Investigation of the interaction between curcumin and nucleic acids in the presence of CTAB

Spectrochimica Acta Part A 67 (2007) 385–390

Investigation of the interaction between curcumin and nucleic acids in the presence of CTAB Feng Wang a,b , Jinghe Yang a,∗ , Xia Wu a , Fei Wang a , Honghong Ding a a

Key Lab of Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China b Department of Chemistry, Zaozhuang University, Zaozhuang 277160, PR China Received 30 April 2006; received in revised form 15 July 2006; accepted 19 July 2006

Abstract It is found that nucleic acid can enhance the resonance light scattering (RLS) enhancement effect of curcumin (CU) in the presence of cationic surfactant cetyltrimethylammonium bromide (CTAB). The investigation indicates that in BR (pH 4.3) buffer, both the positive CTAB and negative yeast RNA (yRNA) combine and form a positive large association, then which is bound on the two carbon atoms of the carbonyls of CU through hydrogen bond and hydrophobic force and form CU–CTAB–yRNA ternary complex, resulting in the RLS enhancement of this system. Based on it, a sensitive method for determination of nucleic acids at ng ml−1 is established. © 2006 Elsevier B.V. All rights reserved. Keywords: Curcumin; Nucleic acids; CTAB; Interaction

1. Introduction Investigations of the interaction between nucleic acids and small molecules such as dyes, drugs and toxic chemicals have been the focus of extensive research in recent years. Direct use of the intrinsic fluorescence and ultraviolet absorption of nucleic acids for the investigation has been severely limited by low sensitivity [1,2]. Therefore, some probes have been employed for the investigation such as fluorescence probes [3–5] and resonance light scattering method [6–9], chemiluminescence probes [10,11], molecular beacons probes [12,13], and nanoparticle probes [14,15], etc. Curcumin [1,7-bis(4-hydroxy-3methoxyphenyl)-1,6-heptadiene-3,5-dione] (CU), the main constituent of the rhizomes of the plant curcumin longa, is a common ingredient used in spices, cosmetics and traditional Chinese medicine. It is a natural antioxidant, which is considered to be a very useful compound in health matters, and is employed in the treatment of cardiovascular and arthritic illnesses. Presently, it is also used as anti-inflammatory and anticarcinogenic [16–20].

∗

Corresponding author. Fax: +86 531 8564464. E-mail address: [email protected] (J. Yang).

1386-1425/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2006.07.027

The experiments indicate that the RLS intensity of CU–CTAB system is greatly enhanced by nucleic acids, the enhanced RLS intensity is in proportion to the concentration of nucleic acids; the detection limit of fsDNA has reached 0.1 ng ml−1 . So a sensitive RLS method for the determination of nucleic acids at nanogram levels has been proposed. Especially, in comparison with most RLS methods reported, this method is quick and simple, and has good sensitivity and stability. The mechanism study indicates that both yRNA and CTAB are combined at the conjugate chain of the ␤-diketone of CU. 2. Experimental 2.1. Chemicals Stock solutions of DNA (1.00 × 10−4 g ml−1 ) and RNA (1.00 × 10−4 g ml−1 ) were prepared by dissolving commercial yeast RNA (yRNA) (Institution Biochemistry, Chinese Academy of Sciences), fish sperm DNA (fsDNA) (Hua-mei Biochemical Reagent Co., China) and calf thymus DNA (ctDNA) (Hua-mei Biochemical Reagent Co., China) in 0.05 mol l−1 sodium chloride solution. These stocks needed to be stored at 0–4 ◦ C. The working solution was prepared by diluting the stock solution to the proper concentration. The purity of DNA was

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checked by measuring the radio of the absorbance of 260 nm to that of 280 nm. A stock solution of CU (1 × 10−3 mol l−1 ) was prepared by dissolving CU in ethanol, and then diluted to 5 × 10−5 mol l−1 with ethanol as the working solution. A series of Briton–Robinson (BR) buffer solutions (H3 PO4 + H3 BO3 + HAc + NaOH) was used for the pH adjustment. A stock solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) (1 × 10−3 mol l−1 ) was prepared by dissolving CTAB in deionized water and then diluted to1 × 10−4 mol l−1 as the working solution. All the chemicals used were of analytical reagents grade and doubly deionized water was used throughout. 2.2. Apparatus The spectrum and intensity of RLS were measured with a Hitachi F-2500 spectrofluorometer (Japan). An UV-4100 (Hitachi, Japan) spectrophotometer was employed in all absorption spectra recordings. The surface tension (σ) was measured with a Kr˝uss Kizmks program surface tension instrument (Kr˝uss GmbH) by using the suspended plate method. Zeta potentials (ξ) were measured with a DXD-Iimicro-television electrophoretic instrument (Jiangsu optical plant). All pH measurements were made with a pHS-2F digital acidity meter (Leici, Shanghai). 2.3. Analytical procedure To a dry 10 ml test-tube, solutions were added as the following order: 1.5 ml BR (pH 4.3), 0.5 ml of CU (5 × 10−5 mol l−1 ), definite standard nucleic acids (or sample solution) and 0.7 ml of CTAB (1 × 10−4 mol l−1 ). The mixture was diluted to volume with water and allowed to stand for 15 min. All RLS spectrum obtained by scanning the excitation and emission simultaneously monochromators (namely, λ = 0) of spectrofluorometer from 220 to 600 nm. The intensity of RLS was measured at λ = 392 nm in a 1 cm quartz cell with slit at 5.0 nm for the excitation and emission. The enhanced RLS intensity of CU–CTAB system by 0 . Here, nucleic acids was represented as IRLS = IRLS − IRLS 0 IRLS and IRLS were the intensities of the systems with or without nucleic acids. All the UV absorption spectrum was measured by a UV-4100 spectrophotometer at the same time.

Fig. 1. Resonance light scattering spectra: (1) CU–CTAB–yRNA; (2) CU–CTAB; (3) CTAB–yRNA; (4) CU–yRNA; (5) CU; (6) CTAB; (7) yRNA. Conditions—CU: 2.5 × 10−6 mol l−1 ; CTAB: 7 × 10−6 mol l−1 ; BR: 0.04 mol l−1 (pH 4.3); yRNA: 1 ␮g ml−1 .

By comparing the light scattering and absorption spectra (Fig. 2), it can be seen that the peaks of light scattering at 285, 392, and 500 nm appear at the red side of the absorption bands at 250, 365 and 428 nm, respectively. According to the theory of RLS [21–23], the peaks of light scattering at 285, 392, and 500 nm are ascribed to the absorption bands of CU at 250, 365, and 428 nm, respectively. Since the light scattering spectrum not only depends on the nature of the system, but also reflects the characteristics of the instrument [24]. The RLS peak around 470 nm is considered to be possibly the emission of the xenon lamp in this region [8]. Since RLS intensity at 392 nm is the maximum in CU–CTAB–nucleic acids, so 392 nm was selected in the further study.

3. Results and discussion 3.1. Features of RLS spectra The scattering peaks of the systems (Fig. 1) are localized near 285, 392, 470, and 500 nm. As can be seen, the light scattering intensity of CU is weak, but the intensity of CU can be enhanced by yRNA or CTAB. This indicates an interaction of CU with yRNA or CTAB. Moreover, when yRNA and CTAB are together added into the system, the scattering light intensity of system was much higher than that of CU–CTAB and CU–yRNA systems. This proved that a large congeries formed in CU–CTAB–yRNA system.

Fig. 2. The absorption spectra: (1) CU–CTAB–yRNA; (2) CU–CTAB; (3) CU; (4) CU–yRNA; (5) CTAB; (6) yRNA. Conditions—CU: 2.5 × 10−6 mol l−1 ; CTAB: 7 × 10−6 mol l−1 ; pH 4.3; yRNA: 1 ␮g ml−1 .

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Table 1 Interference from foreign substances Foreign substances K+ ,

Cl−

Mg2+ , Cl− NH4 + , Cl− Citric acid Glucose Fe3+ , NO3 Cu2+ , Cl− Cd2+ , Cl− Ca2+ , Cl− BSA

Concentration coexing (␮g ml−1 )

IRLS (%)

Foreign substances

7 7 8 5 10 7 5 5 6 0.25

−4.2 −5.3 −4.8 −4.9 −4.7 4.3 4.6 −5.3 −4.2 −3.0

Al3+ ,

Cl−

Cr3+ , Cl− Co2+ , Cl− Vitamin C l-Arg l-Pro l-␣-Ala l-Leu d-l-Ala Asp

Concentration coexing (␮g ml−1 )

IRLS (%)

5 5 8 5 9 8 11 15 9 5

−4.7 −4.5 −5.2 −3.3 −4.5 −4.7 −4.1 −4.0 −4.3 −3.9

CU: 2.5 × 10−6 mol l−1 ; CTAB: 7 × 10−6 mol l−1 ; pH 4.3; yRNA: 10−7 g ml−1 . Table 2 Analytical parameters of this method Nucleic acids

Linear range (␮g ml−1 )

Correlation coefficient, r

Limit of detection (ng ml−1 )

yRNA ctDNA fsDNA

1.0 × 10−3 to 1.0 3.0 × 10−3 to 1.0 1.0 × 10−3 to 1.0

0.9990 0.9997 0.9992

0.5 0.3 0.1

3.2. Analytical application The experiments show that the optimum conditions for the determination of nucleic acids are as follows: 2.5 × 10−6 mol l−1 CU; 7 × 10−6 mol l−1 CTAB; 0.04 mol l−1 (pH 4.3) BR. The interference of various ions was tested and shown in Table 1. It is found that these foreign ions have little effect on the determination of 1.0 × 10−7 g ml−1 yRNA within the permissible ±5% error. Under the optimum conditions defined, the calibration graphs for yRNA and DNA were obtained as shown in Fig. 3 and Table 2. It can be seen that there is a linear relationship between the IRLS of the system and the concentration of nucleic acids. The standard addition method was used for the determination of nucleic acids. From Table 3, it can be seen that the results obtained were satisfactory. A synthetic sample, prepared based on the interference of foreign substances (Table 3) was analyzed. As Table 3 shows, the method is reliable. A comparison of this method with other well-known RLS methods for nucleic acids, in terms of sensitivity is summarized in Table 4. It can be seen that this method has a higher sensitivity

Fig. 3. The calibration plot of nucleic acids. The linear regression equations are log IRLS = 6.22 + 0.71 log C for yRNA, log IRLS = 8.72 + 1.12 log C for ctDNA and log IRLS = 5.30 + 0.56 log C for fsDNA.

than most of other RLS methods for the determination of nucleic acids. 4. The interaction mechanism 4.1. ξ potential assay From Fig. 1, it can be seen that there is the interaction of yRNA with CU and CTAB in the CU–CTAB–yRNA system,

Table 3 Recoveries of nucleic acids in synthetic sample Nucleic acids

Added (␮g ml−1 )

yRNA

0.050

fsDNA

0.110

ctDNA

0.07

Environment (␮g ml−1 )

Found (␮g ml−1 )

KCl (0.1) Fe(NO3 )3 (0.1) Leu (0.1) KCl (1) Fe(NO3 )3 (1) Leu (1) KCl (0.1) Fe(NO3 )3 (0.1) Leu (0.1)

0.0515, 0.0499 0.0505, 0.0500 0.04975 0.100, 0.0966 0.0971, 0.101 0.103 0.0714, 0.0689 0.0673, 0.0680 0.0707

Recovery (%)

R.S.D. (%)

100.7

1.4

99.5

2.7

99.0

2.5

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Table 4 Compared with other methods in sensitivity Probe

Nucleic acids

Limit of detection (ng ml−1 )

References

Azur A TAALPca Acridine red Morin-CTMAB Berberine TAPPb This probe

ctDNA/fsDNA ctDNA/fsDNA/yRNA ctDNA/fsDNA/yRNA ctDNA/fsDNA/yRNA ctDNA/fsDNA/yRNA ctDNA/fsDNA/yRNA ctDNA/fsDNA/yRNA

19.9/12.6 0.4/1.4/2.7 0.95/1.3/8.5 3.4/6.2/4.1 6.5/2.1/3.5 4.1/4.6/6.7c 0.30/0.10/0.25

[25] [26] [9] [27] [28] [6]

a b c

Tetra-substituted amino aluminum phthalocyanine. ␣,␤,␥,␦-tetrakis[4-(trimethylammoniumyl)] phenylporphine. ×10−8 g ml−1 .

which is also proved by the ξ of CU–CTAB–yRNA system at different concentrations of yRNA. It can be seen from Fig. 4 that the ξ of CU–CTAB system is about 120 mV. With the addition of yRNA to this system, the ξ decreases linearly and reaches 0 mV at 2.1 ␮g ml−1 yRNA. This indicates that electroneutralization occurs when the molar ratio of CU:CTAB:yRNA is about 1712:47962:1, then increases to the positive ξ, which means the formation of a large aggregate. It is well known that yRNA is negative charged. Therefore, we consider that in the solution of pH 4.3, positively charged CTAB can aggregate on

the surface of yRNA through a combination of electrostatic and hydrophobic interactions and form the association complex of CTAB–yRNA, and then which binds with curcumin molecules by hydrophobic forces. The formation of the large CU–CTAB–yRNA complex can result in the great enhancement of RLS.

Fig. 4. The ξ relationship between CU–CTAB and yRNA. Conditions—CU: 2.5 × 10−6 mol l−1 ; CTAB: 7 × 10−6 mol l−1 ; yRNA: 1 ␮g ml−1 ; pH 4.3.

Fig. 5. The absorption spectra of yRNA: (1) CTAB–yRNA; (2) CU–yRNA; (3) yRNA; (4) CU–CTAB–yRNA. Conditions—CU: 2.5 × 10−6 mol l−1 ; pH 4.3; CTAB: 7 × 10−6 mol l−1 ; yRNA: 1 ␮g ml−1 .

4.2. UV Absorption spectroscopy Curcumin is a diferuloyl mechane molecule containing two ferulic acid residues joined by a methylene bridge. Due to its ␤diketone moiety, curcumin undergoes keto-enol tautomerism, which is as follows:

And curcumin exists entirely as enol form both in solution and solid phase [29–30]. From the enol form of CU, it can be seen that CU has a symmetrical conjugated ␲-bond system. Fig. 2 exhibits that CU has a maximum absorption peak at 425 nm and a shoulder peak at 365 nm, corresponding to the absorption of conjugated ␲-bond system and symmetrical structure unit, respectively [31]. Under the adding CTAB and yRNA into CU solution, the absorption peak at 428 nm has a blueshift and its intensity decreases; whereas peak of 365 nm has a redshift and its absorbance increases. The above fact indicates that CTAB and yRNA are combined on the center of CU through hydrogen bond and hydrophobic force [32]. The experiment indicates that the surfactant CTAB’s CMC in the system is 4.3 × 10−4 mol l−1 , which is much larger than the optimum of CTAB in the system. This means that CTAB in the system exists as the premicelle clusters or the individual monomers. We consider that the cationic surfactant CTAB can combine with yRNA through electronic attraction and form ion association, which is combined on the center of CU through hydrogen bond and hydrophobic force because that the polyphenolic type molecule such as CU is able to interact strongly with biomacromolecules and there non-covalent interaction may play a decisive role in its mechanism of action [33]. It can be seen from Fig. 5 that the absorption of yRNA decreases a little when CU or CTAB added into RNA, respectively, but the position of absorption peak of yRNA dose not

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Fig. 6. The effect of ionic strength of the system. Conditions—CU: 2.5 × 10−6 mol l−1 ; pH 4.3; CTAB: 7 × 10−6 mol l−1 ; yRNA: 1 ␮g ml−1 . Fig. 7. The NMR spectra of the system: (1) CU; (2) CU + CTAB + RNA.

change; Whereas CU and CTAB are together added into yRNA, the absorption peak of yRNA at 260 nm has a redshift and the absorbance increases. The enhancement of the absorption of nucleic acids at 260 nm indicates that the extension of the nucleic acid chain caused by the weak hydrogen bonds decrease in nucleic acids [31]. So, we believe that curcumin and cationic surfactant destroy the helix structure of yRNA and aggregate in the extended yRNA by the electrostatic force and hydrogen bond, and then form the CU–CTAB–yRNA congeries resulting in the enhancement of RLS. This argument can also be proved by the control assay to investigate the salt effect on the formation of the complex. From Fig. 6, it can be seen that NaCl has obvious effect on the RLS intensity of this system, which indicates that the charges of the system can be screened by NaCl with higher concentration, and the interaction via electric attraction will be weakened, resulting in the decrease of RLS intensity of the system. 4.3. The NMR spectra of the system In order to understand further the interaction between CU and CTAB (or yRNA), NMR spectra at optimum experimental conditions are measured, as shown in Fig. 7 and Table 5. Corresponding to CU, there the chemical shift signals of all hydrogen atoms are broadened and moved downfield significantly when CTAB and yRNA are added into CU solution. The broadening of peaks indicates a significant reduction in CU mobility because it binds with CTAB and yRNA. The motion to downfield of the chemical shift is attributed to the reduction of the electron cloud density. It is considered that the decrease of electron cloud density of hydrogen atoms is attributed to that CU comTable 5 Chemical shifts (ppm) of individual protons of CU in the systems

CU CU–CTAB–yRNA

a

b

c

d

e

7.23 7.25

7.12 7.16

6.84 6.91

6.65 6.69

5.98 6.05

bines with nucleic acid and CTAB. This is consistent with above studies. 5. Conclusion A new RLS assay of nucleic acids is proposed. In the BR (pH 4.3) buffer, the RLS of CU–CTAB system can be greatly enhanced by nucleic acids, owing to the interaction between CU–CTAB and nucleic acids. The enhanced RLS is in proportion to the concentration of nucleic acids in the range 1.0 × 10−3 to 1.0 ␮g ml−1 for fsDNA, 3.0 × 10−3 to 1.0 ␮g ml−1 for ctDNA and 1.0 × 10−3 to 1.0 ␮g ml−1 for yRNA, the detection limits are 0.10 ng ml−1 for fsDNA, 0.30 ng ml−1 for ctDNA and 0.25 ng ml−1 for yRNA. The interaction mechanism is investigated using multi-techniques such as the absorption spectroscopy, potential assay and 1 HNMR spectroscopy. It is considered that CTAB and yRNA form a complex with positive charge, then which is combined on the two carbon atoms of the carbonyls of CU through hydrogen bond and hydrophobic force and form CU–CTAB–nucleic acid ternary complex, resulting in the RLS enhancement of this system. Acknowledgements The Natural Science Foundations of China (20575035) and Shandong Province (Y 2003B02) supported this work. References [1] [2] [3] [4] [5] [6] [7] [8]

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