Syntheses, characterization and biological activities of rare earth metal complexes with curcumin and 1,10-phenanthroline-5,6-dione

Journal of Inorganic Biochemistry 103 (2009) 396–400

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Syntheses, characterization and biological activities of rare earth metal complexes with curcumin and 1,10-phenanthroline-5,6-dione Yu-Min Song a,*, Jun-Peng Xu a, Lan Ding b, Qian Hou b, Jing-Wang Liu c, Zao-Long Zhu a a

College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730 070, China College of Life Sciences, Northwest Normal University, Lanzhou 730 070, China c College of Chemistry and Life Science, Tianjin Normal University, Tianjin 300 073, China b

a r t i c l e

i n f o

Article history: Received 2 September 2008 Received in revised form 30 November 2008 Accepted 1 December 2008 Available online 13 December 2008 Keywords: Rare earth complexes Curcumin 1,10-Phenanthroline-5,6-dione Antibacterial activity

a b s t r a c t Three new solid complexes have been synthesized by the reaction of rare earth(III) nitrate with the first ligand curcumin (HL) and the second ligand 1,10-phenanthroline-5,6-dione (L0 ) in alcohol solution (pH = 6.5–7.0). The composition of the complexes has been characterized by elemental analysis, molar conductivity, thermogravimetric analysis, IR, UV–vis methods. The results reveal that b-diketone group of the first ligand to coordinates with rare earth ions in bidentate mode after deprotonated. But the second ligand uses its two N atoms coordinates with rare earth ions in bidentate mode. The general formula of the complexes is REL3L0 (RE = Sm, Eu, Dy). The results of antibacterial activity indicated that the complexes have excellent antibacterial ability for the testing bacterium than that of curcumin. The result of agarose gel electrophoresis suggested that the complex of SmL3L0 can cleave the plasmid DNA at physiological pH and temperature. And it was found that the cleavage process of plasmid DNA was sensitive to pH, however, adding radical scavengers almost had no effect on the DNA cleavage reaction, therefore, the cleavage of DNA by SmL3L0 does not produce diffusible hydroxyl radicals via the Fenton reaction. Ó 2008 Published by Elsevier Inc.

1. Introduction Curcumin [1,7-bis(4-hydroxy-3methoxyphenyl)-1,6-heptadiene-3,5-dione], which is a naturally occurring yellow pigment obtainable from the rhizomes of turmeric (Curcuma longa Linn.), is a common ingredient used in spices, cosmetics and traditional Chinese medicine. The medicinal activity of curcumin has been known since ancient times and this molecule has been the object of several investigations in the field of biology, medicine and pharmacology [1–3] over the last decades, such as antioxygenation, antibiosis and antitumor activities. From the structure of curcumin (Scheme 1), it can be said that curcumin has a highly conjugated bdiketone moiety. b-Diketones are long known to form complexes with almost every metal and metalloid [4]. Several metal complexes of curcumin have been synthesized, characterized and evaluated for various biological activities. Au(curcumin)2Cl, a five coordinate of gold complex [5], has the properties of anti-arthritic and is assessed in an adjuvant-induced rat polyarthritis model. The Cu(curcumin)2 complexes [6] are most cytotoxic in cultured L929 cells and show significant reduction in solid tumor volume in ascites tumor bearing mice. Orvig et al. have reported vanadyl, gallium and indium complexes of curcumin for medicinal applications [7], corroborating the importance of curcumin’s free phenolic OH * Corresponding author. Fax: +86 0931 7971989. E-mail address: [email protected] (Y.-M. Song). 0162-0134/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.jinorgbio.2008.12.001

groups for scavenging oxidants and correlated with reduced cytotoxic potential. And curcumin with other transition metals ions Ni2+, Zn2+, Pd2+, Fe3+ can form strong chelates [8–10]. However, studies on Biological activities of the rare earth metal complexes with Curcumin and 1,10-phenanthroline-5,6-dione have been rarely reported. In view of that the salt and complexes of rare earth metal have been tested by experiment and clinical use to possess antiinflammation, sterilization and antitumour activities [11–13], a need is appearing to synthesize the complex of rare earth with curcumin which has better biological activity than that of metal or ligand, so we focused on the development of ternary earth metal(III) complexes with curcumin and 1,10-phenanthroline-5,6dione and investigated their biological activities. Moreover, the selection of 1,10-phenanthroline-5,6-dione as the second ligand in the complexes may increase the biocompatibility of these complexes [14]. In this paper, the rare earth metal(III) complexes were synthesized and characterized by elemental analysis, thermogravimetric analysis, molar conductivity, IR, UV–vis methods. The antibacterial activity of the complexes is stronger than that of the ligand of curcumin and they all possessed strong inhibitory action against G(+) Hay bacillus and G() Escherichia coli. In order to gain a better understanding of the biological activity of this complexes, we have investigated the cleaving double stranded DNA (interaction of complex with DNA). The results of the studies show that the REL3L0 complex is capable of cleaving double stranded DNA at physiological pH and temperature. This study for further design

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2. Experimental

sen strains include E. coli which belong to G() strains and H. bacillus which is a G(+) strains. Small circular scraps of filter paper of diameter 5 mm were prepared for the purpose of bacteriostatic slices. Drug (5 mg) (curcumin and its complexes) was dissolved in 10 mL DMSO (1%) to make concentrations of 0.5 mg/mL. The solution (0.1 mL) was poured into a small bottle containing ten paper slices and it was ensured that all the solution was blotted up. The bottle was covered by gauze, sterilized for 20 min under a steam pressure of 15 lb/in.2, and then kept in an oven at 80 °C for subsequent testing. Bacterial strains were inoculated onto the medium plates of corn meal agar with absorbent cotton, and two previously prepared bacteriostatic slices containing the same drug were put on a medium plate. One sample was inoculated in parallel on two medium plates. Finally, all plates were incubated at 35 °C in 5% CO2 for 24 h and then examined.

2.1. Materials and measurement

2.5. Gel electrophoresis experiments

The rare earth(III) nitrates were derived from their oxide (99.9%, Sm2O3, Eu2O3, Dy2O3) acquired from Shanghai Yuelong Chemical Plant (China). Plasmid pBR322 DNA was obtained from Huamei Chemical Co. (Beijing China). Agarose was purchased from Xiasi Biotechnology Co. LTD (Beijing, China). Buffer (50 mM NaCl, 5 mM Tris, pH = 7.40) was used for all experiments. Solutions of complex were prepared by firstly dissolving in DMSO and then diluting by distilled water and buffer. DNA concentrations were determined spectrophotometrically with an extinction coefficient of 6600 M1 cm1 at 260 nm [15]. Rare earth contents of the complexes were determined by EDTA titration using xylenol orange as indicator. All chemicals used in this work were of analytical reagent grade. The contents of C, H and N were determined using a Vario EL elemental analyzer. Molar conductance at room temperature were measured in 1  103 M DMSO (1%) solution using a DSS-11A digital molar conductometer. IR spectra were recorded in the range of 4000–400 cm1 on a Nicolet NEXUS 670 FT-IR spectrometer using KBr pellets. TG–DTA (thermogravimetric and differential thermal analysis) were performed on a Perkin–Elmer TGA 7 instrument in flowing N2 with a heating rate of 10 °C/min. The UV–vis spectra were recorded on a Varian Cary 100 Conc spectrophotometer.

Gel electrophoresis experiments were carried out with pBR322 DNA, in 0.8% agarose at 80 mV with TAE (45 mM Tris, 1 mM EDTA (Disodium salt), pH = 7.40) as the electrolyte. The cleavage reaction was quenched by the addition of EDTA and bromphenol blue.

O

O

R2

R3

OR1

R1O Curcumin (HL)

R1= H, R2 = R3= OCH3

Demethoxycurcumin

R1 = R3= H, R2= OCH3

Bisdemethoxycurcumin

R1= R2= R3= H

Scheme 1. The structure of curcumin and curcumin derivatives.

new drugs of curcumin, and as a sequence-selective cleavage agents for DNA itself is essential.

3. Results and discussion 3.1. Elemental analysis The compositions of these prepared complexes were determined by elemental analysis which exhibited a good agreement with the stoichiometry of REL3L0 (RE = Sm, Eu, Dy, HL = curcumin, L0 = 1,10-phenanthroline-5,6-dione), as shown in Table 1. The molar conductance measurements of the complexes were performed in DMSO solution (with a concentration of 1  103 M) at room temperature. The values varied between 13 and 19 S cm2 mol1, indicating that they are nonelectrolytes. 3.2. FT-IR spectra

1,10-Phenanthroline-5,6-dione was synthesized according to the literature [18]. The syntheses of EuL3L0 were accomplished by adding a stoichiometric quantity of an ethanolic solution of RE (NO3)36H2O (1 mmol) dropwise with stirring to an ethanolic solution containing the anion of the curcumin (3 mmol) (prepared by neutralization with triethylamine) and 1,10-phenanthroline-5,6dione (1 mmol) over a 2 h period under N2 atmosphere, and then cooling to room temperature. The compounds precipitated was separated by filtration and washed with a small amount of ethanol, and dried under vacuum, yield, 84–89%.

Table 2 shows the IR spectra date of the ligands and rare earth ternary complexes. The IR spectra of the three complexes are similar, but different from that of the free ligands. IR spectra of ligand curcumin showed mC@O in 1629 cm1, which shifted to lower energy in the complexes of the same ligand. And the complexes also had no broad band in the 2600–3400 cm1 range, related to the stretching of intramolecular H in the enol function [19], which indicated that the carbonyl group of the ligand coordinated to RE (III). New bands occurring in the region 521–525 cm1 (mRE–O) further supported the formation of RE–O band, the date agree with the literature [20]. A significant feature observed in the IR spectrum of the complex is the C@O stretch band at 1685 cm1, and its peak appears at approximately the same frequency to that observed in the free ligand of 1,10-phenanthroline-5,6-dione (1685 cm1), indicating that the C@O group is uncoordinated [21]. The bands at 1557 cm1 is the stretching vibrations of the C@N group of the 1,10-phenanthroline-5,6-dione ligand, which shift to lower energy owing to coordination and the new band, i.e. 459 cm1, in the EuL3L0 spectrum. These results show that both the nitrogens of 1,10-phenanthroline-5,6-dione are coordinated to rare earth ion.

2.4. Test of antibacterial activity

3.3. UV–vis spectra

In vitro antibacterial activity of curcumin and its complexes was studied by using the filter paper scraps diffusion method. The cho-

Fig. 1 shows the UV–vis spectra of curcumin and its complexes, respectively. The shape of the peaks being basically similar to that

2.2. Separation of curcumin derivatives Curcumin was separated into individual components by a previous method [16]. Purity of the three separated components was verified by TLC (thin layer chromatography), NMR, IR and ESI-MS (electrospray ionization mass spectrometry), and the date of UV– visible (UV–vis) absorption agreed with literature values [16,17]. 2.3. Complexes synthesis

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Table 1 Elemental analysis and molar conductance date of complexes.

Km (S cm2 mol1)

Complex

Found (Calcd.) % C

H

N

RE

EuL3L0 SmL3L0 DyL3L0

61.96(61.52) 60.57(61.60) 60.63(61.08)

4.06(4.31) 4.20(4.31) 4.16(4.28)

1.85(1.91) 2.06(1.92) 1.95(1.90)

10.95(10.39) 9.84(10.25) 11.38(11.03)

15 19 13

Table 2 IR spectrum data of ligands and complexes. Ligand and complexes

HL

mO–H

mC@O

Ligand EuL3L0 SmL3L0 DyL3L0

3425 3423 3421 3426

1629 1622 1620 1623

mRE–O

L0

mC@C

mC@O

mC@N

1589,1512 1591,1504 1590,1502 1592,1504

522 521 525

1685 1688 1690 1688

1557 1504 1502 1504

1.5

c

Absorption

459 463 455

Table 3 TG–DAT Analysis date of complexes.

d 1.0

b

Complexes

Exothermic peak of fusion (°C)

Main exothermic peak of decomposition (°C)

Weightlessness (%)

EuL3L0 SmL3L0 DyL3L0

216 210 219

495 459 470

12.31(2.03) 12.13(1.91) 13.35(2.64)

3.5. DNA cleavage

0.5

0.0 300

mRE–N

a

350

400

450

500

550

600

Wavelength (nm) Fig. 1. UV spectrum of complexes and ligand: (a): L; (b): SmL3L0 ; (c): DyL3L0 and (d): EuL3L0 .

for the free ligand curcumin, which implies that the absorption of complex depends mainly on the ligand. The curcumin ligand showed a main absorption band in the UV–vis is a region of p ? p* transition at 415–430 nm, which shifted to slightly higher energy and a new band at about 475 nm can be observed on the complexes, indicative of involvement of the carbonyl group of curcumin in metal complexation.

Some of rare earth complexes are capable of cleaving double strand DNA at the physiological pH and temperature. When the rare earth complexes were incubated with pBR322 DNA, the supercoiled (ccc) DNA is degraded to relaxed circular (oc) DNA and then slowly to liner [22]. Agarose gel electrophoresis patterns for the cleavage of pBR322 DNA by various concentrations of SmL3L0 was showed in Fig. 2, the concentration of DNA is 0.37 lM and the concentration of SmL3L0 was varied from 0 to 0.08 mM. It can be seen from Fig. 2 that plasmid DNA can be degraded to relaxed DNA as the concentration of the complex increased but the linear did not appear. As shown in Fig. 3, the process of cleavage is sensitive to

O

OH OCH 3

H3 CO

N

3.4. TG–DTA The TG–DTA curves of the three ternary complexes are similar when heated in N2 from 25 to 800 °C. The weightlessness of these prepared complexes exhibited a good agreement with the stoichiometry. It can be seen from Table 3 that there is no weightlessness in the TG curve around 25–210 °C suggesting that the complexes do not contain crystal water. The decomposition of these complexes is through two stages, and the second refers to the decomposition of the complexes to be oxide and is accompanied by a strong exothermic effect with one peak. The complexes decomposed completely at around 605 °C and the residues are rare earth oxide. Based on the above studies, a tentative coordination structure for the complexes is proposed (Scheme 2) .

O

HO

N O

O O

RE

O

H3 CO

O

OCH 3

O

OH

HO HO OCH 3

OH OCH 3

Scheme 2. The proposed structure of rare earth complexes.

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Y.-M. Song et al. / Journal of Inorganic Biochemistry 103 (2009) 396–400 Table 4 Antibacterial activities data (ring of antibacterial diameter mm).

Fig. 2. Agarose gel electrophoresis patterns for the cleavage of pBR322 DNA by various concentrations of SmL3L0 . Cleavage conditions: 37 lV DNA; 50 mM NaCl in 5 mM Tris–HCl buffer; pH 7.40; 37 °C for 3 h. Lane 1: DNA control; Lane 2: DNA + 0.02 mM SmL3L0 ; Lane 3: DNA + 0.04 mM SmL3L0 ; Lane 4: DNA + 0.06 mM SmL3L0 and Lane 5: DNA + 0.08 mM SmL3L0 .

Ligand and complexes

E. coli, Bacillus

H. bacillus

L EuL3L0 SmL3L0 DyL3L0

6 17 17 19

6 19 20 23

the change of pH. When the pH from 6.40 to 7.40, the complexes could cleavage DNA but not completely. The optimal pH of Double strand DNA cleavage was found to be the value of pH 7.40–8.05 (Fig. 3: Lanes 4 and 5). Under the same concentrations of the Sm3+ produced no cleavage of pBR322 DNA at the given conditions, but the ligand HL has a little lower cleavage activity than that of the complex (Fig. 4). As a mean for the mechanism of DNA cleavage, methanol and hydrogen peroxide are used as the radical scavengers. If the cleavage via the mechanism of Fenton reaction, the present of the scavengers will have an obviously effect on the result. From Fig. 5, it can be seen the present of scavengers has a slightly influence on the extent of cleavage, the result suggests the mechanism is contrast to the Fenton reaction, but the cleavage mechanism need to be further study. We tested the temperature sensitivity of the DNA relaxation activity of SmL3L0 complex, the result showed that when the temperature at 30, 33, 37 and 41 °C, Fig. 6. Antibacterial diameter of curcumin and its ternary complexes of rare earth on H. bacillus (1: HL; 2: EuL3L0 ; 3: SmL3L0 and 4: DyL3L0 ).

Fig. 3. Agarose gel electrophoresis patterns for the cleavage of pBR322 DNA by various pH. Cleavage conditions: 37 lV DNA; 50 mM NaCl in 5 mM Tris–HCl buffer; 0.04 mM SmL3L0 , 37 °C for 3 h. Lane 1: DNA control at pH 7.40; Lane 2: pH 6.40; Lane 3: pH 6.94; Lane 4: pH 7.40 and Lane 5: pH 8.05.

Fig. 4. Agarose gel electrophoresis patterns for the cleavage of pBR322 DNA. Cleavage conditions: 37 lV DNA; 50 mM NaCl in 5 mM Tris–HCl buffer; pH 7.40; 37 °C for 3 h. Lane 1: DNA control; Lane 2: DNA + 0.08 mM Sm3+; Lane 3: DNA + 0.08 mM HL and Lane 4: DNA + 0.08 mM SmL3L0 .

plasmid DNA can be cleavage. But only under 37 °C, the process of cleavage is more effect and plasmid DNA can be degraded to relaxed DNA and linear. So the physiological temperature (37 °C) as the reaction temperature of cleaving double strand DNA. 3.6. Antibacterial activity of the ligand and its complexes The antibacterial activity results of the complexes expressed as the diameter of growth inhibition area in mm are listed in Table 4 and Fig. 6, from which the following conclusions are obtained: (1) DyL3L0 exhibits in good antimicrobial activities against E. coli and H. bacillus especially against H. bacillus and have great potential in the exploration of new chemotherapy agents. The other two complexes also had such antibacterial activity, but a little weaker than that of DyL3L0 ; (2) the antibacterial activity of the complexes are better than that of ligand curcumin. The antibacterial mechanism is presumably that the compounds affect the functions associated with cell division of fungi such as cell wall, protein, and/or DNA biosyntheses or kill the exponentially growing cells. 4. Conclusion

Fig. 5. Agarose gel electrophoresis patterns for the cleavage of pBR322 DNA by SmL3L0 in the presence of methanol and hydrogen peroxide. Cleavage conditions: 37 lM DNA; 0.04 mM SmL3L0 50 mM NaCl in 5 mM Tris–HCl buffer; pH 7.40; 37 °C for 3 h. Lane 1: DNA control; Lane 2: SmL3L0 + MeOH; Lane 3: SmL3L0 + H2O2 and Lane 4: SmL3L0 .

The ternary complexes REL3L0 (RE: Sm3+, Eu3+, Dy3+) were synthesis and characterization. The result of agarose gel electrophoresis suggested that the complex of SmL3L0 can cleave the plasmid DNA at physiological pH and temperature. And it was found that the process of pBR322 DNA cleavage was sensitive to pH, however, these radical scavengers almost had no effect on the DNA cleavage reaction. The antibacterial activities testing shows that all these complexes exhibit excellent antibacterial ability against E. coli and H. bacillus. The complexes belong to broad spectrum antibacterial agents.

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5. Abbreviations ccc DMF TG–DTA oc TAE ESI-MS TLC

covalently closed circular dimethylformamide thermogravimetric and differential thermal analysis open circular tris-acetate-EDTA electrospray ionization mass spectrometry thin layer chromatography

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