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The present article describes the syntheses as well as complexation study between morin (3,5,7,2',4'-pentahydroxyflavone) and metal ions of Fe(II), Co(II) and Ni(II), in methanol. The characterization of resulting complexes was carried out by spectral (UV-Vis., IR, 1H NMR, 13C NMR and AAS), elemental, thermal analyses and cyclic voltammetry. The determination of stoichiometric composition and the behavior of ligand coordinating sites with respect to the metal ions have been discussed. The antioxidant activity (evaluated by DPPHâ€¢ radical scavenging method) of all the complexes is higher as compared to the morin and among complexes Co complex shows more activity than others. Cyclic voltammetry also supports the antioxidant activity because the oxidation potentials for complexes are fairly low as compared to morin. Antibacterial study shows that all the three complexes are more bioactive relative to the morin against m. flavus and s. aureus.
Keywords: Morin; complex; flavonoids; metal ions; synthesis; stoichiometry
Flavonoids are polyphenolic organic compounds possessing several potential applications in various fields of medical science. They are present naturally as well as can be synthesized by different methods in laboratory. They act as dyes for vegetables and contribute many colors to the flowering parts of plants. The nature of each flavonoid depends upon its structural class, functional substitution, conjugation, degree of hydroxylation and polymerization [Masek, Zaborski, Chrzescijanska, 2011]. Flavonoids are non-nutritious compounds; however they are very important constituent of human diet. They can be obtained from different sources i.e. foods, beverages, different herbal drugs, and related phytomedicines. Such type of dietary vegetables and fruits are scientifically proved to be protective against cancer [Montoro, Braca, Pizza, De Tommasi, 2005]. Flavonoids possess several properties which make the basis of continuous research in multiple areas of biology and chemistry. In plants, they act as defensive molecules due to their astringent taste and protect them from herbivorous attack. Moreover, the flavonoids show a significant range of biological and pharmacological activities like anticancer, anti-inflammatory, antimicrobial and cardiovascular protection [Ansari, 2008]. They are highly useful in pharmaceuticals and food preparations due to their biological properties [Salem, Chevalot, Harscoat-Schiavo, Paris, Fick, Humeau, 2011]. There are different classes of flavonoids based on the arrangement of phenolic groups, for example flavones, flavonols, flavanones and flavanes, which show the functional varieties and complexities [Xu, In, Yuan, Lee, & Kim, 2007; Sancho, Blanco, & Ferretti, 2006]. The foremost property possessed by flavonoids is that they are natural antioxidants, they protect from the oxidative stress; hence much of the work is underway at the moment on their antioxidant nature. The antioxidant activity of the flavonoids may be increased by chelation with metal ions. The chelates may act as stronger free radical scavengers as compared to the flavonoids alone [MaleÅ¡ev, & KuntiÄ‡, 2007]. Flavonoids are versatile ligands for chelation, for that reason, presently they are highly concentrated to design and synthesize different metal coordination complexes. The presence of hydroxyl and ketonic groups in flavonoids offer the better chelating ability to bind the metal ions. There are many reports regarding the metal complexes of flavonoids with transition as well as non-transition metal ions [Ansari, 2008]. It is learned from the literature that morin (2',3,4',5,7-pentahydroxyflavone) (Fig. 1a) has fewer number of the solid metal complexes [Qi, Liufang, & Xiang, 1996].
From the literature survey, it has been observed that the data on composition and structure of the complexes is ambiguous and contradictory; because morin has three possible chelating sites to interact with metal ions. The proper complexation site and its conditions are still questionable. There is also an indefinite number of stoichiometric compositions previously reported for the different types of morin metal complexes.
Flavonoids have an interesting coordination chemistry, which is widely studied because of their significant exploitation in biomedical sciences [Ansari, 2008]. Metals of Fe, Co and Ni have horizontal similarities and form chelates with flavonoids. Big number of stable chelates forms 5- or 6-membered ring systems. But chelates of 5-membered ring systems are slightly more stable [Iqbal, 2001; Lee, 1996]. Therefore, herein, we report the syntheses of solid metal chelates of morin forming stable 5-membered ring systems, and their electrochemical, antioxidant and antibacterial studies are also explored.
2. Materials and Methods
2.3. Syntheses of metal complexes
Morin [0.0756 g, 0.01M] was added to 100 ml of round bottom flask mounted with an electromagnetic stirrer, containing 25 ml methanol. After the complete dissolution of morin till 15 min. stirring, 150 Î¼l of sodium methoxide was added for deprotonation. As soon as the metal salts [FeCl2Â·4H2O (0.0398g, 0.01M), CoCl2.6H2O (0.0476g, 0.01M) and NiCl6.6H2O (0.0475 g, 0.01 M)] were added in individual flasks [Panhwar, Memon, & Bhanger, 2010], the color of solutions changed. The contents were refluxed for one and half an hour, then filtered the solution to remove unreacted morin. Evaporated the solvent on rotary evaporator, washed the contents with t-butanol, dried in vacuum dessicator and finally calculated the yield (Table 1A) [Bukhari, Memon, Mahroof-Tahir, & Bhanger, 2008].
2.4. Stoichiometry of the complexes
Job's method of continuous variation was employed to determine the stoichiometric ratios for complexation between metal ions and flavonoids, in methanol. Solutions of both the components were prepared by mixing their equimolar concentrations (1 x 10-4 M) in varying ratios of 1:9 to 9:1 [De Souza, & De Giovani, 2005]. Finally, the sufficient stoichiometric amounts of morin and analogous metal salts were mixed in methanol to measure the absorbance. From the results, molar ratio (metal:ligand) was found as 1:2 in all the three cases (Fig. 2) [Bukhari, Memon, Mahroof-Tahir, & Bhanger, 2009; Tan et al., 2009].
2.5. Physical properties of the complexes
Analytical data that also includes the physical properties of the title complexes is well illustrated in Table 1A. It is taken to be granted that morin is a bidentate ligand (Fig. 1b) in which a metal ion binds two ligands simultaneously and form mononuclear complexes. This statement is in good agreement with the elemental analysis, TGA, IR, UV-Vis, 1H NMR and 13C NMR. In the data obtained from results, the general formula for the complexes is deduced as [ML2.(H2O)2].nH2O, where M = ferrous(II), cobalt(II) and nickel(II), L = morin (3-OH group deprotonated) and n = 1 or 2 [Zhou, Wang, Wang, & Tang, 2001]. Here, it is also noted that the elemental analysis data is also in good agreement with theoretical information. The complexes are highly stable in air for longer times [Tan et al., 2009] and don't change their composition while they are stored [De Souza, & De Giovani, 2004]. They are frequently soluble in MeOH, EtOH, DMSO and DMF, slightly soluble in Me2CO, but barely soluble in H2O and CCl4 [Zhou, Wang, Wang, & Tang, 2001]. Molar conductivities of 10-3 M solutions of all the complexes were measured at room temperature, by dissolving them in DMSO. The molar conductance of complexes was found in the range of 2-4 Î¼S/cm [Imran, Kokab, Latif, Liviu, & Mahmood, 2010], it suggested that these are very low conductance values; hence the complexes are regarded as non-electrolytes [Qi, Liufang, & Xiang, 1996].
2.6. Antioxidant activity
The antioxidative activity of morin and the complexes was evaluated by radical scavenging effect of the stable DPPHâ€¢ free radical. It has the maximum absorption at 517 nm, where morin and its complexes were subjected to screening for their possible antioxidant activity, in methanol. While assessing the DPPHâ€¢ radical scavenging capabilities for standard and the complexes, it has been examined that the exchange of protons takes place between the antioxidant (standard/complexes) and the free radical. Basically, DPPHâ€¢ radical is reduced to the analogous hydrazine, which is indicated in the form of color change from violet to yellow, when examined spectrophotometrically [Silva, Santos, Caroço, Rocha, Justino, & Mira, 2002; StanojeviÄ‡ et al., 2009]. The degree of decolourisation is the measure of reducing capacity of the morin and complexes that enables to evaluate the antioxidant activity. In experimental procedure, the standard solutions of morin were prepared in the concentration range of 1, 2, 4, 6, 8, 10, 15, 20, 30 and 40 Î¼M in methanol, subsequently; 0.1 ml of standard solution of each concentration was added to 3.9 ml of newly prepared DPPH of 57.65 Î¼M concentration, in methanol. On the addition of morin and its complexes, the decrease in absorbance was observed and it was monitored up to 30 min until the plateau was achieved for the reaction [Panhwar, Memon, & Bhanger, 2010]. The absorbance for all the samples (As) as well as pure DPPHâ€¢ solution (Ac) was recorded at 517 nm; it was also used as a control. DPPHâ€¢ radical scavenging ability for all the complexes specified by decrease in the absorbance was then calculated by using the equation (1): Scavenging activity (%) = 100(Ac-As)/Ac [Chen, Sun, Cao, Liang, & Song, 2009]
Morin A517 = 0.009 Ã- 10-6 Ã-XDPPH - 0.586, r2 = 0.9992 (1)
2.7. Antibacterial studies
Antimicrobial activity of the complexes was found [Agarwal, & Prasad, 2005] in vitro by using different microorganisms i.e. Micrococcus flavus (G+ve) and Staphylococcus aureus (G+ve) through well diffusion method. Both the bacterial species were obtained from the Department of Microbiology, University of Karachi [Canpolat, & Kaya, 2004]. Both the bacterial cultures were kept under the temperature of -20Â°C for preservation. In order to carry out the experiment, the cultures were reactivated in brain heart infusion (BHI) agar [Pereira et al., 2007]. For this, at first sterilized the petri dishes, applied the BHI agar medium for pouring [Canpolat, & Kaya, 2004], and then incubated the petri dishes for 24 hrs at 37Â°C, after that the bacteria were grown on agar plates. Inoculation of the cultures was made either by using platinum wire loop (that was first made red hot in the flame, cooled and then used for the application of bacterial species) or a cotton swab in the fashion of lawning. All the compounds were dissolved in DMSO and then examined. Wells of 8 mm size were cut in the medium and they were filled with the solutions of ligand and the complexes using micropipette [Mahmud, Iqbal, Bhatti, Tariq, & Gulzar, 2009]. DMSO solvent was exercised as a control. Again the petri dishes were put into the incubator for 24 h at 37Â°C and finally assessed the bactericide action of compounds [Canpolat, & Kaya, 2004]. Experiments were performed in triplicate [Pereira et al., 2007].
3. Results and discussion
3.1. UV-Vis studies
UV-Vis spectra of all the complexes and morin were recorded in MeOH (Fig. 3a). The electronic spectrum of only morin illustrates two major peaks one at 375 nm (band I) and another at 263 nm (band II). It suggested that the peak one belongs to the ring B (cinnamoyl system) and peak two pertains to the ring A (benzoyl system). As soon as the complex formation takes place, it represents the bathochromic shifts in the spectra of morin (Table 1B). The red shift in band II is very small (ca. 0-3 nm); but in Band I its very high (ca. 0-47nm) which implies that there is formation of metal-oxygen bond in ring B (in cinnamoyl system). This information at first verifies the role of participation of 3-OH group in complex formation [Zhou, Wang, Wang, & Tang, 2001]. This is also confirmed by the work of [Zhou, Wang, Wang, & Tang, 2001; De Souza, & De Giovani, 2004; Kopacz, & WoÅºnicka, 2004] that the metal ions most probably replace the proton at this position, which is responsible for the red shift of bands in complexes [Tan et al., 2009]. This study examines the chelation efficacy of different number of metal ions with morin at various pH values i.e. 6.5, 7.3 and 7.4, in which all the metal ions caused strong bathochromic shifts nearly equally with negligible difference to each other in band I maxima but Band II experiences a very low shift in the value [Mira, Fernandez, Santos, Rocha, Florêncio, & Jennings, 2002].
3.2. pH studies
Flavonoids are weakly polybasic acids that have a tendency to protonate the species; therefore they experience the substantial effect of pH on the formation of their complexes. Our results show that the most suited pH to form the complexes of highest coordination numbers lies in the slightly acidic or neutral range, but hardly they are formed in basic media. However, pH 6 is most favorable in the formation of complexes, though it is enormously reliant on the characteristics of the metal ions.
Flavonoids remain in undissociated form at the pH values below 3.0; hence complexes are hardly formed in this condition. But at higher pH values they are deprotonated considerably and in result there may be the possibility of the formation of more complex species, another problem is that the side reactions can also take place at higher pH values for metals and possibly they can develop the hydroxo-complexes.
When flavonoids undergo the complexation, they either act as monodentate or bidentate. At the time of complex formation, protons are also present besides the metal ions and ligand (and they also can form protonated complexes) that undergo dissociation at higher pH values. Thus, at higher pH, absorption spectra of such complexes show bathochromic shift upon the dissociation of protonated complexes than to form complexes of different stoichiometric compositions [MaleÅ¡ev, & KuntiÄ‡, 2007].
In the formation of Fe(II), Co(II) and Ni(II)-morin complexes, the pH of their solutions was noted as 6.5, 7.3 and 7.4 respectively, in methanol. For stability purpose, the Î»max of the complexes was examined in the range of 2.0 to 12 pH that was obtained by adding HCl and NaOH (0.01M) [Pereira et al., 2007]. It was ensured that nearly all the complexes found sufficiently stable over this pH range.
3.3. Vibrational spectra
From the tentative assignments (Table 2A) of IR spectra (Fig. 3b) of morin and all the three complexes only the major peaks were analyzed, which undergo the frequency changes. When the results were compared for both the complexes as well as ligand (morin. 2H2O), it imparted the most important knowledge It clarifies that Ï…(Mâˆ’O) peaks at 598-640 cm-1 in the complexes are not found for ligand. It is also observed that there is a little shift in the frequency of Ï…(Câˆ’Oâˆ’C) and Ï…(C=C), which infers that the ring oxygen is not involved in the formation of metal-oxygen bond. On the other hand, the carbonyl group present in morin has the characteristic frequency of 1662 cm-1, which drops off to still lower value in the case of complexes by ca. 16, 14 and 11 cm-1 having the characteristic frequencies at 1646, 1648 and 1651 cm-1 for Fe(II), Co(II) and Ni(II) complexes, respectively. It verifies that it is the oxygen of carbonyl group, which is mainly involved in the metal-oxygen bond formation to give metal chelates after deprotonation through 3-OH or 5-OH group of morin ligand. Another informative frequency of Ï…(Oâˆ’H) comes into view as a broad band covering the range of 3055-3460 cm-1 clearly witness the presence of water molecules, which is more analogous to thermal analysis results [Zhou, Wang, Wang, & Tang, 2001].
The peak for hydroxyl groups of free ligand at 3200 cm-1 demonstrates the considerable change in spectral value of complexes due to their involvement in chelation with metal ions [Tan et al., 2009]. Consequently, it may be revealed that -OH and C=O groups are the main sites of oxygen that cause the coordination of M(II) ions. This argument is further supported when peaks near 600cm-1 were found for Ï…(M-O) in the far IR region of frequency for respective complexes.
3.4. 1H NMR studies
The 1H NMR data for complexes indicate that hydrogen of 3-OH group was not found due to complexation [Zhou, Wang, Wang, & Tang, 2001]. The data also show that the Î´ values of complexes are shifted to lower field as compared to pure morin due to increase of conjugation caused by the effect of coordination, after the complexes are formed, it increases the planarity of the flavonoid molecules. Upon complexation, the metal ions remove phenolic hydrogen from the morin. The complexes are paramagnetic in nature due to the availability of unpaired electrons, which are localized in the complexes. This information indicates that during complex formation, only one proton of free ligand is deprotonated and ligand behaves in a monobasic bidentate fashion.
The data further indicates that all the ring protons are shifted to lower field in the case of complexes. They also contain the 5-OH, 7-OH, 2'-OH and 4'-OH group protons, which are also shifted to lower field relative to the free morin but among them the shift of 5-OH is biggest. It is because the 5-OH group is close to the oxygen of 3-OH group in space; therefore, it forms a weak hydrogen bond with it. This is also in agreement with the considerable bathochromic shifts observed in UV-Vis spectra in band I of the complexes. The 1H NMR data also allowed some conclusions about the chelation sites, i.e. morin is able to sequester metal ions via 3 phenolic and 4 carbonyl groups; therefore, upon complexation, the metal ions displace hydrogen from the morin [De Souza, & De Giovani, 2005].
3.5. 13C NMR studies
13C NMR spectra were performed in DMSO-d6 solvent at room temperature, for morin and M(II)-morin complexes. The assignments of morin carbons are also available in the literature data. The main feature of the spectra of complexes is that, once the morin undergoes the complex formation with metal ions, the resonance of each carbon atom is changed in it due to structural modification. In the Fe(II), Co(II) and Ni(II) complexes, C(3) and C(4) impart major differences relative to the morin by undergoing significant downfield shift in the orders of 10.46, 5.04 and 6.5 ppm for C(3), and 1.96, 1.39, and 2.16 ppm for C(4), respectively. This is for the reason that C(3) and C(4) carbons may experience the electronic deficiency. Which is because of the decrease of bond orders for the C(4)=O and C(2)=C(3) after complexation [Kopacz, & WoÅºnicka, 2004]. Hence, both the C(3) and C(4) are the major sites in morin to be involved in complexation with metal ions.
3.6. Thermal analysis
All the three complexes show nearly same type of thermal behavior. The complexes display two main steps of weight loss in their TG-DTA curves. Step one quantitatively specifies the weight loss for water molecules (dehydration); another step is related to the decomposition, which corresponds to weight loss around 89% [De Souza, & De Giovani, 2005; Qi, Liufang, & Xiang, 1996]. From the results (Table 2B) it has been deduced that the Fe-morin complex undergoes the thermal degradation in three steps within 40-550Â°C. In the first step, dehydration occurs at 100-200Â°C due to decomposition of water molecules present outside the coordination sphere. Second step dehydration is also because of water molecules but those inside the coordination sphere, which is actually difficult task to differentiate between two types of water molecules directly from graphic data. This temperature range corresponding to the dissociation of two coordinated water molecules is at 250-300Â°C. Final step of decomposition presents the indication of break down of ring in the ligand. In the Co-morin complex, the decomposition of ligand molecules occurs at the temperature range of 320-450Â°C. The complex also undergoes the dissociation at the temperatures of 100-160 Â°C and 260-320Â°C to evidence the hydrated and coordinated water molecules. The Ni-morin complex follows the same way of degradation as Fe-morin. It undergoes the decomposition at 270Â°C illustrating the decomposition for the two inner sphere coordinated water molecules [Tan et al., 2009].
Based on the above studies, we have proposed a tentative coordination structure for the complexes (Fig. 4a).
3.7. DPPHâ€¢ free radical scavenging assay
The antioxidant activities of all the three complexes as well as morin were determined by DPPHâ€¢ assay. Both the morin and complexes scavenge the DPPHâ€¢ radical effectively. The results of DPPHâ€¢ radical scavenging analysis showed that complexes exhibited stronger antioxidant activities than morin; in addition, cobalt and nickel complexes had high antioxidant potential and can be used as a useful antioxidant source conversely, the iron complex showed weak activity than the former complexes. DPPHâ€¢ scavenging activity of the investigated complexes do not differ from each other significantly. But it is higher in the case of complexes relative to the morin, indicating that the complexes are much stronger free radical scavengers and antioxidants than morin following the order below.
Co(II) complex > Ni(II) complex > Fe(II) complex > morin
3.8. Electrochemical properties of the compounds
Morin and its complexes with Fe(II) and Co(II) show distinct oxidation peaks and almost no reverse reduction peaks. It points out that the EC processes involve two electrons, which is well illustrated in the cyclic voltammograms (Fig. 4b). Moreover, in the case of Ni(II) complex two oxidation peaks are observed with no corresponding reduction peaks. This shows that EC processes are two irreversible oxidations with one electron transition [Zhou, Wang, Wang, & Tang, 2001].
Morin itself represents only one oxidation process at + 668 mV that is characteristic to the 3-OH group oxidation [De Souza, & De Giovani, 2005]. In the case of metal complexes (Table 3A), the oxidation peak potential for Co complex appears at + 653 mV but Ni complex shows two peak potentials one at + 648 mV and another at 880 mV, which are fairly low as compared to the free morin + 668 mV. Fe complex shows an exceptional case for which the peak potential is + 1000 mV that may be due to change in interaction which is specific to the nature of metal with respect to the ligand. When the coordination of metal ions takes place with ligand it facilitates more the oxidation process because of the decline of flavonoid structure.
Hydroxyl groups and the conjugation of flavonoid both are equally attributable to the Epa values; hence the extended conjugation causes the decrease in peak potentials for complexes. Complexation shows the negative shift of peak potentials due to stability of intermediate radical by means of electrostatic interaction with metal center [PÄ™kal, Biesaga, & Pyrzynska, 2010].
Redox potential values also evaluate the antioxidant activity of the title complexes, lower the potential, higher the antioxidant activity. Thus, the Co(II) and Ni(II) complexes of morin are essentially more antioxidants than the morin alone [De Souza, & De Giovani, 2004] that is also supported by DPPH free radical scavenging assay.
3.9. Antibacterial studies
Under identical conditions, study of all the metal complexes, in vitro, exhibit moderately appreciable antibacterial activity (Table 3B) against M. flavus and S. aureus. This enhanced antibacterial activity can be explained on the basis of Overtone concept and chelation theory. The complexes inhibited the bacterial growth considerably [Agarwal, & Prasad, 2005].
Morin was found active against s. aureus but not effective against m. flavus. In the case of complexes, Co(II) showed higher activity against m. flavus than Fe(II) and Ni(II) complexes. Conversely, the Fe(II) complex was more promising against s. aureus than the other two complexes. Moreover, all the three complexes are relatively more antibacterial agents than morin against aforementioned species of bacteria.
It has been concluded from the above study that the complexes undergo 1:2 stoichiometry, which is proved from the Job's method as well as characterization of title complexes. This study also made it possible to establish the appropriate chelating site of morin with respect to Fe(II), Co(II) and Ni(II) metal ions. Moreover, due to solubility reasons, this study was carried out in methanol.
UV-Vis spectra show that the complex formation takes place via 3-OH group displaying remarkable bathochromic shifts in this ring portion, it is also supported by 1H NMR and 13C NMR, which show the displacement 3-OH proton and significant differences of C(3) and C(4) signals relative to morin, respectively. IR spectra also illustrate the bonding of metal ions through oxygen atom of the carbonyl group. Thermal analysis confirms the presence of coordinated as well as hydrated water molecules that is also supported by IR. The complexes show higher antioxidant activity relative to the morin that is also aided by cyclic voltammetry. The biological study shows that the complexes demonstrate appreciable activity as compared to the morin.