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Synthesis, characterization and study of antioxidant activity of quercetin–magnesium complex
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 807–813
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Synthesis, characterization and study of antioxidant activity of quercetin–magnesium complex Nilanjan Ghosh a, Tania Chakraborty a, Sougata Mallick a, Supriya Mana a, Deepanwita Singha b, Balaram Ghosh c, Souvik Roy a,⇑ a b c
NSHM Knowledge Campus Kolkata, Group of Institutions, 124-BL Saha Road, Kolkata, West Bengal 700053, India Nightingale Diagnostic & Medicare Centre Pvt Ltd., 11, Shakespeare Sarani, Kolkata, West Bengal 700071, India Midnapore Medical College and Hospital, Vidyasagar Road, Medinipur, West Bengal 721101, India
h i g h l i g h t s Quercetin is able to chelate metal ions. We synthesize and characterize quercetin–magnesium (II) complex. Antioxidant activity of quercetin increased after chelation with magnesium. Chelation of magnesium induced a bathochromic shift in absorbance band. Peak at 596.40 cm
a r t i c l e
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in infrared spectroscopy represented the formation of M–O.
i n f o
Article history: Received 19 September 2014 Received in revised form 7 July 2015 Accepted 8 July 2015 Available online 9 July 2015 Keywords: Quercetin–magnesium complex Flavonoids Quercetin Spectral characterisation Antioxidant activity Radical scavenging activity
a b s t r a c t Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone) one of the most abundant dietary flavonoids, has been investigated in the presence of magnesium (II) in methanol. The complex formation between quercetin and magnesium (II) was examined under UV–visible, Infra-red and 1H NMR spectroscopic techniques. The spectroscopic data denoted that quercetin can reacts with magnesium cation (Mg+2) through the chelation site in the quercetin molecule. The free radical antioxidant activity of the complex with respect to the parent molecule was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. It was observed that the free radical scavenging activity of quercetin was increased after complexation of magnesium (Mg+2) cation. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Flavonoids (2-phenyl-benzo-c-pyrones, Fig. 1A) are a large group of polyphenolic natural compounds that are extensively distributed in plant-based foods [1]. A multitude of substitution patterns in the two benzene rings (A and B) of the basic structure occur in nature. Variations in their heterocyclic rings give rise to flavonols, flavones, catechins, flavanones, anthocyanidins and isoflavones. Over 4000 different naturally occurring flavonoids have been described [2]. Flavonoids are mostly present in human diet, such as fruits, vegetables and plant-derived beverages such
⇑ Corresponding author at: Department of Pharmacology, NSHM Knowledge Campus, Kolkata, India. E-mail address: [email protected] (S. Roy). http://dx.doi.org/10.1016/j.saa.2015.07.050 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
as tea and red wine [3]. It is estimated that the human intake of all flavonoids is a few hundreds of milligrams per day [4]. Human being obtains bioflavonoids through diet since they do not have the capacity to synthesize these polyphenols. The consumed bioflavonoids have been shown to be absorbed through the stomach as well as the small intestine, and they are mainly metabolized by the liver and intestinal mucosa and excreted in the urine and feces [5]. Interest in the therapeutic applications of flavonoids and their derivatives (glycosylated flavonoids) for the treatment and prevention of human diseases has increased in recent years. Such flavonoids have many biological activities like anti-cancer, anti-inflammatory, anti-diabetic, antiviral, anti-allergic etcetera. Some epidemiological studies support a protective role of flavonoids rich diets in developing cancer and cardiovascular diseases [6–11]. Most of the bioflavonoids are strong natural antioxidant and having free radical scavenging activity along with potent metal
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Fig. 1. (A) Basic structure of flavonoid. (B) Basic structure of quercetin (3,30 ,40 ,5,7-pentahydroxyflavone).
chelators [12]. Flavonoids also play an important role in both the bioavailability for metal ions which are in low amount in our body and toxicity of a variety of toxic metals like Pb (II) [13]. Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone), (Fig. 1B) is one of the most common flavonoids present in nature that has attracted the attention of many researchers because of its biological properties [14]. Quercetin is very effective metal chelators which possesses three possible chelating sites in competition: the 3-hydroxy-4-carbonyl, the 5-hydroxy-4-carbonyl, and the 30,40-dihydroxyl (catechol) groups (Fig. 1B). Complexation of metal cations by Q has already been reported for a large number of metals (Mo(VI), Fe(II), Fe(III), Cu(II), Zn(II), Al(III), Tb(III), Pb(II), Co(II) and many other cations) and in these studies different stoichiometric ratios can be seen between different metals and quercetin as a ligand [15–22]. Metal chelation is widely considered as another mechanism of the antioxidant activity of flavonoids. The interaction of flavonoids with metal ions may also change the antioxidant properties and also biological effects of the flavonoids [23]. It is suggest that the biological activity of an organic ligand can be increased when co-ordinated or mixed with suitable metal ion; because of its ability to act as free radical acceptor [24–26]. Magnesium is an alkaline earth metal and the fourth most common cation in the body and the second most common intracellular cation after potassium. It has fundamental role as a co-factor in more than 300 enzymatic reactions involving energy metabolism and nucleic acid synthesis. It is also involved in several process including: hormone receptor binding; gating of calcium channels; transmembrane ion flux and regulation of adenylated cyclase; muscle contraction; neuronal activity; control of vasomotor tone; cardiac excitability; and neuro-transmitter release. In many of its action it has been linked to a physiological calcium antagonist [27]. Epidemiological studies trace the prevalence of cardiovascular disease and cardiac deaths to the degree of magnesium depletion induced by a diet and drinking water low in magnesium [28]. The three areas of particular relevance of magnesium are myocardial infarction, arrhythmia and cardiac surgery. As metal ions play a vital role in the initiation of free radical processes, metal chelation is widely considered as another mechanism of the antioxidant activity of flavonoids. The interaction of flavonoids with metal ions may change the antioxidant properties and some biological effects of the flavonoids [23]. The purpose of our investigation was to examine the complexation process between quercetin2H2O and magnesium (II) (Scheme 1), characterization and also to investigate the variation of antioxidant properties of quercetin after chelation with magnesium which is represented in Scheme 2.
2. Experimental 2.1. Material All reagent used for experimental purpose were of analytical reagent grade. Extra pure methanol, quercetin2H2O ([2-(3,4-dihy droxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4one]), DPPH (2,2-diphenyl-1-picryhydrazyl), magnesium sulfate2H2O were purchased from Sigma Aldrich Chemical Co. (St, Louis, MO, USA). 2.2. Physical measurement UV–visible spectroscopy study of chelation was performed using UV–visible spectrophotometer (UV-1800 Shimadzu) by following of titration of quercetin2H2O with different concentration of MgSO42H2O salt in methanolic solution. IR spectra were recorded in KBr pellets with a FT-IR (ALPHA-T, Bruker, Rheinstetten, Germany) spectrophotometer in the range 500–4000 cm1. Elemental analyses were performed using a CHNS instrument model Flash EA 1112 elemental analyzer. 1H NMR spectra in DMSO were obtained on a Bruker 600 MHz spectrometer. The X-ray powder diffraction patterns were collected on a DRON-3 diffractometer with a nickel filtered Cu Ka radiation (k = 1.5418 Å) in a 2 (range of 5–70°), step width of 0.05° and an acquisition time of 2 s on each step. The morphological characteristics of sample was investigated using a scanning electron microscope (SEM, S-4100, Hitachi, Japan) at an accelerating voltage of 17 kV. Double-sided sticky tape with lightly sprinkled sample was affixed to aluminum stub and made electrically conductive with gold coating (3–5 nm/min; 100 s; 30 W) in vacuum prior to observation under SEM. Micrographs were recorded at different magnifications to study the surface and morphological characteristics. 2.3. Stoichiometric ratio of the metal and ligand in the complex Job’s method [29] (continual variation method) was used to determine the stoichiometric ratio between the quercetin and the metal ion for their complexation in methanol; by mixing solutions of both chemicals having equimolar concentration (4 104 molar) in different ratios varying from 1:9 to 9:1. Then the absorbance was measured at 427 nm. 2.4. Synthesis of the complex In a 100-cm3 two necked round bottomed flask provided with electromagnetic stirrer and thermometer were placed, solid quercetin2H2O (0.17 g, 0.01 mol) and HPLC grade methanol
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Scheme 1. Schematic representation of the synthesis of quercetin–magnesium complex.
reduction of DPPH absorbance was followed by monitoring at 517 nm every 5 min for about 30–35 min (As) [12]. As a control the absorbance of blank solution of DPPH was also determined at 517 nm (Ac). The percentage of radical scavenging activity (RSA%) was calculated according to the following equation:
RSA % ¼ 100ðAc AsÞ=Ac
3. Results and discussion 3.1. Synthesis and characterization
Scheme 2. Proposed quercetin–metal complex oxidation pathway by DPPH radical via semiquinone radical intermediate.
(20 ml). Start the starring until the solid quercetin2H2O was completely dissolved the color of the solution was yellow. Methanolic NaOH (MeONa) (150 ll) was added for the deprotonation of the ligand after 5 min. The pH was adjusted to 6 by adding 1 M H2SO4 solution. Then solid MgSO42H2O salt was added into the flask (0.225 gm or 0.25 gm or 0.02 mol) and the color of the solution change to dark yellow with continuous stirring for one and half hour at room temp. The reaction mixture was filtered, and the filtrate was evaporated slowly at room temp. Collect the dark yellow product and washed with methanol to remove the uncreative part of the reagent, dried the product in vacuum desiccators, yielded the brown yellowish product. Analysis found: Mg, 6.23%; C, 51.51%; H, 2.34%; calculated for C15H8O7Mg [atomic mass 324.305]: Mg, 7.49%; C, 55.50%; H, 2.46%. 2.5. Antioxidant activity of the complex by DPPH method The antioxidant activity of free quercetin and quercetin– magnesium complex was evaluated using, DPPH radical scavenging activity. In the DPPH radical scavenging method, different concentrations (20 lg, 40 lg, 50 lg, 70 lg, 90 lg/ml) of quercetin and quercetin–magnesium complex were taken. Then 1 ml of the sample and 3 ml of DPPH solution and the reaction mixture was shaken vigorously and kept in completely dark for a better reaction. The
The effect of the pH was studied by adding 2 ml of buffer solution (pH range 1–9) to 10 ml of an equimolar (4 104 molar) mixture of quercetin and magnesium sulfate. It was observed that starting with pH 8 the complex formed was insoluble in the water– methanol mixture. So all other tests were done without any added buffer because pH of the system was maintained at 6.5. Previous survey shows that most of results of the stoichiometric composition of bioflavonoid complexes were obtained by the method of continual variation of equimolar solutions [29]. Consequently, the stoichiometry of the complex was investigated using water and methanol by both Job’s method [30] (spectra recorded at total constant concentration) and the slope ratio method. It was noticed that the intensity of the absorption at 427 nm was a function of both magnesium and quercetin concentration. When plotting the absorbance at 427 nm versus mole fraction of ligand, the results indicated that 1:1 chelate is formed whenever the solvent was a mixture of water and methanol (Fig. 2). The dissociation degree a was estimated from the absorbance of the solution when all the magnesium present, is complexed (Am) and the absorbance at the stoichiometric molar ratio (As):
a ¼ Am As =Am K was computed from the instability constant Ki:
Ki ¼ ða cÞ2 =ð1 aÞ c The average value of the stability constant was calculated as 1.62 104 (log b = 4.21). The results indicate that the 1:1 complex is predominant in methanol-containing solutions and this complex is moderately stable. Job’s method has been also performed in order to investigate the stoichiometry of the complex in water and methanol at pH 6 (phosphate buffer).
3.2. Physical properties of the complex The dark brown complex is stable in room temperature and soluble in methanol–water solution, DMSO, but insoluble in water and carbon tetra chloride.
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Fig. 2. Method of continuous variation. The plot of the absorbance at 427 nm (kmax of chelate) versus mole fraction of quercetin.
3.3. UV–vis spectroscopic study of the complex Like quercetin, most flavones and flavonols, shows two major absorption bands in the UV–vis region. Band I designated cinnamoyl group located in the wavelength of 300–400 nm, was attached with the nearby conjugated with ring B and carbonyl ring C, and band II located in the UV range of 240–300 nm, called benzoyl ring. It is related to conjugated system between ring A and carbonyl of ring C (Fig. 1B). While band II is interpreted for benzoyl system (A ring). From the molecular structure of quercetin, it can also be inferred that quercetin can chelate the metal ions via two site either 3-hydroxy-4-oxo system and 30 , 40 -dihydroxy system. When MgSO4 solution was added to a methanolic solution of quercetin, it caused the significant change in the quercetin spectrum due to the appearance of new peak at 427 nm kmax. It shows that the bathochromic shift of about 55 nm takes place. The spectral change can be easily observed in (Fig. 3). It confirms that complex formation takes place between quercetin and magnesium. That change takes place in band I; conversely the shift of band II at 264 nm is relatively insignificant (band IV). Therefore, it supports the clue that newly appeared peak at 427 nm in magnesium complex of quercetin may arise due to complexation of magnesium at 3-OH and 4-C@O of quercetin. High delocalization of oxygen electrons of 3-hydroxyl group, facilitate the p electrons delocalization. Thus, complex formation at band I caused by the interaction of 3-hydroxyl group of quercetin with magnesium subsequently follows the electronic redistribution between the magnesium and quercetin to form a big extended p bond system. It changes the electronic distribution in quercetin from p–p⁄ transition of lower energy. Hence, this information is supportive in the sense that the chelation ability of quercetin is attributable to the presence of 3-OH and 4–C@O groups in ring C or 30 , 40 -dihydroxy in ring B. Thus the red shift in the quercetin spectrum is highly informative for the coordination site in ligand having chelating site and due to the acidic nature of 3-OH proton and more suitable location of 4– C@O, they may be a proper site to be involved in complex formation. 3.4. 1H NMR 1 H NMR spectra of free quercetin and quercetin–magnesium (II) complex were obtained by using DMSO as a solvent and the main data are reported here:
3.4.1. Quercetin d 12.50 (1H, 5-OH); d 10.88 (1H, 7-OH); d 9.62 (1H, 3-OH); d 9.39 (1H, 40 -OH); d 9.29 (1H, 30 -OH); d 7.69 (1H, H-20 ); d 7.58 (1H, H-60 ); d 6.89 (1H, H-50 ); d 6.40 (1H, H-8); d 6.18 (1H, H-6).
Fig. 3. UV–Vis spectra of (a) quercetin and (b) quercetin–magnesium (II) complex.
3.4.2. Quercetin–magnesium (II) complex: d 12.44 (1H, 5-OH); d 10.86 (1H, 7-OH); d 9.36 (1H, 40 -OH); d 7.67 (1H, H-20 ); d 7.55 (1H, H-60 ); d 6.82 (1H, H-50 ); d 6.40 (1H, H-8); d 6.19 (1H, H-6). 1 H NMR studies of the quercetin and its complex show that, the quercetin is able to chelate metal ions via 30 or 40 phenolic groups. Upon complexation, the metal ion removes one hydrogen atom from the flavonoid [31]. 1H NMR data shows that, during complex formation one H atom has been removed easily, whereas the other H atom is intra molecularly bonded. The 1H NMR spectra of the quercetin–magnesium complex reveal the absence of hydrogen of 3-OH group. The other proton signals of the complex are slightly shifted as compared to the free flavonoid, this is probably due to the increase of the conjugation caused by the effect of coordination when the complex is formed. The complex is paramagnetic in nature due to availability of the unpaired electron which is localized in the complex. This information clearly indicates that during complex formation, two protons of free ligand are deprotonated. These data from 1H NMR fulfill the data of UV–visible and IR Spectroscopic studies.
3.5. IR study of the complex The coordination sites and the binding properties of quercetin were determined by using IR spectroscopy. Fig. 4 depicted IR of quercetin and quercetin–magnesium (II) complex and the data were analyzed in Table 1. Important information can be obtained by comparing the IR spectra of quercetin with
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Fig. 4. Infra-red (IR) spectra of (A) quercetin and (B) quercetin–magnesium (II) complex.
Table 1 Assignment of the IR spectra of the quercetin and the complex (band position in cm1). Compound
m(C@O)
m(C–O–H)
m(C@C)
m(O–H)
m(C–O–C)
m (M–O)
Quercetin Quercetin–magnesium complex
1664.76 1632.24
1318.95 1360.02
1609.19 1612.89
3408.41–3321.28 3289.51
1261.23 1240.96
Absent 596.40
Fig. 5. Diffractogram pattern of quercetin–magnesium (II) complex.
quercetin–magnesium (II) complex. The C@O stretching mode of the free quercetin occurs at 1664.76 cm1, which has been shifted to 1632.24 cm1 by the formation of complex. This shift suggests the coordination of carbonyl oxygen with metal ion [32–34,21]. The band situated at 1609.19 cm1 and 1261.23 cm1 are related
to m(C@C) and m(C–O–C) frequencies, which are slightly shifted by complex. The m(C–O–H) deformation mode observed at 1318.95 cm1 in the ligand (Fig. 4A), is shifted to 1360.02 cm1 in the complex (Fig. 4B), indicating an increase in bond order, which is normally observed when metal coordination involves
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were found to be in good agreement which refers the crystal nature of the complex.
Table 2 X-ray diffraction data for complexes. Compound
d/Å
I/I0 %
Quercetin–magnesium complex
27.26 27.24 27.22 27.20 27.18 27.16 20.14 20.10 20.08 20.06 20.04 20.02 20.00 19.89 19.69
73.60 68.86 74.46 70.86 85.26 64.46 68.26 95.00 98.60 95.00 83.60 85.00 79.46 77.53 63.06
with the ortho-phenolic m(O–H) group on the quercetin B ring. Moreover, the presence of m(M–O) stretching vibration at 569.40 cm1 indicates the formation of metal complex while the ligand exhibits no such bond. m(O–H) frequencies appear as broad bans (quercetin 3408.41–3321.28, quercetin–magnesium complex 3289.51) may be assigned for the presence of water molecule. 3.6. X-ray diffraction study of the complex The physical state i.e. crystallinity and crystal orientation of the complex was investigated by powder X-ray diffraction studies. Fig. 5 shows typical anticipation of the complex in which sharp peaks appeared due to its distinctive crystalline structure at diffraction angles of 19.89 Å, 20.00 Å, 20.02 Å, 20.04 Å, 20.06 Å, 20.08 Å, 20.10 Å and 27.18 Å. The d values and the relative intensity of peaks are summarized in (Table 2) which concludes the crystalline nature of the complex as because of the appearance of sharp peaks instead of diffused peaks. Powder X-ray diffraction pattern of the complexes are being studied in order to test the degree of crystallinity of the complexes. Diffraction pattern showed 15 distinct reflections in the range of 0–70° (2h), which are arised from diffraction of X-ray by the planes of complex. The interplanar spacing (d) has been calculated by using Bragg’s equation, nk = 2d sin h. The calculated interplaner d-spacing together with relative intensities with respect to most intense peak have been recorded and depicted in (Table 2). The observed interplaner d-spacing values and calculated relative intensities
3.7. Scanning electron microscopy Fig. 6 shows surface morphology of quercetin–magnesium complex as examined by scanning electron microscope (SEM) in two different magnifications. Fig. 6A was observed in 1000 magnification which represents the shape of particles and surface morphology of the compound. Fig. 6A illustrates SEM micrograph of randomly oriented structures with some well developed faces and others appearing as irregular arrangement. Those particles pose rod like or needle shaped structure with smooth surface. Fig. 6B was observed in 1500 magnification which confirms the needle shaped acicular structure with regular size and shape. The surface morphology of the particles revealed by these images indicates the un-fractured smooth surface area. 3.8. Antioxidant activity of quercetin–magnesium complex by DPPH radical scavenging method Quercetin is a bioflavonoid and well known for its good antioxidant and radical scavenging activity and to form chelate with metals. Formation of metal and flavonoid complex cations changes its natural free radical scavenging ability signifying that the complex has increased ability to scavenge oxidants and free radicals. DPPH test shows that the antioxidants have inheritant potential to reduce the DPPH radical from violet to yellow colored diphenyl-picrylhydrazine. In the chemical reaction antioxidants donate the hydrogen DPPH and convert it into DPPH-H. Actually antioxidant activity of quercetin and magnesium (II) complex depends upon their structure, especially their hydrogen donating ability. The reaction between quercetin and DPPH occurs in two several steps, they are as follows: in the fast step the DPPH absorbance diminishes very quickly and in the next slow step in which the absorbance of DPPH diminishes slowly in near about 1 h to reach a fixed or constant value. Fast step corresponds to abstraction of most liable H-atom, whereas slow step shows the oxidative degradation in the remaining product. However, the antioxidant activity of the compounds depends upon their molecular structures, but complexation made with the metal ions may affect the chemical properties of ligand molecules hence variation in the activity [35]. Our recent study has also in an agreement with the previous findings that our coordination complex may affect the
Fig. 6. (A and B) Scanning electron micrographs of quercetin–magnesium complex 1000 magnification in 10 lm range at an accelerating voltage of 17 kV.
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would like to thank Dr. Santanu Sannigrahi for editing and proof reading of the manuscript and acknowledged to Mr. Subhash Kumar Manna for technical help and Mr. Lal Mohan Masanta for providing laboratory support. References
Fig. 7. Kinetic behavior of DPPH free radical scavenging activity (RSA%) of different concentration of quercetin–magnesium complex. The radical scavenging activity were carried out for 30 min with increasing concentration (lg/ml) for 5 min interval.
ability of parent antioxidants. The antioxidant activities of the compounds have been studied in a concentration dependent manner (Fig. 7). Thus the plot illustrated that quercetin–magnesium complex scavenged the free radical about 55% at 70 mcg level. It could be due to the coordination of magnesium in 3 and 4 positions in C ring and 30 and 40 positions of B ring there by to scavenge free radicals (Fig. 1). 4. Conclusion In this study the interaction of quercetin with MgSO47H2O has been examined. The UV–visible spectra of quercetin in methanol– water exhibit two main absorption bands at 372 and 256 nm; but on the complexation the k max have been shifted towards the higher wavelength and new bands were appeared at 427 and 264 nm. The Job’s method was applied to validate the stoichiometric composition of the complex. The spectroscopic data show the importance of the 3-OH group which is coordination site of the ligand. The antioxidant activity of flavonoid depends on the number and positions of OH groups present in the flavonoid structure. The complex has been characterized on the basis of analytical and spectral data. The complex shows higher antioxidant activity as compared to the pure quercetin. This suggests that the metal ions significantly change the chemical properties of the quercetin. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements The authors are indebted to the Hari Charan Garg charitable trust for the financial support to carry this work. The authors
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Synthesis, characterization and study of antioxidant activity of quercetin–magnesium complex Nilanjan Ghosh a, Tania Chakraborty a, Sougata Mallick a, Supriya Mana a, Deepanwita Singha b, Balaram Ghosh c, Souvik Roy a,⇑ a b c
NSHM Knowledge Campus Kolkata, Group of Institutions, 124-BL Saha Road, Kolkata, West Bengal 700053, India Nightingale Diagnostic & Medicare Centre Pvt Ltd., 11, Shakespeare Sarani, Kolkata, West Bengal 700071, India Midnapore Medical College and Hospital, Vidyasagar Road, Medinipur, West Bengal 721101, India
h i g h l i g h t s Quercetin is able to chelate metal ions. We synthesize and characterize quercetin–magnesium (II) complex. Antioxidant activity of quercetin increased after chelation with magnesium. Chelation of magnesium induced a bathochromic shift in absorbance band. Peak at 596.40 cm
a r t i c l e
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in infrared spectroscopy represented the formation of M–O.
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Article history: Received 19 September 2014 Received in revised form 7 July 2015 Accepted 8 July 2015 Available online 9 July 2015 Keywords: Quercetin–magnesium complex Flavonoids Quercetin Spectral characterisation Antioxidant activity Radical scavenging activity
a b s t r a c t Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone) one of the most abundant dietary flavonoids, has been investigated in the presence of magnesium (II) in methanol. The complex formation between quercetin and magnesium (II) was examined under UV–visible, Infra-red and 1H NMR spectroscopic techniques. The spectroscopic data denoted that quercetin can reacts with magnesium cation (Mg+2) through the chelation site in the quercetin molecule. The free radical antioxidant activity of the complex with respect to the parent molecule was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. It was observed that the free radical scavenging activity of quercetin was increased after complexation of magnesium (Mg+2) cation. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Flavonoids (2-phenyl-benzo-c-pyrones, Fig. 1A) are a large group of polyphenolic natural compounds that are extensively distributed in plant-based foods [1]. A multitude of substitution patterns in the two benzene rings (A and B) of the basic structure occur in nature. Variations in their heterocyclic rings give rise to flavonols, flavones, catechins, flavanones, anthocyanidins and isoflavones. Over 4000 different naturally occurring flavonoids have been described [2]. Flavonoids are mostly present in human diet, such as fruits, vegetables and plant-derived beverages such
⇑ Corresponding author at: Department of Pharmacology, NSHM Knowledge Campus, Kolkata, India. E-mail address: [email protected] (S. Roy). http://dx.doi.org/10.1016/j.saa.2015.07.050 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
as tea and red wine [3]. It is estimated that the human intake of all flavonoids is a few hundreds of milligrams per day [4]. Human being obtains bioflavonoids through diet since they do not have the capacity to synthesize these polyphenols. The consumed bioflavonoids have been shown to be absorbed through the stomach as well as the small intestine, and they are mainly metabolized by the liver and intestinal mucosa and excreted in the urine and feces [5]. Interest in the therapeutic applications of flavonoids and their derivatives (glycosylated flavonoids) for the treatment and prevention of human diseases has increased in recent years. Such flavonoids have many biological activities like anti-cancer, anti-inflammatory, anti-diabetic, antiviral, anti-allergic etcetera. Some epidemiological studies support a protective role of flavonoids rich diets in developing cancer and cardiovascular diseases [6–11]. Most of the bioflavonoids are strong natural antioxidant and having free radical scavenging activity along with potent metal
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Fig. 1. (A) Basic structure of flavonoid. (B) Basic structure of quercetin (3,30 ,40 ,5,7-pentahydroxyflavone).
chelators [12]. Flavonoids also play an important role in both the bioavailability for metal ions which are in low amount in our body and toxicity of a variety of toxic metals like Pb (II) [13]. Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone), (Fig. 1B) is one of the most common flavonoids present in nature that has attracted the attention of many researchers because of its biological properties [14]. Quercetin is very effective metal chelators which possesses three possible chelating sites in competition: the 3-hydroxy-4-carbonyl, the 5-hydroxy-4-carbonyl, and the 30,40-dihydroxyl (catechol) groups (Fig. 1B). Complexation of metal cations by Q has already been reported for a large number of metals (Mo(VI), Fe(II), Fe(III), Cu(II), Zn(II), Al(III), Tb(III), Pb(II), Co(II) and many other cations) and in these studies different stoichiometric ratios can be seen between different metals and quercetin as a ligand [15–22]. Metal chelation is widely considered as another mechanism of the antioxidant activity of flavonoids. The interaction of flavonoids with metal ions may also change the antioxidant properties and also biological effects of the flavonoids [23]. It is suggest that the biological activity of an organic ligand can be increased when co-ordinated or mixed with suitable metal ion; because of its ability to act as free radical acceptor [24–26]. Magnesium is an alkaline earth metal and the fourth most common cation in the body and the second most common intracellular cation after potassium. It has fundamental role as a co-factor in more than 300 enzymatic reactions involving energy metabolism and nucleic acid synthesis. It is also involved in several process including: hormone receptor binding; gating of calcium channels; transmembrane ion flux and regulation of adenylated cyclase; muscle contraction; neuronal activity; control of vasomotor tone; cardiac excitability; and neuro-transmitter release. In many of its action it has been linked to a physiological calcium antagonist [27]. Epidemiological studies trace the prevalence of cardiovascular disease and cardiac deaths to the degree of magnesium depletion induced by a diet and drinking water low in magnesium [28]. The three areas of particular relevance of magnesium are myocardial infarction, arrhythmia and cardiac surgery. As metal ions play a vital role in the initiation of free radical processes, metal chelation is widely considered as another mechanism of the antioxidant activity of flavonoids. The interaction of flavonoids with metal ions may change the antioxidant properties and some biological effects of the flavonoids [23]. The purpose of our investigation was to examine the complexation process between quercetin2H2O and magnesium (II) (Scheme 1), characterization and also to investigate the variation of antioxidant properties of quercetin after chelation with magnesium which is represented in Scheme 2.
2. Experimental 2.1. Material All reagent used for experimental purpose were of analytical reagent grade. Extra pure methanol, quercetin2H2O ([2-(3,4-dihy droxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4one]), DPPH (2,2-diphenyl-1-picryhydrazyl), magnesium sulfate2H2O were purchased from Sigma Aldrich Chemical Co. (St, Louis, MO, USA). 2.2. Physical measurement UV–visible spectroscopy study of chelation was performed using UV–visible spectrophotometer (UV-1800 Shimadzu) by following of titration of quercetin2H2O with different concentration of MgSO42H2O salt in methanolic solution. IR spectra were recorded in KBr pellets with a FT-IR (ALPHA-T, Bruker, Rheinstetten, Germany) spectrophotometer in the range 500–4000 cm1. Elemental analyses were performed using a CHNS instrument model Flash EA 1112 elemental analyzer. 1H NMR spectra in DMSO were obtained on a Bruker 600 MHz spectrometer. The X-ray powder diffraction patterns were collected on a DRON-3 diffractometer with a nickel filtered Cu Ka radiation (k = 1.5418 Å) in a 2 (range of 5–70°), step width of 0.05° and an acquisition time of 2 s on each step. The morphological characteristics of sample was investigated using a scanning electron microscope (SEM, S-4100, Hitachi, Japan) at an accelerating voltage of 17 kV. Double-sided sticky tape with lightly sprinkled sample was affixed to aluminum stub and made electrically conductive with gold coating (3–5 nm/min; 100 s; 30 W) in vacuum prior to observation under SEM. Micrographs were recorded at different magnifications to study the surface and morphological characteristics. 2.3. Stoichiometric ratio of the metal and ligand in the complex Job’s method [29] (continual variation method) was used to determine the stoichiometric ratio between the quercetin and the metal ion for their complexation in methanol; by mixing solutions of both chemicals having equimolar concentration (4 104 molar) in different ratios varying from 1:9 to 9:1. Then the absorbance was measured at 427 nm. 2.4. Synthesis of the complex In a 100-cm3 two necked round bottomed flask provided with electromagnetic stirrer and thermometer were placed, solid quercetin2H2O (0.17 g, 0.01 mol) and HPLC grade methanol
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Scheme 1. Schematic representation of the synthesis of quercetin–magnesium complex.
reduction of DPPH absorbance was followed by monitoring at 517 nm every 5 min for about 30–35 min (As) [12]. As a control the absorbance of blank solution of DPPH was also determined at 517 nm (Ac). The percentage of radical scavenging activity (RSA%) was calculated according to the following equation:
RSA % ¼ 100ðAc AsÞ=Ac
3. Results and discussion 3.1. Synthesis and characterization
Scheme 2. Proposed quercetin–metal complex oxidation pathway by DPPH radical via semiquinone radical intermediate.
(20 ml). Start the starring until the solid quercetin2H2O was completely dissolved the color of the solution was yellow. Methanolic NaOH (MeONa) (150 ll) was added for the deprotonation of the ligand after 5 min. The pH was adjusted to 6 by adding 1 M H2SO4 solution. Then solid MgSO42H2O salt was added into the flask (0.225 gm or 0.25 gm or 0.02 mol) and the color of the solution change to dark yellow with continuous stirring for one and half hour at room temp. The reaction mixture was filtered, and the filtrate was evaporated slowly at room temp. Collect the dark yellow product and washed with methanol to remove the uncreative part of the reagent, dried the product in vacuum desiccators, yielded the brown yellowish product. Analysis found: Mg, 6.23%; C, 51.51%; H, 2.34%; calculated for C15H8O7Mg [atomic mass 324.305]: Mg, 7.49%; C, 55.50%; H, 2.46%. 2.5. Antioxidant activity of the complex by DPPH method The antioxidant activity of free quercetin and quercetin– magnesium complex was evaluated using, DPPH radical scavenging activity. In the DPPH radical scavenging method, different concentrations (20 lg, 40 lg, 50 lg, 70 lg, 90 lg/ml) of quercetin and quercetin–magnesium complex were taken. Then 1 ml of the sample and 3 ml of DPPH solution and the reaction mixture was shaken vigorously and kept in completely dark for a better reaction. The
The effect of the pH was studied by adding 2 ml of buffer solution (pH range 1–9) to 10 ml of an equimolar (4 104 molar) mixture of quercetin and magnesium sulfate. It was observed that starting with pH 8 the complex formed was insoluble in the water– methanol mixture. So all other tests were done without any added buffer because pH of the system was maintained at 6.5. Previous survey shows that most of results of the stoichiometric composition of bioflavonoid complexes were obtained by the method of continual variation of equimolar solutions [29]. Consequently, the stoichiometry of the complex was investigated using water and methanol by both Job’s method [30] (spectra recorded at total constant concentration) and the slope ratio method. It was noticed that the intensity of the absorption at 427 nm was a function of both magnesium and quercetin concentration. When plotting the absorbance at 427 nm versus mole fraction of ligand, the results indicated that 1:1 chelate is formed whenever the solvent was a mixture of water and methanol (Fig. 2). The dissociation degree a was estimated from the absorbance of the solution when all the magnesium present, is complexed (Am) and the absorbance at the stoichiometric molar ratio (As):
a ¼ Am As =Am K was computed from the instability constant Ki:
Ki ¼ ða cÞ2 =ð1 aÞ c The average value of the stability constant was calculated as 1.62 104 (log b = 4.21). The results indicate that the 1:1 complex is predominant in methanol-containing solutions and this complex is moderately stable. Job’s method has been also performed in order to investigate the stoichiometry of the complex in water and methanol at pH 6 (phosphate buffer).
3.2. Physical properties of the complex The dark brown complex is stable in room temperature and soluble in methanol–water solution, DMSO, but insoluble in water and carbon tetra chloride.
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Fig. 2. Method of continuous variation. The plot of the absorbance at 427 nm (kmax of chelate) versus mole fraction of quercetin.
3.3. UV–vis spectroscopic study of the complex Like quercetin, most flavones and flavonols, shows two major absorption bands in the UV–vis region. Band I designated cinnamoyl group located in the wavelength of 300–400 nm, was attached with the nearby conjugated with ring B and carbonyl ring C, and band II located in the UV range of 240–300 nm, called benzoyl ring. It is related to conjugated system between ring A and carbonyl of ring C (Fig. 1B). While band II is interpreted for benzoyl system (A ring). From the molecular structure of quercetin, it can also be inferred that quercetin can chelate the metal ions via two site either 3-hydroxy-4-oxo system and 30 , 40 -dihydroxy system. When MgSO4 solution was added to a methanolic solution of quercetin, it caused the significant change in the quercetin spectrum due to the appearance of new peak at 427 nm kmax. It shows that the bathochromic shift of about 55 nm takes place. The spectral change can be easily observed in (Fig. 3). It confirms that complex formation takes place between quercetin and magnesium. That change takes place in band I; conversely the shift of band II at 264 nm is relatively insignificant (band IV). Therefore, it supports the clue that newly appeared peak at 427 nm in magnesium complex of quercetin may arise due to complexation of magnesium at 3-OH and 4-C@O of quercetin. High delocalization of oxygen electrons of 3-hydroxyl group, facilitate the p electrons delocalization. Thus, complex formation at band I caused by the interaction of 3-hydroxyl group of quercetin with magnesium subsequently follows the electronic redistribution between the magnesium and quercetin to form a big extended p bond system. It changes the electronic distribution in quercetin from p–p⁄ transition of lower energy. Hence, this information is supportive in the sense that the chelation ability of quercetin is attributable to the presence of 3-OH and 4–C@O groups in ring C or 30 , 40 -dihydroxy in ring B. Thus the red shift in the quercetin spectrum is highly informative for the coordination site in ligand having chelating site and due to the acidic nature of 3-OH proton and more suitable location of 4– C@O, they may be a proper site to be involved in complex formation. 3.4. 1H NMR 1 H NMR spectra of free quercetin and quercetin–magnesium (II) complex were obtained by using DMSO as a solvent and the main data are reported here:
3.4.1. Quercetin d 12.50 (1H, 5-OH); d 10.88 (1H, 7-OH); d 9.62 (1H, 3-OH); d 9.39 (1H, 40 -OH); d 9.29 (1H, 30 -OH); d 7.69 (1H, H-20 ); d 7.58 (1H, H-60 ); d 6.89 (1H, H-50 ); d 6.40 (1H, H-8); d 6.18 (1H, H-6).
Fig. 3. UV–Vis spectra of (a) quercetin and (b) quercetin–magnesium (II) complex.
3.4.2. Quercetin–magnesium (II) complex: d 12.44 (1H, 5-OH); d 10.86 (1H, 7-OH); d 9.36 (1H, 40 -OH); d 7.67 (1H, H-20 ); d 7.55 (1H, H-60 ); d 6.82 (1H, H-50 ); d 6.40 (1H, H-8); d 6.19 (1H, H-6). 1 H NMR studies of the quercetin and its complex show that, the quercetin is able to chelate metal ions via 30 or 40 phenolic groups. Upon complexation, the metal ion removes one hydrogen atom from the flavonoid [31]. 1H NMR data shows that, during complex formation one H atom has been removed easily, whereas the other H atom is intra molecularly bonded. The 1H NMR spectra of the quercetin–magnesium complex reveal the absence of hydrogen of 3-OH group. The other proton signals of the complex are slightly shifted as compared to the free flavonoid, this is probably due to the increase of the conjugation caused by the effect of coordination when the complex is formed. The complex is paramagnetic in nature due to availability of the unpaired electron which is localized in the complex. This information clearly indicates that during complex formation, two protons of free ligand are deprotonated. These data from 1H NMR fulfill the data of UV–visible and IR Spectroscopic studies.
3.5. IR study of the complex The coordination sites and the binding properties of quercetin were determined by using IR spectroscopy. Fig. 4 depicted IR of quercetin and quercetin–magnesium (II) complex and the data were analyzed in Table 1. Important information can be obtained by comparing the IR spectra of quercetin with
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Fig. 4. Infra-red (IR) spectra of (A) quercetin and (B) quercetin–magnesium (II) complex.
Table 1 Assignment of the IR spectra of the quercetin and the complex (band position in cm1). Compound
m(C@O)
m(C–O–H)
m(C@C)
m(O–H)
m(C–O–C)
m (M–O)
Quercetin Quercetin–magnesium complex
1664.76 1632.24
1318.95 1360.02
1609.19 1612.89
3408.41–3321.28 3289.51
1261.23 1240.96
Absent 596.40
Fig. 5. Diffractogram pattern of quercetin–magnesium (II) complex.
quercetin–magnesium (II) complex. The C@O stretching mode of the free quercetin occurs at 1664.76 cm1, which has been shifted to 1632.24 cm1 by the formation of complex. This shift suggests the coordination of carbonyl oxygen with metal ion [32–34,21]. The band situated at 1609.19 cm1 and 1261.23 cm1 are related
to m(C@C) and m(C–O–C) frequencies, which are slightly shifted by complex. The m(C–O–H) deformation mode observed at 1318.95 cm1 in the ligand (Fig. 4A), is shifted to 1360.02 cm1 in the complex (Fig. 4B), indicating an increase in bond order, which is normally observed when metal coordination involves
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were found to be in good agreement which refers the crystal nature of the complex.
Table 2 X-ray diffraction data for complexes. Compound
d/Å
I/I0 %
Quercetin–magnesium complex
27.26 27.24 27.22 27.20 27.18 27.16 20.14 20.10 20.08 20.06 20.04 20.02 20.00 19.89 19.69
73.60 68.86 74.46 70.86 85.26 64.46 68.26 95.00 98.60 95.00 83.60 85.00 79.46 77.53 63.06
with the ortho-phenolic m(O–H) group on the quercetin B ring. Moreover, the presence of m(M–O) stretching vibration at 569.40 cm1 indicates the formation of metal complex while the ligand exhibits no such bond. m(O–H) frequencies appear as broad bans (quercetin 3408.41–3321.28, quercetin–magnesium complex 3289.51) may be assigned for the presence of water molecule. 3.6. X-ray diffraction study of the complex The physical state i.e. crystallinity and crystal orientation of the complex was investigated by powder X-ray diffraction studies. Fig. 5 shows typical anticipation of the complex in which sharp peaks appeared due to its distinctive crystalline structure at diffraction angles of 19.89 Å, 20.00 Å, 20.02 Å, 20.04 Å, 20.06 Å, 20.08 Å, 20.10 Å and 27.18 Å. The d values and the relative intensity of peaks are summarized in (Table 2) which concludes the crystalline nature of the complex as because of the appearance of sharp peaks instead of diffused peaks. Powder X-ray diffraction pattern of the complexes are being studied in order to test the degree of crystallinity of the complexes. Diffraction pattern showed 15 distinct reflections in the range of 0–70° (2h), which are arised from diffraction of X-ray by the planes of complex. The interplanar spacing (d) has been calculated by using Bragg’s equation, nk = 2d sin h. The calculated interplaner d-spacing together with relative intensities with respect to most intense peak have been recorded and depicted in (Table 2). The observed interplaner d-spacing values and calculated relative intensities
3.7. Scanning electron microscopy Fig. 6 shows surface morphology of quercetin–magnesium complex as examined by scanning electron microscope (SEM) in two different magnifications. Fig. 6A was observed in 1000 magnification which represents the shape of particles and surface morphology of the compound. Fig. 6A illustrates SEM micrograph of randomly oriented structures with some well developed faces and others appearing as irregular arrangement. Those particles pose rod like or needle shaped structure with smooth surface. Fig. 6B was observed in 1500 magnification which confirms the needle shaped acicular structure with regular size and shape. The surface morphology of the particles revealed by these images indicates the un-fractured smooth surface area. 3.8. Antioxidant activity of quercetin–magnesium complex by DPPH radical scavenging method Quercetin is a bioflavonoid and well known for its good antioxidant and radical scavenging activity and to form chelate with metals. Formation of metal and flavonoid complex cations changes its natural free radical scavenging ability signifying that the complex has increased ability to scavenge oxidants and free radicals. DPPH test shows that the antioxidants have inheritant potential to reduce the DPPH radical from violet to yellow colored diphenyl-picrylhydrazine. In the chemical reaction antioxidants donate the hydrogen DPPH and convert it into DPPH-H. Actually antioxidant activity of quercetin and magnesium (II) complex depends upon their structure, especially their hydrogen donating ability. The reaction between quercetin and DPPH occurs in two several steps, they are as follows: in the fast step the DPPH absorbance diminishes very quickly and in the next slow step in which the absorbance of DPPH diminishes slowly in near about 1 h to reach a fixed or constant value. Fast step corresponds to abstraction of most liable H-atom, whereas slow step shows the oxidative degradation in the remaining product. However, the antioxidant activity of the compounds depends upon their molecular structures, but complexation made with the metal ions may affect the chemical properties of ligand molecules hence variation in the activity [35]. Our recent study has also in an agreement with the previous findings that our coordination complex may affect the
Fig. 6. (A and B) Scanning electron micrographs of quercetin–magnesium complex 1000 magnification in 10 lm range at an accelerating voltage of 17 kV.
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would like to thank Dr. Santanu Sannigrahi for editing and proof reading of the manuscript and acknowledged to Mr. Subhash Kumar Manna for technical help and Mr. Lal Mohan Masanta for providing laboratory support. References
Fig. 7. Kinetic behavior of DPPH free radical scavenging activity (RSA%) of different concentration of quercetin–magnesium complex. The radical scavenging activity were carried out for 30 min with increasing concentration (lg/ml) for 5 min interval.
ability of parent antioxidants. The antioxidant activities of the compounds have been studied in a concentration dependent manner (Fig. 7). Thus the plot illustrated that quercetin–magnesium complex scavenged the free radical about 55% at 70 mcg level. It could be due to the coordination of magnesium in 3 and 4 positions in C ring and 30 and 40 positions of B ring there by to scavenge free radicals (Fig. 1). 4. Conclusion In this study the interaction of quercetin with MgSO47H2O has been examined. The UV–visible spectra of quercetin in methanol– water exhibit two main absorption bands at 372 and 256 nm; but on the complexation the k max have been shifted towards the higher wavelength and new bands were appeared at 427 and 264 nm. The Job’s method was applied to validate the stoichiometric composition of the complex. The spectroscopic data show the importance of the 3-OH group which is coordination site of the ligand. The antioxidant activity of flavonoid depends on the number and positions of OH groups present in the flavonoid structure. The complex has been characterized on the basis of analytical and spectral data. The complex shows higher antioxidant activity as compared to the pure quercetin. This suggests that the metal ions significantly change the chemical properties of the quercetin. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements The authors are indebted to the Hari Charan Garg charitable trust for the financial support to carry this work. The authors
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