Food Chemistry 146 (2014) 472–478
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Analytical Methods
Antioxidant study of quercetin and their metal complex and determination of stability constant by spectrophotometry method R. Ravichandran, M. Rajendran ⇑, D. Devapiriam Department of Chemistry, Centre for Research and Post Graduate Studies in Chemistry, N.M.S.S. Vellaichamy Nadar College, Nagamalai, Madurai 625 019, Tamilnadu, India
a r t i c l e
i n f o
Article history: Received 24 May 2012 Received in revised form 9 September 2013 Accepted 13 September 2013 Available online 23 September 2013 Keywords: Free radical Antioxidants Quercetin–cadmium complex Chelation therapy
a b s t r a c t Quercetin found chelate cadmium ions, scavenge free radicals produced by cadmium. Hence new complex, quercetin with cadmium was synthesised, and the synthesised complex structures were determined by UV–vis spectrophotometry, infrared spectroscopy, thermogravimetry and differential thermal analysis techniques (UV–vis, IR, TGA and DTA). The equilibrium stability constants of quercetin–cadmium complex were determined by Job’s method. The determined stability constant value of quercetin–cadminum complex at pH 4.4 is 2.27 106 and at pH 7.4 is 7.80 106. It was found that the quercetin and cadmium ion form 1:1 complex in both pH 4.4 and pH 7.4. The structure of the compounds was elucidated on the basis of obtained results. Furthermore, the antioxidant activity of the free quercetin and quercetin–cadmium complexes were determined by DPPH and ABTS assays. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone) is one of the most common flavonols present in nature that has attracted the attention of many researchers because of its biological and pharmaceutical properties (Cornard & Merlin, 2002). A multitude of substitution patterns in the two benzene rings (A and B) of the basic structure occur in nature and variations in their heterocyclic rings give rise to flavonols, flavones, catechins, flavanones, anthocyanidins and isoflavones. Over 4000 different naturally occurring flavonoids have been described and this list was still growing. Quercetin (C15H10O7) is a flavonoid of the flavonol type that contains five hydroxyl groups in positions 3,30 ,40 ,5,7, and a carbonyl group in fourth position. Owing to these features, quercetin easily forms complexes with many metals. A great number of flavonoids, especially flavones, can efficiently chelate metals like Al(III), Fe(II), Fe(III), Cu(II), or Zn(II). Formation of metal complexes play important and multiple roles in biological systems, and provide sensitive colour stabilisation mechanisms in vivo in higher plants (Kuntic, Blagojevic, MalesÏev, Radovic, & Bogavac, 1998; Markovic, Markovic, Veselinovic, Krstic, & Simovic, 2009; Panhwar et al., 2010; Sun, Chen, Cao, Zhang, Song, & Tian, 2008). Quercetin complexing capacity, widely used for elucidating the structure of natural flavonoids, can also contribute to the bioactivity of these compounds, by acting as carri-
⇑ Corresponding author. Tel.: +91 452 2459187; fax: +91 452 2458356. E-mail address: [email protected] (M. Rajendran). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.080
ers and regulators of metal concentration (Castro & Blanco, 2004; Marinic, Piantanida, Rusak, & Zinic, 2006). Concentrations of cadmium are increasing in the biosphere mainly as a result of its industrial uses. Formation of reactive oxygen species (ROS) involving cadmium suggests that DNA can also be taken into account as a potential target of this metal (Gao, Huang, Xu 2001). Intraperitoneal administration of a soluble cadmium salt results mainly in acute hepatotoxicity in rodents, which is a well-studied experimental animal toxicological model (Wena, Zhaoa, Bhadauriab, & Niralab, 2013). Metal complexation by organic ligands changes the speciation of Cd(II) and influences its toxicity (Wena et al., 2013). Cadmium was absorbed by inhalation and ingestion and has very long biological half-life. It was classified in group 1 of the International Agency for Research on cancer categories of carcinogens (Blasiak, 2001). The ability of cadmium to generate oxidative stress has been well documented (Wen et al., 2013). Redox reactions are also observed through the change of the oxidation state of the metal, jointly with the oxidation of the flavonoids by loss of hydrogen (Panhwar et al., 2010). In our studies, a number of direct methods of observing the binding of cadmium ion to flavonoids, such as UV–vis, infra red spectroscopies have revealed information about coordination of the metal ion binding sites. The aim of this study was to investigate the interaction between Cd(II) ions and quercetin. The objective of this paper is a detailed in vitro study of the ability of quercetin to chelate cadmium under different pH conditions. The antioxidant activity of quercetin and quercetin–cadmium complex was determined using DPPH and ABTS+ radicals scavenging methods.
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2. Experimental
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in absorbance at 734 nm is in accordance with the reduction ABTS+ ion.
2.1. Chemicals and reagents 2.7. DPPH assay Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone), 2,20 -diphenyl-1picrylhydrazyl radical (DPPH) and 2,20 -azinobis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) were purchased from Sigma Aldrich chemicals, USA. Cadmium nitrate-4 hydrate was obtained from Merck, India. All the reagents and solvents were of analytical grade and chemically pure and were used as received. 2.2. Buffer and stock solutions The pH of acetate and phosphate buffers were 4.4 and 7.4 respectively (Green, 1933). Stock solutions of quercetin (0.01 mol l1) in ethanol were prepared and diluted to obtain different concentrations. The stock solutions of cadmium nitrate (0.01 mol l1) prepared in doubly distilled water was diluted to 50 l mol l1 for stability constant studies.
DPPH radical scavenging analysis was performed according to the method of Williams, Cuvelier, and Berset (1995). Quercetin and quercetin–cadmium complex were dissolved in DMSO and added to 50 l mol l1 of DPPH solution in cuvette, which was placed in the spectrophotometer immediately and monitored for 20 min. The absorbance at 517 nm (AT) was recorded for every 2 min. exactly. Pure DPPH solution was used as a control. The decrease of in absorbance equates the DPPH radical scavenging capacity. The above process was repeated three times for quercetin and quercetin–cadmium complex (Chen, Sun, cao, Liang, & Song, 2009). The radical scavenging ability was calculated according to:
Radical scavenging activityð%Þ ¼ ½A0 AT 100=A0 3. Results and discussion
2.3. Instrumental study UV-visible spectra of free and complexed quercetin were recorded using SHIMADZU-1800 model UV–visible spectrophotometer (S. no. A11454806363). FTIR spectroscopic studies of the quercetin and its complex were determined by SHIMADZU, spectrophotometer within the range of 400–4000 cm1. Thermogravimetric analysis and differential thermal analysis of quercetin metal complex was analysed using an SII Exstar 6000 model TGA/DTA analyser.
In this study, we have evaluated the stability constant, coordination aspects of quercetin in presence of Cd(II) ion and its antioxidant behaviour. UV–vis study of the complex was performed to validate the stoichiometric composition of the chelate using Job’s method. The UV–visible spectrum of quercetin showed two major absorption bands at 372 (band I) and 256 nm (band II). Band I was considered as to be associated with the absorption due to the B ring and Band II with the absorption of due to the A ring (Bukhari, Memona, Tahir, & Bhanger, 2008). 3.1. Quercetin–cadmium complex and their stability
2.4. Determination of stability constant Job’s method was used to determine the stoichiometric ratio and stability constant of complex (Avinash & Maruti, 2012). The solutions were prepared by mixing solutions of both components with equal molar concentration (1 103 mol l1) in ratio varying from 1:9 to 9:1. The absorbance of quercetin was measured at 372 nm (Souza & Giovani, 2005).
Spectroscopic studies indicate that quercetin is a ligand capable of chelating in wide range of pH values, from acidic to slightly basic. The pH of the medium has considerable impact on quercetin and cadmium chelation. At the pH = 7.4 the absorption spectrum of quercetin showed three bands positioned at 250, 324 and 384 nm (Fig. 1) (Pusz, Nitka, Zielinska, & Wawer, 2000). The addition of cadmium to quercetin solution decreased the intensity of
2.5. Synthesis of quercetin–cadmium complex In a 50 ml round bottom flask, with an electromagnetic stirrer, solid quercetin (0.01 mol l1) and 20 ml ethanol, were combined until the quercetin was completely dissolved. To the yellow-coloured solution, solid cadmium nitrate (0.02 mol l1) was added quickly; the colour of the solution changed to dark green. Stirring continued for 2 h at room temperature. After stirring, the reaction mixture was filtered and the filtrate was evaporated slowly at room temperature to obtain a solid product. Unreative part reagents were removed by washing with water (Bukhari, Memonb, Tahir, & Bhanger, 2009). 2.6. ABTS antioxidant assay The antioxidant activity was measured using the method of Roberta et al. (1999). A mixture of ABTS (7 l mol l1) and potassium persulphate (2.45 l mol l1) was prepared in water, and incubated in room temperature for 12–16 h. The radical cation produced (ABTS+) is stable for more than 2 days when stored in dark at room temperature. The product ABTS+ (e734 = 15000 M1 cm1) was diluted to 50 in 10 l mol l1 phosphate buffer (pH 7.4) the reduction of ABTS+ by antioxidant flavonoids and other flavonoid metal complex was monitored at 734 nm. The decrease
Fig. 1. Absorption spectra of free quercetin and their metal complex in 10 l mol l1 phosphate buffer (pH = 7.4).
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quercetin absorption and simultaneously a bathochromic shift is observed due to the complex formation at pH 7.4. But at pH 4.4 only the intensity of the peak decreased without showing any bathochromic shift (figure not shown). The earlier results were also supporting that the 3 and 4 sites are the first preference binding site. It was reported that in quercetin–iron complexes, the iron binding site was 30 and 40 and is 0.5–1.0 eV less stable than the binding at the 3, 4 and 4, 5 sites (Leopoldini, Russo, Chiodo, & Toscano, 2006; Ren, Meng, Lekka, & Kaxiras, 2008). We are also suggesting that binding at the 3, 4 sites are stronger than at the 30 , 40 sites. Our earlier prediction based on Mulliken spin density calculation shows that fourth position oxygen in the flavones have higher charges than the other position oxygen, and hence chelation by metal mainly occur in the keto group at fourth position as a first preference (Rajendran, Mahalakshmi, Ramya, & Devapiriam, 2011).
Table 1 The Maximum temperature Tmax (°C), and weight loss values of the decomposition stage for the quercetin–cadmium complex in TGA and DTA. Compound
Steps
Decomposition temperature Tmax (°C)
Eliminated species
Quecertin–cadmium complex
First step Second step Third step Fourth step Total loss Residue
62
2H2O
129
4H2O
304
C6H3O
374
C9H4O4 + SO4
529
Cadmium oxide Nil
–
110.0
5.000 800.0
80.00
100.0
4.500
A
700.0
70.00
90.0 4.000
600.0
80.0
60.00
3.500 70.0
500.0 DTG ug/min 400.0
50.00 DTA uV
3.000 TG mg
40.00
2.500
50.0
30.00
2.000
40.0
1.500
30.0
300.0
60.0 TG %
200.0 20.00
100.0
20.0
1.000 10.00
0.0
10.0 0.500 0.00
-100.0
0.0 0.000 -10.0 100.0
50.00 40.00
300.0
30.00
200.0 150.0
300.0
400.0
500.0
600.0
5.000
100.0
4.500
90.0
Temp Cel
B
4.000
20.00
3.500
10.00
3.000
0.00
2.500
100.0 2.000
0.0
-20.00
1.500
-50.0
-30.00
-100.0
70.0 60.0 50.0
-10.00
50.0
80.0
40.0 30.0 1.000 20.0
-40.00
0.500 100.0
200.0
300.0
400.0
500.0
600.0
o
Temp C Fig. 2. TGA and DTA curve of the (A) quercetin, (B) quercetin–cadmium complex. (Sample mass 5 mg; heating rate 20 C min1).
TG %
350.0
DTA µV
DTG ug/min
400.0
250.0
200.0
TG mg
450.0
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Fig. 3. The concentration dependent decrease in UV–vis spectrum of (A) ABTS+ (50 l mol l1) radical cation as a function of increasing concentration of quercetin (B) 5; (C) 10; (D) 15 l mol l1) measured after 1 min of initial mixing of quercetin in 10 l mol l1 phosphate buffer (pH = 7.4).
Fig. 4. (A) Reaction between ABTS+ and quercetin in 10 l mol l1 phosphate buffer (pH = 7.4), Decrease in absorbance of ABTS+ at 734 nm in the presence of different concentration of Quercetin (l mol l1); 10 (NNN), 15 (jjj), 25 (⁄⁄⁄), 30 (ddd), 35 (+++), control ABTS .+ (). (B) Reaction between DPPH and Quercetin in phosphate buffer (pH = 7.4). Decrease in absorbance (k 517 nm) of DPPH, in the presence of various concentration of quercetin 5 l mol l1 (jjj) and 12 l mol l1 (NNN), (control: DPPH 50 l mol l1 ()).
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In both acidic pH as well as alkaline pH, the mole fraction ratio of quercetin and cadmium was found to be 1:1 (figure not shown) indicating that cadmium preferentially binding only at one site i.e. 3, 4 or 5, 4 position of the quercetin. To understand the complexing behaviour of quercetin and cadmium, stability constant was calculated by Job’s method. At pH 4.4 the stability constant value was found to be 2.27 106, which was lower than the value determined at pH 7.4. The stability constant at pH = 7.4 is 7.80 106, these values once again proves that the complex was more stable at basic medium. Therefore, pH has a considerable impact on the complex formation. The complex formation at pH values lower than three is difficult because the flavonoids were predominantly present in their undissociated form. Although high pH values favour deprotonation of flavoniods and, consequently, more complex species, at high pH values metal ions were often involved in hydrolysis and hydro-complexes were formed (Malsev & Kuntic, 2007).
The spectra were recorded under conditions generally applied in quantitative work. The spectral data showed the evidence for the coordination between the cadmium metal ions and quercetin molecule. Some features of the spectra are discussed below. The appearance of peak at 464.8 cm1 in IR spectrum of the complex indicates the existence of O–Cd bond in the complex, while the free quercetin exhibits no such band. The C@O stretching mode of the free quercetin occurs at 1670.24 cm1. Due to the interaction of quercetin with cadmium the absorption band of C@O stretching mode has been shifted to 1668.31 cm1. The appearance of new strong and sharp absorption band at 1668.31 cm1 in the complex stands as an evidence for the binding quercetin to cadmium through carbonyl oxygen. The bands located at 1317.29 (m) and 1244 cm1 (m), were related to (C–OH) deformations vibrations. The broad band of (O–H) vibration frequency (from 3296.12 to 3359.7 cm1) indicates the existence of water in the complex and free quercetin. The presence of coordinated water was also supported by thermal analysis.
3.2. IR spectral analysis of quercetin–cadmium complex 3.3. Thermal analysis of quercetin–cadmium complex IR spectra were recorded by using SHIMADZU FT-IR Spectrometer. The spectra were recorded in the 4000–400 cm1 range with the spectral resolution of 2 cm1. Pure quercetin was studied in potassium bromide matrix with a ratio of 1:150 mg (sample:KBr).
Thermogravimetric analysis (TGA) was carried out for quercetin–cadmium complex under the flow of nitrogen. Fig. 2 showed two distinct breaks one at dehydration and another at decomposi-
Fig. 5. (A) ABTS+ radical scavenging activities evaluated through the absorbance decrease at 734 nm caused by addition of quercetin () and quercetin–cadmium (jjj), in 10 l mol l1 phosphate buffer (pH = 7.4). (B) Dependence of the quercetin () and quercetin–cadmium (jjj) in DPPH 50 l mol l1, scavenging activities calculated through the absorbance decrease at 517 nm in phosphate buffer (pH = 7.4).
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tion. TGA results showed that the complex was stable up to 99 °C and the mass loss starts at the temperature range of 100–122 °C owing to the elimination of water molecules. Differential thermal analysis (DTA) peaks for the complex showed exothermic peaks that agree with mass losses observed in TGA and DTA. The TGA and DTA plot of quercetin–cadmium complex showed that it was decomposed four steps given in Table 1. The dehydrated complex was stable over the temperature range of 122–300 °C confirming the above result. The complex decomposes at the 300–500 °C temperature range of causing 70% weight loss showing the decomposition of organic matter. In DTA analysis below 100 °C an exothermic peak was obtained and is believed to be the loss of two water molecules. The temperature range 100–200 °C three exothermic peaks are present, showing the loss four water molecules. The fifth and sixth exothermic peaks correspond to decomposition of organic matter (Zhou, Wang, Wang, & Tang, 2001) (see Fig. 3). 3.4. Total antioxidant activity The ABTS+ radical cation assay was useful to study the total antioxidant activity (TAA) of antioxidants. The antioxidant activity of quercetin was determined by following the decolourisation (reduction) of ABTS+ radical cation. The suppression of the absorbance of ABTS+ in a concentration-dependent manner was typically observed. Fig. 4A showed the decrease in the absorbance of ABTS+ radical cation at 734 nm for various quercetin concentrations. The experimental results demonstrated that the reaction with ABTS+ was completed within 2 min. The ABTS+ radical cation reduction by quercetin and quercetin– cadmium complex were shown in Fig. 5A. The percentage of ABTS+ scavenging efficiency with respect to concentration showed that quercetin–cadmium complex radical scavenging efficiency was lower than molecular quercetin. The higher antioxidant activity of molecular quercetin may be attributed to the number of hydroxyl groups present and their lower oxidation potential. The lower antioxidant activity of quercetin–cadmium complex may be due to their higher oxidation potential than free quercetin. Fresh ABTS+ solution was prepared for each assay (Asghar, Khan, Zia, Ahmad, & Qureshi, 2008; Guha, Rajkumar, Mathew, & Kumar, 2011; Khan, Khan, Sahreen, & Ahmed, 2012; Thaipong, Boonprakob, Crosby, Zevallos, & Byrne, 2006). The decrease (Fig. 5A) in ABTS+ absorbance was converted into percentage by using the following equations: þ
þ
þ
þ
% of ABTS remaining ¼ ð½ABTSInitial ½ABTST 100=½ABTSInitial Þ where ABTS+ T is the time needed to reach the steady state. The values were plotted against the complex concentration (Fig. 5A).
477
4. Conclusions As a part of research project devoted to developing a new complex of Cd(II) with quercetin have been prepared and characterised. The antioxidant activity of quercetin and their complex were elucidated by UV–vis spectroscopic technique using ABTS+ and DPPH radicals. The job’s method was applied to validate the stoichiometric composition of the complex. The experiments were performed under different pH conditions and the stoichiometric composition of the complex was found to be 1:1 in both pH 4.4 and pH 7.4. The spectroscopic data showed the 3–OH and the carbonyl group in the ring C are the main metal complexing domain which interacts with cadmium ion. The quercetin–cadmium complex showed lower antioxidant activity as compared to the free quercetin. This suggested that the metal ion Cd(II) significantly change the chemical properties of the quercetin. The results obtained have also demonstrated that the chelation of metal cadmium with quercetin may influence the higher oxidation potentials relative to those of free quercetin. Medicinal treatment of acute and chronic metal toxicity was treated by chelating agents. Chelation was one of the chemical functions that take place in the bodies of almost all living organisms. Most of the currently used chelating agents have serious side effects (Angle, 1996). Cadmium is a soft metal can bind with any soft base biological protein system and inhibit or destroy the activity of protein and enzyme. Cadmium, unlike other heavy metals was unable to generate free radicals by itself, however, reports have indicated superoxide radical, hydroxyl radical and nitric oxide radicals could be generated indirectly (Galan, Garcia, Troyano, Vilaboa, Fernandez, Blas, et al., 2001). According to reviews (Crinnion, 2011; Fatemi, Saljooghi, Balooch, Iranmanesh, & Golbafan, 2011, 2012) only a limited knowledge was presently available concerning the possibility for chelate treatment of cadmium intoxification in human. Cadmium is a potent human carcinogen and has been associated with cancers of the lung, prostate, pancreas, and kidney. Because of its carcinogenic properties, cadmium has been classified as a number one category human carcinogen by the International Agency for Research on Cancer of USA. The goal of this study was to determine the stability constant of metal cadmium with natural chelating agent quercetin. The natural ligands were widely used to remove and to minimise the unwanted excretion of essential metal ions. Quercetin was one of the natural ligand largely present plant derivative foods, and complexes with metal ion to remove the hazardous metals. Quercetin– cadmium complex results show higher stability constant (Kf) value, so flavonoids may be used as best chelating agent and to remove the toxic metal ions. The determined stability constant value of this investigation was much useful in the chelation therapy treatment of cadmium and also other toxic metals. Acknowledgement
3.5. DPPH radical scavenging activity Antioxidant property of flavonoids was more significantly related to their molecular structure. There are two main mechanisms through which phenolic compounds can exert their antioxidant functions, hydrogen atom transferring and electron donation. Fig. 4B showed the decrease in absorbance at (k = 517 nm) of DPPH ethanolic solution in the presence of different concentration of quercetin. When the concentration of quercetin increases the quercetin radical scavenging efficiency also increased. Free quercetin showed a more antioxidant activity than the quercetin–cadmium complex (Fig. 5B). The higher antioxidant activity of molecular quercetin may be attributed to the number of hydroxyl groups (Dehghan & Khosham 2011) while the lower antioxidant activity of quercetin–cadmium complex may be due to lowering of number of hydroxyl groups.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Antioxidant study of quercetin and their metal complex and determination of stability constant by spectrophotometry method R. Ravichandran, M. Rajendran ⇑, D. Devapiriam Department of Chemistry, Centre for Research and Post Graduate Studies in Chemistry, N.M.S.S. Vellaichamy Nadar College, Nagamalai, Madurai 625 019, Tamilnadu, India
a r t i c l e
i n f o
Article history: Received 24 May 2012 Received in revised form 9 September 2013 Accepted 13 September 2013 Available online 23 September 2013 Keywords: Free radical Antioxidants Quercetin–cadmium complex Chelation therapy
a b s t r a c t Quercetin found chelate cadmium ions, scavenge free radicals produced by cadmium. Hence new complex, quercetin with cadmium was synthesised, and the synthesised complex structures were determined by UV–vis spectrophotometry, infrared spectroscopy, thermogravimetry and differential thermal analysis techniques (UV–vis, IR, TGA and DTA). The equilibrium stability constants of quercetin–cadmium complex were determined by Job’s method. The determined stability constant value of quercetin–cadminum complex at pH 4.4 is 2.27 106 and at pH 7.4 is 7.80 106. It was found that the quercetin and cadmium ion form 1:1 complex in both pH 4.4 and pH 7.4. The structure of the compounds was elucidated on the basis of obtained results. Furthermore, the antioxidant activity of the free quercetin and quercetin–cadmium complexes were determined by DPPH and ABTS assays. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone) is one of the most common flavonols present in nature that has attracted the attention of many researchers because of its biological and pharmaceutical properties (Cornard & Merlin, 2002). A multitude of substitution patterns in the two benzene rings (A and B) of the basic structure occur in nature and variations in their heterocyclic rings give rise to flavonols, flavones, catechins, flavanones, anthocyanidins and isoflavones. Over 4000 different naturally occurring flavonoids have been described and this list was still growing. Quercetin (C15H10O7) is a flavonoid of the flavonol type that contains five hydroxyl groups in positions 3,30 ,40 ,5,7, and a carbonyl group in fourth position. Owing to these features, quercetin easily forms complexes with many metals. A great number of flavonoids, especially flavones, can efficiently chelate metals like Al(III), Fe(II), Fe(III), Cu(II), or Zn(II). Formation of metal complexes play important and multiple roles in biological systems, and provide sensitive colour stabilisation mechanisms in vivo in higher plants (Kuntic, Blagojevic, MalesÏev, Radovic, & Bogavac, 1998; Markovic, Markovic, Veselinovic, Krstic, & Simovic, 2009; Panhwar et al., 2010; Sun, Chen, Cao, Zhang, Song, & Tian, 2008). Quercetin complexing capacity, widely used for elucidating the structure of natural flavonoids, can also contribute to the bioactivity of these compounds, by acting as carri-
⇑ Corresponding author. Tel.: +91 452 2459187; fax: +91 452 2458356. E-mail address: [email protected] (M. Rajendran). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.080
ers and regulators of metal concentration (Castro & Blanco, 2004; Marinic, Piantanida, Rusak, & Zinic, 2006). Concentrations of cadmium are increasing in the biosphere mainly as a result of its industrial uses. Formation of reactive oxygen species (ROS) involving cadmium suggests that DNA can also be taken into account as a potential target of this metal (Gao, Huang, Xu 2001). Intraperitoneal administration of a soluble cadmium salt results mainly in acute hepatotoxicity in rodents, which is a well-studied experimental animal toxicological model (Wena, Zhaoa, Bhadauriab, & Niralab, 2013). Metal complexation by organic ligands changes the speciation of Cd(II) and influences its toxicity (Wena et al., 2013). Cadmium was absorbed by inhalation and ingestion and has very long biological half-life. It was classified in group 1 of the International Agency for Research on cancer categories of carcinogens (Blasiak, 2001). The ability of cadmium to generate oxidative stress has been well documented (Wen et al., 2013). Redox reactions are also observed through the change of the oxidation state of the metal, jointly with the oxidation of the flavonoids by loss of hydrogen (Panhwar et al., 2010). In our studies, a number of direct methods of observing the binding of cadmium ion to flavonoids, such as UV–vis, infra red spectroscopies have revealed information about coordination of the metal ion binding sites. The aim of this study was to investigate the interaction between Cd(II) ions and quercetin. The objective of this paper is a detailed in vitro study of the ability of quercetin to chelate cadmium under different pH conditions. The antioxidant activity of quercetin and quercetin–cadmium complex was determined using DPPH and ABTS+ radicals scavenging methods.
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2. Experimental
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in absorbance at 734 nm is in accordance with the reduction ABTS+ ion.
2.1. Chemicals and reagents 2.7. DPPH assay Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone), 2,20 -diphenyl-1picrylhydrazyl radical (DPPH) and 2,20 -azinobis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) were purchased from Sigma Aldrich chemicals, USA. Cadmium nitrate-4 hydrate was obtained from Merck, India. All the reagents and solvents were of analytical grade and chemically pure and were used as received. 2.2. Buffer and stock solutions The pH of acetate and phosphate buffers were 4.4 and 7.4 respectively (Green, 1933). Stock solutions of quercetin (0.01 mol l1) in ethanol were prepared and diluted to obtain different concentrations. The stock solutions of cadmium nitrate (0.01 mol l1) prepared in doubly distilled water was diluted to 50 l mol l1 for stability constant studies.
DPPH radical scavenging analysis was performed according to the method of Williams, Cuvelier, and Berset (1995). Quercetin and quercetin–cadmium complex were dissolved in DMSO and added to 50 l mol l1 of DPPH solution in cuvette, which was placed in the spectrophotometer immediately and monitored for 20 min. The absorbance at 517 nm (AT) was recorded for every 2 min. exactly. Pure DPPH solution was used as a control. The decrease of in absorbance equates the DPPH radical scavenging capacity. The above process was repeated three times for quercetin and quercetin–cadmium complex (Chen, Sun, cao, Liang, & Song, 2009). The radical scavenging ability was calculated according to:
Radical scavenging activityð%Þ ¼ ½A0 AT 100=A0 3. Results and discussion
2.3. Instrumental study UV-visible spectra of free and complexed quercetin were recorded using SHIMADZU-1800 model UV–visible spectrophotometer (S. no. A11454806363). FTIR spectroscopic studies of the quercetin and its complex were determined by SHIMADZU, spectrophotometer within the range of 400–4000 cm1. Thermogravimetric analysis and differential thermal analysis of quercetin metal complex was analysed using an SII Exstar 6000 model TGA/DTA analyser.
In this study, we have evaluated the stability constant, coordination aspects of quercetin in presence of Cd(II) ion and its antioxidant behaviour. UV–vis study of the complex was performed to validate the stoichiometric composition of the chelate using Job’s method. The UV–visible spectrum of quercetin showed two major absorption bands at 372 (band I) and 256 nm (band II). Band I was considered as to be associated with the absorption due to the B ring and Band II with the absorption of due to the A ring (Bukhari, Memona, Tahir, & Bhanger, 2008). 3.1. Quercetin–cadmium complex and their stability
2.4. Determination of stability constant Job’s method was used to determine the stoichiometric ratio and stability constant of complex (Avinash & Maruti, 2012). The solutions were prepared by mixing solutions of both components with equal molar concentration (1 103 mol l1) in ratio varying from 1:9 to 9:1. The absorbance of quercetin was measured at 372 nm (Souza & Giovani, 2005).
Spectroscopic studies indicate that quercetin is a ligand capable of chelating in wide range of pH values, from acidic to slightly basic. The pH of the medium has considerable impact on quercetin and cadmium chelation. At the pH = 7.4 the absorption spectrum of quercetin showed three bands positioned at 250, 324 and 384 nm (Fig. 1) (Pusz, Nitka, Zielinska, & Wawer, 2000). The addition of cadmium to quercetin solution decreased the intensity of
2.5. Synthesis of quercetin–cadmium complex In a 50 ml round bottom flask, with an electromagnetic stirrer, solid quercetin (0.01 mol l1) and 20 ml ethanol, were combined until the quercetin was completely dissolved. To the yellow-coloured solution, solid cadmium nitrate (0.02 mol l1) was added quickly; the colour of the solution changed to dark green. Stirring continued for 2 h at room temperature. After stirring, the reaction mixture was filtered and the filtrate was evaporated slowly at room temperature to obtain a solid product. Unreative part reagents were removed by washing with water (Bukhari, Memonb, Tahir, & Bhanger, 2009). 2.6. ABTS antioxidant assay The antioxidant activity was measured using the method of Roberta et al. (1999). A mixture of ABTS (7 l mol l1) and potassium persulphate (2.45 l mol l1) was prepared in water, and incubated in room temperature for 12–16 h. The radical cation produced (ABTS+) is stable for more than 2 days when stored in dark at room temperature. The product ABTS+ (e734 = 15000 M1 cm1) was diluted to 50 in 10 l mol l1 phosphate buffer (pH 7.4) the reduction of ABTS+ by antioxidant flavonoids and other flavonoid metal complex was monitored at 734 nm. The decrease
Fig. 1. Absorption spectra of free quercetin and their metal complex in 10 l mol l1 phosphate buffer (pH = 7.4).
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quercetin absorption and simultaneously a bathochromic shift is observed due to the complex formation at pH 7.4. But at pH 4.4 only the intensity of the peak decreased without showing any bathochromic shift (figure not shown). The earlier results were also supporting that the 3 and 4 sites are the first preference binding site. It was reported that in quercetin–iron complexes, the iron binding site was 30 and 40 and is 0.5–1.0 eV less stable than the binding at the 3, 4 and 4, 5 sites (Leopoldini, Russo, Chiodo, & Toscano, 2006; Ren, Meng, Lekka, & Kaxiras, 2008). We are also suggesting that binding at the 3, 4 sites are stronger than at the 30 , 40 sites. Our earlier prediction based on Mulliken spin density calculation shows that fourth position oxygen in the flavones have higher charges than the other position oxygen, and hence chelation by metal mainly occur in the keto group at fourth position as a first preference (Rajendran, Mahalakshmi, Ramya, & Devapiriam, 2011).
Table 1 The Maximum temperature Tmax (°C), and weight loss values of the decomposition stage for the quercetin–cadmium complex in TGA and DTA. Compound
Steps
Decomposition temperature Tmax (°C)
Eliminated species
Quecertin–cadmium complex
First step Second step Third step Fourth step Total loss Residue
62
2H2O
129
4H2O
304
C6H3O
374
C9H4O4 + SO4
529
Cadmium oxide Nil
–
110.0
5.000 800.0
80.00
100.0
4.500
A
700.0
70.00
90.0 4.000
600.0
80.0
60.00
3.500 70.0
500.0 DTG ug/min 400.0
50.00 DTA uV
3.000 TG mg
40.00
2.500
50.0
30.00
2.000
40.0
1.500
30.0
300.0
60.0 TG %
200.0 20.00
100.0
20.0
1.000 10.00
0.0
10.0 0.500 0.00
-100.0
0.0 0.000 -10.0 100.0
50.00 40.00
300.0
30.00
200.0 150.0
300.0
400.0
500.0
600.0
5.000
100.0
4.500
90.0
Temp Cel
B
4.000
20.00
3.500
10.00
3.000
0.00
2.500
100.0 2.000
0.0
-20.00
1.500
-50.0
-30.00
-100.0
70.0 60.0 50.0
-10.00
50.0
80.0
40.0 30.0 1.000 20.0
-40.00
0.500 100.0
200.0
300.0
400.0
500.0
600.0
o
Temp C Fig. 2. TGA and DTA curve of the (A) quercetin, (B) quercetin–cadmium complex. (Sample mass 5 mg; heating rate 20 C min1).
TG %
350.0
DTA µV
DTG ug/min
400.0
250.0
200.0
TG mg
450.0
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Fig. 3. The concentration dependent decrease in UV–vis spectrum of (A) ABTS+ (50 l mol l1) radical cation as a function of increasing concentration of quercetin (B) 5; (C) 10; (D) 15 l mol l1) measured after 1 min of initial mixing of quercetin in 10 l mol l1 phosphate buffer (pH = 7.4).
Fig. 4. (A) Reaction between ABTS+ and quercetin in 10 l mol l1 phosphate buffer (pH = 7.4), Decrease in absorbance of ABTS+ at 734 nm in the presence of different concentration of Quercetin (l mol l1); 10 (NNN), 15 (jjj), 25 (⁄⁄⁄), 30 (ddd), 35 (+++), control ABTS .+ (). (B) Reaction between DPPH and Quercetin in phosphate buffer (pH = 7.4). Decrease in absorbance (k 517 nm) of DPPH, in the presence of various concentration of quercetin 5 l mol l1 (jjj) and 12 l mol l1 (NNN), (control: DPPH 50 l mol l1 ()).
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In both acidic pH as well as alkaline pH, the mole fraction ratio of quercetin and cadmium was found to be 1:1 (figure not shown) indicating that cadmium preferentially binding only at one site i.e. 3, 4 or 5, 4 position of the quercetin. To understand the complexing behaviour of quercetin and cadmium, stability constant was calculated by Job’s method. At pH 4.4 the stability constant value was found to be 2.27 106, which was lower than the value determined at pH 7.4. The stability constant at pH = 7.4 is 7.80 106, these values once again proves that the complex was more stable at basic medium. Therefore, pH has a considerable impact on the complex formation. The complex formation at pH values lower than three is difficult because the flavonoids were predominantly present in their undissociated form. Although high pH values favour deprotonation of flavoniods and, consequently, more complex species, at high pH values metal ions were often involved in hydrolysis and hydro-complexes were formed (Malsev & Kuntic, 2007).
The spectra were recorded under conditions generally applied in quantitative work. The spectral data showed the evidence for the coordination between the cadmium metal ions and quercetin molecule. Some features of the spectra are discussed below. The appearance of peak at 464.8 cm1 in IR spectrum of the complex indicates the existence of O–Cd bond in the complex, while the free quercetin exhibits no such band. The C@O stretching mode of the free quercetin occurs at 1670.24 cm1. Due to the interaction of quercetin with cadmium the absorption band of C@O stretching mode has been shifted to 1668.31 cm1. The appearance of new strong and sharp absorption band at 1668.31 cm1 in the complex stands as an evidence for the binding quercetin to cadmium through carbonyl oxygen. The bands located at 1317.29 (m) and 1244 cm1 (m), were related to (C–OH) deformations vibrations. The broad band of (O–H) vibration frequency (from 3296.12 to 3359.7 cm1) indicates the existence of water in the complex and free quercetin. The presence of coordinated water was also supported by thermal analysis.
3.2. IR spectral analysis of quercetin–cadmium complex 3.3. Thermal analysis of quercetin–cadmium complex IR spectra were recorded by using SHIMADZU FT-IR Spectrometer. The spectra were recorded in the 4000–400 cm1 range with the spectral resolution of 2 cm1. Pure quercetin was studied in potassium bromide matrix with a ratio of 1:150 mg (sample:KBr).
Thermogravimetric analysis (TGA) was carried out for quercetin–cadmium complex under the flow of nitrogen. Fig. 2 showed two distinct breaks one at dehydration and another at decomposi-
Fig. 5. (A) ABTS+ radical scavenging activities evaluated through the absorbance decrease at 734 nm caused by addition of quercetin () and quercetin–cadmium (jjj), in 10 l mol l1 phosphate buffer (pH = 7.4). (B) Dependence of the quercetin () and quercetin–cadmium (jjj) in DPPH 50 l mol l1, scavenging activities calculated through the absorbance decrease at 517 nm in phosphate buffer (pH = 7.4).
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tion. TGA results showed that the complex was stable up to 99 °C and the mass loss starts at the temperature range of 100–122 °C owing to the elimination of water molecules. Differential thermal analysis (DTA) peaks for the complex showed exothermic peaks that agree with mass losses observed in TGA and DTA. The TGA and DTA plot of quercetin–cadmium complex showed that it was decomposed four steps given in Table 1. The dehydrated complex was stable over the temperature range of 122–300 °C confirming the above result. The complex decomposes at the 300–500 °C temperature range of causing 70% weight loss showing the decomposition of organic matter. In DTA analysis below 100 °C an exothermic peak was obtained and is believed to be the loss of two water molecules. The temperature range 100–200 °C three exothermic peaks are present, showing the loss four water molecules. The fifth and sixth exothermic peaks correspond to decomposition of organic matter (Zhou, Wang, Wang, & Tang, 2001) (see Fig. 3). 3.4. Total antioxidant activity The ABTS+ radical cation assay was useful to study the total antioxidant activity (TAA) of antioxidants. The antioxidant activity of quercetin was determined by following the decolourisation (reduction) of ABTS+ radical cation. The suppression of the absorbance of ABTS+ in a concentration-dependent manner was typically observed. Fig. 4A showed the decrease in the absorbance of ABTS+ radical cation at 734 nm for various quercetin concentrations. The experimental results demonstrated that the reaction with ABTS+ was completed within 2 min. The ABTS+ radical cation reduction by quercetin and quercetin– cadmium complex were shown in Fig. 5A. The percentage of ABTS+ scavenging efficiency with respect to concentration showed that quercetin–cadmium complex radical scavenging efficiency was lower than molecular quercetin. The higher antioxidant activity of molecular quercetin may be attributed to the number of hydroxyl groups present and their lower oxidation potential. The lower antioxidant activity of quercetin–cadmium complex may be due to their higher oxidation potential than free quercetin. Fresh ABTS+ solution was prepared for each assay (Asghar, Khan, Zia, Ahmad, & Qureshi, 2008; Guha, Rajkumar, Mathew, & Kumar, 2011; Khan, Khan, Sahreen, & Ahmed, 2012; Thaipong, Boonprakob, Crosby, Zevallos, & Byrne, 2006). The decrease (Fig. 5A) in ABTS+ absorbance was converted into percentage by using the following equations: þ
þ
þ
þ
% of ABTS remaining ¼ ð½ABTSInitial ½ABTST 100=½ABTSInitial Þ where ABTS+ T is the time needed to reach the steady state. The values were plotted against the complex concentration (Fig. 5A).
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4. Conclusions As a part of research project devoted to developing a new complex of Cd(II) with quercetin have been prepared and characterised. The antioxidant activity of quercetin and their complex were elucidated by UV–vis spectroscopic technique using ABTS+ and DPPH radicals. The job’s method was applied to validate the stoichiometric composition of the complex. The experiments were performed under different pH conditions and the stoichiometric composition of the complex was found to be 1:1 in both pH 4.4 and pH 7.4. The spectroscopic data showed the 3–OH and the carbonyl group in the ring C are the main metal complexing domain which interacts with cadmium ion. The quercetin–cadmium complex showed lower antioxidant activity as compared to the free quercetin. This suggested that the metal ion Cd(II) significantly change the chemical properties of the quercetin. The results obtained have also demonstrated that the chelation of metal cadmium with quercetin may influence the higher oxidation potentials relative to those of free quercetin. Medicinal treatment of acute and chronic metal toxicity was treated by chelating agents. Chelation was one of the chemical functions that take place in the bodies of almost all living organisms. Most of the currently used chelating agents have serious side effects (Angle, 1996). Cadmium is a soft metal can bind with any soft base biological protein system and inhibit or destroy the activity of protein and enzyme. Cadmium, unlike other heavy metals was unable to generate free radicals by itself, however, reports have indicated superoxide radical, hydroxyl radical and nitric oxide radicals could be generated indirectly (Galan, Garcia, Troyano, Vilaboa, Fernandez, Blas, et al., 2001). According to reviews (Crinnion, 2011; Fatemi, Saljooghi, Balooch, Iranmanesh, & Golbafan, 2011, 2012) only a limited knowledge was presently available concerning the possibility for chelate treatment of cadmium intoxification in human. Cadmium is a potent human carcinogen and has been associated with cancers of the lung, prostate, pancreas, and kidney. Because of its carcinogenic properties, cadmium has been classified as a number one category human carcinogen by the International Agency for Research on Cancer of USA. The goal of this study was to determine the stability constant of metal cadmium with natural chelating agent quercetin. The natural ligands were widely used to remove and to minimise the unwanted excretion of essential metal ions. Quercetin was one of the natural ligand largely present plant derivative foods, and complexes with metal ion to remove the hazardous metals. Quercetin– cadmium complex results show higher stability constant (Kf) value, so flavonoids may be used as best chelating agent and to remove the toxic metal ions. The determined stability constant value of this investigation was much useful in the chelation therapy treatment of cadmium and also other toxic metals. Acknowledgement
3.5. DPPH radical scavenging activity Antioxidant property of flavonoids was more significantly related to their molecular structure. There are two main mechanisms through which phenolic compounds can exert their antioxidant functions, hydrogen atom transferring and electron donation. Fig. 4B showed the decrease in absorbance at (k = 517 nm) of DPPH ethanolic solution in the presence of different concentration of quercetin. When the concentration of quercetin increases the quercetin radical scavenging efficiency also increased. Free quercetin showed a more antioxidant activity than the quercetin–cadmium complex (Fig. 5B). The higher antioxidant activity of molecular quercetin may be attributed to the number of hydroxyl groups (Dehghan & Khosham 2011) while the lower antioxidant activity of quercetin–cadmium complex may be due to lowering of number of hydroxyl groups.
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