used during preparations. Complexes form clear solutions with water at all dilutions and were observed for one year and found to be remain stable in refrigerator. For identification purposes, complexes prepared in ultrasound bath will be named as “sonicated” and complexes prepared by mixing on a magnetic stirrer will be named as “mixed” in the rest of the paper. Surface charges and sizes of complexes were measured by Malvern zetasizer and nanosizer instruments.
2.3. FTIR Measurements
FTIR spectrum of sonicated complexes are given in Figure 1a and b, for PEG200-curcumin and for PEG600-curcumin samples.
Figure 1a. IR Spectra of Curcumin, PEG200, and PEG200-Curcumin complexes
A detailed study on the vibrational spectra of curcumin has been reported by Kolev et al [29]. The FTIR spectrum of curcumin is shown in Figure’s 1a and 1b as a first spectrum from the bottom. The peak at 1626 cm−1 has a mixed C= C and C= O character. Another band at 1601 cm−1 is attributed to the symmetric aromatic ring stretching vibrations C = C ring. The 1508 cm−1 peak is assigned to the C = O. PEG200 and PEG600 spectra are shown in the middle in both figures. Here CH bending is attributed to the peaks at 1454cm-1 1351cm-1 in PEG 200 and 1464cm-1 1345 cm-1 in PEG600. The spectra of the PEG200-curcumin and PEG600-Curcumin complexes are shown as the top spectra in Figure 1a and 1b. Peaks at 1637 cm-1 in PEG200-curcumin and 1640cm-1 in PEG600-
H. Çatalgil Giz, S. İşçi, F. Bahadori, Z. Kalaycıoğlu, B. Akkurt / Sigma J Eng & Nat Sci 35 (1), 47-58, 2017
curcumin attributed to mixed C=C and C=O character and small shoulder at PEG600-curcumin at 1507cm-1 is attributed to C=O stretching as a part of curcumin structure.
Figure 1b. IR Spectra of Curcumin, PEG600, and PEG600-Curcumin complexes
1. DPPH free radical scavenging assay
The free radical-scavenging activity of PEG-curcumin complexes were determined by the DPPH assay described by Blois [30] with slight modification. 20 mg/L DPPH solution was prepared in methanol. 1980 µl of this DPPH solution was added to 20 μL sample solution. Thirty minutes later the absorbance at 515nm was measured at Shimadzu UV-1800 spectrophotometer. Inhibition of free radical DPPH in percent (I %) was calculated by using the following equation where Asample is the absorbance of the samples and Acontrol is the absorbance of the water, since the complexes were in water. Percentage inhibition (I %) = [(Acontrol-Asample)/Acontrol] x 100
2. FRAP Assay
The ferric-reducing antioxidant powers (FRAP) of complexes were determined, following the method of Benzie and Strain [31]. The FRAP reagent was prepared containing 1:1:10 ratio of 10 mmol/L 2,4,6-tripyridyl-s tri-azine (TPTZ) solution in 40 mmol/L HCl, 20 mmol/L FeCl3 and 0.3 mol/L acetate buffer at pH 3.6, and warmed up to 37 °C, for 10 min prior to use. The mixture which containing 100 µl sample, 100 µl deionized water, and 1.8 ml FRAP reagent incubated at 37 °C for 10 min. The absorbance of the mixture at 593 nm was measured by a Shimadzu UV1800 spectrophotometer. Results were expressed as mM Fe (II)/g curcumin. The calibration equation for FeSO4.7H2O was y = 1.0474x-0.1174, (R2= 0.998).
3. Results And Discussion
The amount of curcumin in sonicated and mixed complexes, are given in Table 1. Amount of curcumin in complexes increased as the PEG % and PEG molecular weight are increased, and sonicated complexes carried more curcumin in all cases. The most efficient results are obtained with PEG2000 and solubilized curcumin in sonicated complexes are three times more than mixed
Curcumin, Increasing its Water Solubility by … / Sigma J Eng & Nat Sci 35 (1), 47-58, 2017
ones. In the literature, curcumin solubility of 8.4×10 −8 M (3.09 10-5 mg/ml) was obtained in phosphate buffer solution containing 10.0% PEG 400. 27 In this work, curcumin solubility obtained in sonicated PEG200-Curcumin, PEG600-Curcumin and PEG2000-Curcumin samples are, 0.0071mg/ml, 0.066mg/ml and 0.038mg/ml respectively. In the case of mixed samples, solubility values obtained are 0.0069mg/ml, 0.011mg/ml and 0.018mg/ml respectively and higher than the literature results in all systems. One possible reason for the low curcumin solubility value in the above work is the phosphate salt used.27 Available curcumin junctions on the PEG chains had saturated by salt and curcumin solubility is decreased in PEG containing phosphate buffer solutions. Table 1. Amount of curcumin in sonicated and mixed PEG-Curcumin complexes Sonicated %PEG in water
Curcumin concentrations obtained from UV results in sonicated and mixed PEG2000Curcumin complexes are shown in Figure 2. As can be seen from the figure, up to a certain PEG percentage, complexes carry only a little amount of curcumin. After this point (approximately 7 % PEG, for sonicated and 15 % PEG for mixed complexes the curcumin concentration increase is rapid. The intersection of the two regimes is identified as the CAC for the PEG2000-curcumin system. To avoid repetition only PEG2000-curcumin system is shown as an example. PEG
H. Çatalgil Giz, S. İşçi, F. Bahadori, Z. Kalaycıoğlu, B. Akkurt / Sigma J Eng & Nat Sci 35 (1), 47-58, 2017
required to reach critical aggregate concentration (CAC) was determined as %30, 25%, %7 for sonicated PEG200, PEG600 and PEG2000-curcumin complexes and as %35, %25, %15 for mixed PEG200, PEG600, PEG2000-curcumin complexes respectively. 0.3
Figure 2. CAC determination in sonicated and mixed PEG2000-Curcumin complexes. Zeta potential measurements of sonicated and mixed PEG2000 complexes are shown in Figure 3. All complexes are negatively charged at low PEG concentrations and converged towards zero as the PEG concentration increases. Zero charge is seen to occur when the polymer fully covers the particle surface at CAC. Sonication results in better dispersion of particles and surface covering occurs faster and at a lower concentration. The CAC, indicated by zero potential, occurred at approximately 12% PEG2000 in sonicated and 17% PEG in mixed samples. All zeta results are shown in Table 2 and CAC concentrations from zeta potential results are found as 30, 30, 12 in sonicated complexes and 40, 30, 17 in mixed complexes for PEG200, PEG600 and PEG2000-curcumin complexes respectively. The approximate coincidence of the “zero zeta potential” concentration with the point of regime change supports identification of this point as the CAC. As the PEG molecular weight increases required amount of PEG to reach the CAC decreased. Less PEG was needed to reach CAC in sonicated samples due to more homogenous mixing.
Curcumin, Increasing its Water Solubility by … / Sigma J Eng & Nat Sci 35 (1), 47-58, 2017
Figure 3. CAC determination from zeta potential measurements for sonicated and mixed PEG2000 complexes. Table 2. Zeta potential of sonicated and mixed PEG-curcumin complexes
Schematic representations of complexes are shown in Figure 4, below critical aggregate concentration (CAC) (a) and above CAC (b). Below CAC, complexes could carry very little amount of curcumin, and above CAC curcumin concentration increased rapidly.
H. Çatalgil Giz, S. İşçi, F. Bahadori, Z. Kalaycıoğlu, B. Akkurt / Sigma J Eng & Nat Sci 35 (1), 47-58, 2017
Figure 4. A graphical representation of PEG-Curcumin complexes, before (a) and after (b) CAC is reached. Sonication process produced well dispersed particles with a homogeneous distribution. Particle sizes of complexes are shown in Table 3. Before CAC sonicated and mixed complexes gave two or three different size distribution. In sonicated complexes after CAC one distribution showed up however in mixed samples size distribution still carried two humps. Particle sizes increased as PEG molecular weight is increased and PEG % increased. Antioxidant capacity of complexes were determined by two different methods and results are given in Table 4. Following the same procedures % DPPH inhibition value of pure curcumin is 25±3.80 and the ferric-reducing antioxidant powers (FRAP) of pure curcumin is found as 1926± 58.95. As can be seen from the table all complexes have higher values than these. Antioxidant capacity of complexes increased as the amount of curcumin in the complex increased. Especially in PEG2000 after CAC has passed antioxidant capacity showed remarkable increase.
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Tablo 3. Particle Size of sonicated and mixed PEG-Curcumin complexes % PEG
H. Çatalgil Giz, S. İşçi, F. Bahadori, Z. Kalaycıoğlu, B. Akkurt / Sigma J Eng & Nat Sci 35 (1), 47-58, 2017
Table 4. Antioxidant capacities of PEG-Curcumin complexes %PEG in water PEG200Curcumin PEG600Curcumin PEG2000Curcumin
DPPH (% inhibition) Sonicated 45.47±3.019 61.13±5.258 47.16±3.874 66.88±5.698 41.85±1.055 42.36±1.736 44.33±2.625 53.96±3.592 Mixed 42.35±2.658 57.74±4.215 43.77±3.756 86.95±5.698 37.36±1.018 39.43±1.387 46.13±1.528 46.69±2.034
FRAP (mM Fe(II)/g curcumin) 4059±52 6814±85 4230±138 18813±225 5628±72.4 7441±85.7 14709±146 18162±125 2079±48.3 9026±126 1832±145 5527±256 1226±25.19 2604±28.87 4863±53.45 5025±46.81
4. Conclusion
The CAC was observed to decrease with increasing PEG molecular weight. In all experiments it is seen that sonicated samples have narrower zeta potential distributions, indicating that the US mixing is much better and the sonicated samples are more homogenous compared to the mixed examples. Antioxidant capacity of complexes were higher than pure curcumin itself in all cases; especially above the CAC. Complexes gave clear solutions at all dilutions with water. Another promising result; the complexes were found to be stable at all dilutions with water. No precipitation or phase separation was observed in either sonicated or mixed samples as long as they were observed (for more than a year). REFERENCES / KAYNAKLAR [1]
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GİZ, H.Ç.; İŞÇİ, S.; BAHADORİ, F.; KALAYCIOĞLU, Z.; AKKURT, B. Curcumin Increasing its Water Solubility by Ultrasound and Peg. Sigma Journal of Engineering and Natural Sciences 2017, Vol. 35, pp. 47-58. https://doi.org/10.62051/ytu.sigma-journal-of-engineering-and-natural-sciences-curcumin-increasing-its-water-solubility-by-ultrasound-and-peg