Chemical modification of curcumin: Solubility and antioxidant capacity

ABSTRACT

The aim of this study was to develop a chemical method for demythylation of curcumin. The methoxy groups reduced solubility and low bioavailability of curcumin. The treatment of curcumin with hydrogen bromide or choline chloride increased cucumin water solubility from 1 mg/mL to 30 or 25 mg/mL, respectively. ¹HNMR spectra showed that the chemical shift of O-methoxy groups at 3.9 ppm disappeared upon chemical treatment of curcumin, and indicated that these groups were removed especially after hydrogen bromide treatment. The antioxidant activity of treated curcumin and untreated curcumin was measured using different in vitro assays (i.e., 1,1-diphenyl-2-picrylhydrazyl and 2,2’-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) radical scavenging, and phosphomolybdenum complex formation). A remarkable increase in 1,1-diphenyl-2-picrylhydrazyl and 2,2’-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) radical scavenging was observed as curcumin-hydrogen bromide > curcumin-Choline chloride > curcumin. The formation of phosphomolybdenum complex was found to increase in the order of curcumin-choline cloride > curcumin-hydrogen bromide > curcumin with EC₅₀ 30, 41, and 114 µg/mL, respectively. In conclusion, hydrogen bromide-treated curcumin could be used as potential antioxidant in new functional foods.

KEYWORDS:

• Curcumin
• Demethylation
• Acid treatment
• Water solubility
• Antioxidant activities

Introduction

Curcumin (diferuoly methane) was reported as a natural antioxidant and possessed therapeutic activities against several diseases such as diabetes, rheumatoid arthritis, multiple sclerosis, Alzheimer’s, and atherosclerosis.^[1–4] It exhibited prevention and treatment of different cancers, including gastrointestinal, melanoma, genitourinary, breast, lung, hematological, head and neck, neurological, and sarcoma.^[5–8] Curcumin had also hepatoprotective, nephroprotective, cardioprotective, neuroprotective, and hypoglycemic activities.^[9,^10]

The limitation of applicability of curcumin as a health promoting agent was due to its poor bioavailability.^[6] To improve bioavailability of curcumin, many technologies were developed and applied to overcome this limitation. Nano-sized delivery systems for curcumin, including liposomes, polymeric nanoparticles and micelles, conjugates, peptide carriers, cyclodextrins, solid dispersions, lipid nanoparticles, and emulsions could be the promising novel formulations, which appeared to provide longer circulation, better permeability, and resistance to metabolic processes.^[11–18] A bioactive compound could be protected by encapsulation from environmental destructive factors, solubilized, and delivered in a controlled manner. The complexation of curcumin with soy protein and its implications on solubility and stability was also studied.^[19]

The enhancement of solubility and bioavailability of curcumin requires more studies because until 2015 the problem was not resolved. It could be hypothesized that the demethylation of methoxy groups presented in curcumin molecule would increase its solubility and phenolic character as well as its bioavailability. Therefore, the aim of this study was to enhance the solubility and bioavailability of curcumin by treatment of curcumin with aqueous acid for splitting the two methyl groups of curcumin. The evaluation of antioxidant capacity of treated curcumin was achieved.

Materials and methods

Curcumin

Curcumin was purchased from Bio Basic Inc.

Hydrogen bromide (HBr) method

Curcumin (0.94 g, 2.55 mmol) was refluxed at 130ºC in 50% (v/v) glacial acetic acid/concentrated HBr solution for 3 h. The mixture was then cooled to room temperature, neutralized with ammonium hydroxide and filtered. The product was washed several times with distilled water and extracted with tetrahydrofuran (THF). THF extract was evaporated by rotavapor at 50ºC and dried to make 0.4 g of a dark product.

Choline chloride method

This method was based on the use of deep eutectic solvent (DES) which was prepared according to the procedure reported in the literature.^[20] DES was obtained by reaction of choline chloride (1 mol) with urea (2 mol) at 90°C until getting a clear solution. Curcumin (0.48 g, 1.3 mmol) and 0.96 g of DES were mixed and heated with stirring for 19 h at 70ºC. The mixture was then dissolved in THF and precipitated with water, filtered off and dried to afford 0.4 g of a dark product.

Determination of antioxidant activity of curcumin

DPPH radical scavenging activity

Free radical scavenging activity of curcumin was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method.^[21] Curcumin solutions (5–25 µg) were added to 3.9 mL of freshly prepared DPPH methanol solution (0.1 mM). An equal amount of methanol was used as a control. After incubation for 30 min at room temperature in the dark, the absorbance was measured at 517 nm using a spectrophotometer. The activity of scavenging (%) was calculated using the following formula:

ABTS radical cation decolorization assay

ABTS (2,2’-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) also forms a relatively stable free radical, which decolorizes in its non-radical form.^[22] The spectrophotometric analysis of ABTS^•+ scavenging activity was determined according to the method of Re.^[23] Curcumin was added to a pre-formed ABTS radical solution and the remaining ABTS^•+ after a fixed time period was quantified spectrophotometrically at 734 nm. ABTS^•+ was produced by reacting 2 mM ABTS in H₂O with 2.45 mM potassium persulfate (K₂S₂O₈), and stored in the dark at room temperature for 4 h. The ABTS^•+ solution was diluted to give an absorbance of 0.750 ± 0.025 at 734 nm in 0.1 M sodium phosphate buffer (pH 7.4). One mL of ABTS^•+ solution was added to 1 mL of curcumin solutions (0.2–1 µg). The absorbance was recorded 30 min after mixing, and the percentage of radical scavenging was calculated relative to a blank containing no scavenger. The extent of decolorization was calculated as percentage reduction of absorbance. The scavenging capability of test compounds was calculated using the following equation:

where AC is absorbance of a control (blank) lacking any radical scavenger and AS is absorbance of the remaining ABTS^•+ in the presence of scavenger.

Phosphomolybdenum complex assay

Spectrophotometric evaluation of antioxidant activity by the formation of a phosphomolybdenum complex was carried out according to Prieto.^[24] Curcumin solutions (40–200 µg) were mixed with 1 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a thermal block at 95ºC for 90 min. The samples were cooled to room temperature, and the absorbance of aqueous solutions of each sample was measured at 695 nm against a blank. The antioxidant activity was expressed as the absorbance of the sample.

NMR analysis

¹HNMR spectra were recorded in a mixture of CDCl₃ and DMSO-d₆ solutions on a Bruker Avance 600 MHz spectrometer.

Statistical analysis

The obtained data were statistically analyzed as a completely randomized design with three replicates by analysis of variance (ANOVA) using the statistical package software SAS (SAS Institute Inc., 2000, Cary, NC, USA). Comparisons between means were made by F-test and the least significant differences (LSD) at p = 5%.

Results and discussion

Curcumin is known to be insoluble in water. In this study, curcumin was treated with some chemical reagents for demythyalation of methoxy groups present in curcumin molecule. Methoxy groups caused the insolubility and low bioavailability of curcumin. The treatment of curcumin with HBr or choline chloride increased the water solubility of cucumin from 1 to 30 or 25 mg/mL, respectively. The structure of curcumin and its treated form could be assisted by ¹HNMR data as shown in Fig. 1. Part of ¹HNMR in this figure where methoxy groups are appearing was only considered. It was clearly observed that the appearance of O-methoxy groups at 3.9 ppm^[25] in curcumin disappeared especially in the case of HBr treatment.

Figure 1. NMR spectra of curcumin, curcumin-HBr and curcumin-choline chloride.

The evaluation of antioxidant activity of untreated curcumin and treated curcumin was conducted by several methods. Assays of DPPH^• and ABTS^•+ radicals are the most popular spectrophotometric methods for determination of the antioxidant capacity of foods, beverages, and vegetables. Such DPPH^• and ABTS^•+ scavenging methods were used to evaluate the antioxidant activity of compounds due to the simple, rapid, sensitive, and reproducible procedures.^[26] It was reported that when the antioxidants were added, DPPH^• and ABTS^•+ radicals converted to DPPH₂ and ABTS⁺ with a degree of decolorization.^[26]

DPPH assay revealed that the antioxidant substances were able to convert the blue-DPPH to the yellow-colored diphenyl-picrylhydrazine. The method is based on the reduction of DPPH in the presence of a hydrogen-donating antioxidant. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule.^[27] A remarkable increase in the percentage of DPPH radical scavenging was detected in the order of curcumin-HBr > curcumin-Choline chloride > curcumin with IC₅₀ 7.4, 11.0, and 16.4 µg/ml, respectively (Fig. 2, Table 1).

Table 1. Antioxidant effect of curcumin and treated curcumin on DPPH and ABTS radical scavenging and formation of phosphomolybdenum complex (PM).

Sample	DPPH	ABTS	PM
	IC50	IC50	EC50
Curcumin	16.4 ± 0.82	0.91 ± 0.031	114 ±5.8
Curcumin-HBr	7.4 ± 0.43	0.42 ± 0.022	41 ± 3.6
Curcumin-choline chloride	11.0 ± 0.54	0.74 ± 0.033	30 ± 2.3

IC₅₀ (µg) is the inhibition concentration of the test sample that decreases 50% initial radical.

EC₅₀ (µg) is the efficient concentration of the test sample that increases O.D 0.5.

Each value is expressed as means ± SD (n = 3).

Figure 2. Correlation between the concentrations of (1) curcumin, (2) curcumin-HBr, and (3) curcumin-choline chloride, and their antioxidant capacity as determined by DPPH assay.

The reactions with ABTS^•+ radicals involve an electron-transfer process. ABTS^•+ radicals are known to be more reactive than DPPH in the evaluation of antioxidant activity of compounds.^[28] All the curcumin samples exhibited ABTS scavenging activity in the concentrations ranged from 0.2 to 1.0 µg. The scavenging effect on ABTS radicals was increased in the order of curcumin-HBr > curcumin-Choline chloride > curcumin with IC₅₀ 0.42, 0.74, and 0.91 µg/mL, respectively (Fig. 3, Table 1). The antioxidant activity of curcumin samples were more reactive 10 times for ABTS^•+ than DPPH according to the amount of curcumin assayed.

Figure 3. Correlation between the concentrations of (1) curcumin, (2) curcumin-HBr, and (3) curcumin-choline chloride and their antioxidant capacity as determined by ABTS assay.

A phosphomolybdenum method was developed for the quantitative determination of total antioxidant capacity. The assay is based on the reduction of Mo(VI) to Mo(V) by the sample analyte and the subsequent formation of a green phosphate/Mo(V) complex at acidic pH. This method was optimized and characterized with respect to linearity interval, repetitively and reproducibility, and molar absorption coefficients for the quantitation of several antioxidants.^[24,^29] In the present study, the total antioxidant capacity of untreated curcumin and treated curcumin was developed spectrophotometrically by phosphomolybdenum method. An exponential increase of activity with tested amount of curcumin samples was shown in Fig. 4. The formation of a phosphomolybdenum complex was found to increase in the order of curcumin-choline chloride > curcumin-HBr > curcumin with EC₅₀ 30, 41, and 114 µg/mL, respectively (Table 1).

Figure 4. Correlation between the concentrations of (1) curcumin, (2) curcumin-HBr, and (3) curcumin-choline chloride and their antioxidant capacity as determined by formation of phosphomolybdenum complex assay.

Conclusions

Different in vitro assays indicated that treated curcumin showed stronger antioxidant activity than untreated curcumin. Higher antioxidant activity of treated curcumin might be related to the demethylation of curcumin upon chemical treatment, which increased its solubility and its phenolic characters. From the above results of ¹HNMR and antioxidant assays, it was also reported that HBr treatment is more potent than choline chloride treatment. HBr-treated curcumin, as potential antioxidant, could be used as a new functional food.

Funding

This article was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant Number 36-130-D1432. The authors are grateful to DSR for the technical and financial support.

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Additional information

Funding

This article was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant Number 36-130-D1432. The authors are grateful to DSR for the technical and financial support.