Synthesis, Spectroscopic and Biological Investigation of a New Ca(II) Complex of Meloxicam as Potential COX-2 Inhibitor

Drug development on basis of coordination compounds provides versatile structural and functional properties as compared to other organic compounds. In the present study, a new Ca(II) complex of meloxicam was synthesized and characterized by elemental analysis, FT-IR, UV–Vis, 13C NMR, SEM–EDX, powder XRD and thermal analysis (TGA). The Ca(II) complex was investigated for its in vitro, in vivo biological activities and in silico docking analysis against COX-1 and COX-2. The spectral analysis indicates that the meloxicam acts as a deprotonated bidentate ligand (coordinated to the metal atom through the amide oxygen and the nitrogen atom of the thiazolyl ring) in the complex. SEM–EDX and powder XRD analysis depicted crystalline morphology of Ca(II) complex with a crystalline size of 32.86 nm. The in vitro biological activities were evaluated by five different antioxidant methods and COX inhibition assay, while in vivo activities were evaluated by carrageenan-, histamine- and PGE2-induced paw edema methods and acetic acid-induced writhing test. The Ca(II) complex showed prominent antioxidant activities and was found to be more selective toward COX-2 (43.77) than COX-1 as compared to meloxicam. It exhibited lower toxicity (LD50 1000 mg/Kg) and significantly inhibited carrageenan- and PGE2-induced inflammation at 10 mg/Kg (P < 0.05), but no significant effect was observed on histamine-induced inflammation. Moreover, Ca(II) complex significantly reduced the number of writhes induced by acetic acid (P < 0.05). The in silico molecular docking data revealed that Ca(II) complex obstructed COX-2 (dock score 6438) more effectively than COX-1 (dock score 5732) as compared to meloxicam alone.


Introduction
In the recent times, the inflammatory diseases such as autoimmune and infectious diseases have exponentially escalated. The management of never-endingly transmitted infectious diseases and various microbial and viral infections like multisystem inflammatory syndrome and Coronavirus Disease 2019 (COVID-19) requires effective anti-inflammatory and analgesic drugs to cure the post-infection inflammatory events [1,2]. Likewise, the severe and prevalent autoimmune diseases such as allergic asthma and rheumatoid arthritis manifest chronic and acute inflammation characterized by pain, edema and redness and are usually treated by non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids and disease-modifying antirheumatic drugs (DMARDs) [3][4][5]. The NSAIDs are widely prescribed and also used over the counter for alleviating the conditions of the different diseases by inhibiting the prostaglandin endoperoxide synthase enzyme also called cyclooxygenase (COX) [6]. Among the isoforms of enzyme, designated as COX-1 and COX-2, COX-1 plays housekeeping actions and is responsible for maintaining the protective lining of the gastrointestinal tract (GIT), while COX-2 is responsible for the synthesis and migration of pro-inflammatory cytokines to the site of inflammation [7,8].
The NSAIDs are designed to suppress inflammation by obstructing the enzyme COX-2 through receptor analogy, thus preventing the synthesis and accumulation of proinflammatory cytokines and macrophages at the site of inflammation [9]. Free radicals triggered by neutrophils and macrophages play a key role in inflammatory reactions as they enhance the inflammatory response by increasing the production of cytokines and chemokines (in the positive feedback mechanism) and also damage local cells through oxidation and nitration [10,11]. Reactive oxygen species (ROS) production typically increased during inflammatory process, and it was shown that some of the NSAIDs have potential to interact with reactive species and therefore preventing oxidative damage [12].
Meloxicam (4-hydroxy-2-methyl-N-(5-methyl-1,3-thiazol-2-yl)-1,1-dioxo-1λ 6 ,2-benzothiazine-3carboxamide, C 14 H 13 N 3 O 4 S 2 , Fig. 1) is NSAID of oxicam family, often used in inflammatory diseases such as rheumatoid arthritis or osteoarthritis [13]. It is designed to selectively inhibit the COX-2; however, the presence of acidic groups in its structure still hinders the proper management of inflammation [14]. Other than its anti-inflammatory and analgesic properties, meloxicam has been reported to reduce the formation of singlet oxygen and other reactive oxidants [15]. Given that inflammation has a complex pathogenesis, complexation of meloxicam with suitable metal ion may increase its anti-radical effectiveness and efficiency of anti-inflammatory treatment. Till date numerous metal complexes of meloxicam have been synthesized to alleviate the inflammation as well as oxidative stress without causing harm to the body. Metal ions perform remarkable roles in biological regulations [16], and the existence of the metallic ions can also affect the bioavailability of drugs [17]. Metal complexes have a greater impact on the target tissues as compared to the sole drugs due to their synergic action [18]. However, most of the reported metal complexes have greater toxicity plausibly due to the poisonous nature of the metals used [19]. Therefore, bio-metals are considered more desirable for preparing such drugs as these metals would not pose toxicity in the body [20].
The present work was designed to synthesize and characterize a new calcium complex of meloxicam through different spectroscopic techniques. The Ca(II) complex was assessed for in vitro antioxidant and COX inhibition assays, in vivo anti-inflammatory activities through carrageenan-, histamine-and prostaglandin E2-induced edema, while analgesic efficacy was evaluated by acetic acid-induced writhings. Inhibitory effect on COX-2 was investigated by in silico molecular docking for visualizing the in vivo anti-inflammatory and analgesic efficiency of new Ca(II) complex of meloxicam.

Synthesis of Ca(II) Complex of Meloxicam
The new calcium metal complex of meloxicam was synthesized by following previously reported protocol with few modifications [21]. Briefly, 0.5 mM ethanolic solution of anhydrous CaCl 2 was treated with 1 mM ethanolic solution of meloxicam at pH 7. The mixture was stirred overnight and was then placed in water bath at 60°C until precipitates were condensed. The precipitates were filtered, washed with hot ethanol and then dried at ambient temperature. The overnight stirring was done to obtain good yield of calcium metal complex which is difficult to achieve due to inert nature of calcium metal as compared to reactive metals we reported in our previous work [22].

Characterization
The UV-visible spectroscopy was performed on T90 + UV/VIS spectrometer PG instrument within the range of 200-450 nm using DMSO as blank. Melting and decomposition temperatures were measured on Gallen Kamp melting point apparatus. FTIR of the dry sample was performed using PerkinElmer Spectrum-100 (KBr crystal). Elemental analysis was performed using a Euro EA elemental analyzer, and 13 C NMR spectra were recorded by Bruker Avance 400. Metal contents were obtained through atomic absorption spectrometry and flame photometry performed by PerkinElmer AA Analyst-100 and Sherwood 410 flame photometer, respectively. Field emission scanning electron microscopy with energy-dispersive spectroscopy analysis (FESEM-EDS) of the sample was done by Nova Nano SEM 450 field emission scanning electron microscope (FESEM). Powder X-ray diffraction (XRD) was carried out with Cu K α between 2θ° 5-50°with a step size of 0.04°using Bruker D8 Discover XRD system. The thermogravimetric analysis (TGA) was performed by SDT Q600 thermogravimetric analyzer to determine the thermal stability of the metal complex. The sample (0.05 g) was subjected to temperature rise from 25 to 1000°C in the presence of N 2 environment, and the rate of temperature rise was kept 10°C per minute. Temperature T max (°C) at each step of degradation was recorded. Coats Redfern equation was used to determine the energy of activation based on the results of TGA [23].

In Vitro Cyclooxygenase Inhibition Assay
The COX inhibitor Screening Assay Kit (Catalog No. 560131, Cayman Chemical, Ann Arbor, MI, USA) was used to measure the cyclooxygenase inhibitory potential of synthesized Ca(II) meloxicam complex. The ovine COX-1 and human recombinant COX-2 activity directly measure PGF 2α produced by SnCl 2 reduction of COX-derived PGH 2 . The prostanoid product is quantified via enzyme immunoassay (EIA) using a broadly specific antibody that binds to all the major prostaglandin compounds. Briefly, control value was obtained in the absence of compound. The COX enzyme was mixed with different concentration of tested compound and heme and incubated for 10 min at 37°C. The reaction was initiated by adding arachidonic acid, and all tubes were incubated for another 2 min at 37°C. The efficacy of compound was determined as the concentration causing 50% enzyme inhibition (IC 50 ). The selectivity index (SI values) was calculated as IC 50 (COX-1)/IC 50 (COX-2) [25,26].

In Vivo Assays
Animals All in vivo experimentation of the synthesized Ca(II) complex of meloxicam was performed using Sprague-Dawley (SD) rats, 10-12 weeks old (150-200 g), maintained at controlled temperature (25 ± 5°C) and humidity (50 ± 10%) in the institutional animal house. The animals were exposed to 12 h light and dark cycle and had free access to autoclaved tap water and pathogen-free feed for 24 h. International ethical guidelines were followed for the care of laboratory animals to provide them with a healthy and clean environment. Experiments were approved by Institutional Ethical Committee, University of the Punjab, Lahore (Approval No. D/025/2018, March 07, 2018).

Acute Toxicity of Ca(II) Complex of Meloxicam
Acute toxicity of the Ca(II) complex was tested, and safe dose was calculated as per organization for economic cooperation and development (OECD) test guideline 425 [27]. Rats were randomly divided into eight groups of either sex (n 5) and were treated with 5, 25, 50, 100, 250, 500, 1000 and 2000 mg/Kg dose of Ca(II) complex orally. The control group received CMC (0.5%) at a dose 10 mL/kg. All the animals were observed randomly for any signs of toxicity during first 4 h, and then, the numbers of dead animals were counted after 24 h. LD 50 was calculated by the previously used method [28,29].
In Vivo Anti-Inflammatory Activity Carrageenan-induced paw edema method was followed to evaluate the antiinflammatory effect of meloxicam and its Ca(II) complex [30]. The rats were randomly divided into five groups (n 6): the Carrageenan group, low (5 mg/Kg), medium (10 mg/Kg) and high (20 mg/Kg) dose Ca(II) complex groups and standard meloxicam (10 mg/Kg) group [31]. Rats in the carrageenan control group received oral gavage of 0.5% CMC and served as control group, while rats in standard meloxicam and low, medium and high dose Ca(II) complex groups received respective compound dosage. All of the groups received 1% carrageenan solution in sub-plantar region of right paw. Thickness of rat paws measured by water displacement after 5 min (t o 0) exhibited the initial paw volume. The thickness of paw volume was measured after 1, 2, 3, 4 and 5 h.

Histamine-and PGE 2 -Induced Paw Edema
The antiinflammatory mechanism of synthesized Ca(II) complex was evaluated using the histamine-and prostaglandin E2 (PGE 2 )-induced paw edema assays. The SD rats were randomly divided into five groups (n 6): the histamine or PGE 2 group, low (5 mg/Kg), medium (10 mg/Kg) and high (20 mg/Kg) dose Ca(II) complex groups and standard meloxicam (10 mg/Kg) group [31]. Rats in the histamine or PGE 2 group received 1 mL oral gavage of 0.5% CMC and served as inflammatory control group, while rats in standard meloxicam and low, medium and high dose Ca(II) complex groups received respective compound dosage. After 1 h, paw edema was induced by sub-planter injection of 0.1 mL of histamine (1 mg/mL) or prostaglandin E2 (0.01 μg/mL). Paw volume of each rat was immediately measured before and after the sub-planter administration of inflammatory agents at 1, 2, 3 and 4 h [32].
Acetic Acid-Induced Analgesic Potential Acetic acidinduced writhing test was performed to evaluate the analgesic potential of meloxicam and its Ca(II) complex. The rats were randomly divided into three groups (n 6): acetic acid control group (0.5% CMC), Ca(II) complex (10 mg/Kg)and meloxicam (10 mg/Kg)-treated groups. Rats in both the meloxicam and complex group (10 mg/Kg body weight) were administered orally as a suspension in 0.5% CMC, to 16 h fasten rats. Later, upon 1 h of treatment, 0.6% acetic acid (10 mL/Kg) was injected intraperitoneally to induce the characteristic writhing in the rats. The number of writhings occurring between 5 and 25 min after the acetic acid injection in control and treated animals was recorded. The anti-nociceptive activity of drugs was calculated by the following formula.
Percentage inhibition of writhing ((mean writhing of control -mean writhing of test /mean writhing of control) × 100

In-Silico Molecular Docking Study
The in silico molecular docking was studied by preparing and optimizing the three-dimensional structure of the drugs meloxicam, diclofenac sodium and calcium complex in software Avogadro, respectively [33]. The protein receptors COX-1 and COX-2 were downloaded from RCSB PDB (ID: 1CQE and 6COX, respectively). The binding pockets of COX-2 and amino acids lining the cavity were identified using software Deepsite-PlayMolecule [34]. The receptor and ligand files were subjected to Patchdock Beta 1.3 software for docking [35,36]. The docking scores, approximate interface protein-ligand complex area and values of atomic contact energy (ACE) (kJ/mol) were recorded for the proposed inhibitor ligands. The docking mode of enzyme inhibition was selected, and the root-mean-square deviations of atomic positions were 1.5 [37]. The results were analyzed by using UCSF Chimera 1.14 [38].

Statistical Analysis
Statistical analyses were performed using one-way ANOVA with Tukey test by GraphPad prism (version 7.03). Data for in vivo study were presented as mean ± SEM values. P value < 0.05 was accepted as statically significant.

Synthesis of Ca(II) Complex of Meloxicam
The synthesis of calcium complex of meloxicam was accomplished by mixing ethanolic solutions of meloxicam and CaCl 2 at pH 7 (Fig. 2). The inert nature of calcium required overnight stirring of the mixture; afterward, the pale white precipitates were obtained after heating the mixture at 60°C. The UV-visible absorbance peaks shifted to 375 and 280 nm for Ca(II) complex from 365 and 275 nm of the ligand, respectively. This shifting showed the involvement of meloxicam in complexation with calcium (Fig. 3a). The UV-Vis spectra of the complex did not change on keeping the DMSO solutions for 48 h, and no precipitation, turbidity or decomposition were observed even after long storage at room temperature (at least 3 months after preparation). This clearly indicates the stability of Ca(II) complex. 13 C NMR spectra in DMSO-D 6 , δ ppm, showed suppression of peaks in C=O and C-OH region (Fig. 3b). The 13 C NMR of meloxicam shows definitive peaks in region > 150 ppm as reported in literature [39]. The diminishment of peaks in 156 and 160 ppm ranges reported for meloxicam in the literature depicts the formation of complex through these groups [40]. The FTIR spectra of meloxicam depicted prominent absorption bands at 3282(sh, s) cm −1 , 1614(sh, s) cm −1 , 1550 (s) cm −1 , 1342(s) cm −1 and 1182(s) cm −1 which can be attributed to the stretching vibrations of ν(N-H) amide , ν(C=O) amide , Ѵ(C=N) thiazolyl ring , ν(SO 2 ) asym and ν(SO 2 ) sym , respectively (Fig. 3c). The FTIR results of synthesized Ca(II) complex showed no absorption peaks in the region of 3200-3300 cm −1 relating to N-H stretching mode because the N-H group of meloxicam is involved in a strong intramolecular hydrogen bond to enolate oxygen. The bands of stretching vibrations for the ν(C=O) amide and Ѵ(C=N) thiazolyl ring are shifted to lower wavenumber in the spectra of the Ca(II) complex which indicates the coordination of H 2 mel through these two groups [21,41]. In comparison with the meloxicam spectrum, the two stretching bands of the SO 2 group (ν as and ν s ) also shift slightly to lower frequencies (Fig. 3d). Flame emission spectroscopy results showed complete absence of sodium metal, while the atomic absorption spectroscopy showed the incorporation of calcium in our synthesized Ca(II) Complex.

FESEM and EDS Analysis
FESEM was employed to study surface and structural morphology of synthesized Ca(II) complex of meloxicam. The SEM images of Ca(II) complex revealed perfectly crystalline morphology (Fig. 4a) with rectangular-shaped crystals, while uncoordinated meloxicam has rock-like morphology, with irregularly shaped crystals of various sizes [42]. SEM analysis depicted significant changes in the shape and surface morphology of Ca(II) complex which occurred due to the complexation, and it might improve the properties of meloxicam. The cavities in the crystal system could be used for the electrostatic drug loading [22]. The EDS is used to calculate the percentage level of the elements present in the metal complexes like C, O, N, S and respective metal present in the complex [43]. This result confirms the presence of calcium along with other elements in the synthesized complex (Fig. 4b). The revealed data are in good agreement with that of the elemental analysis.

Powder XRD Analysis
The crystallinity of Ca(II) complex of meloxicam was evaluated by powder XRD measurement. The X-ray diffractogram of Ca(II) complex exhibited several sharp peaks at different angles (2θ ) 8.04, 9.08, 12.56, 15.04, 16.72, 17.08, 18.28, 19.52, 20.4, 21.96 and 28.4°suggested that the synthesized Ca(II) complex existed as crystalline material (Fig. 5), while the X-ray diffractogram of pure meloxicam exhibited sharp peaks at different angles (2θ ) 13.0, 15.0, 18.5 and 26.0° [44]. It is observed from X-ray diffractogram that complexation of meloxicam with calcium metal enhanced its crystallinity. The average crystalline sizes of the Ca(II) complex (d XRD ) were calculated using Debye-Scherrer equation (D Kλ/β Cos θ ), where D particle size, K dimensionless shape factor, λ X-ray wavelength (0.15406 Å), β full width at half maximum (FWHM) of the diffraction peak, and θ diffraction angle [45]. The synthesized Ca(II) complex has a crystalline size of 32.86 nm suggesting that the complex is in a nanocrystalline phase.

Thermogravimetric Analysis
Thermogravimetric analyses (TGAs) are used to get information about the thermal stability of new complexes and suggested a general scheme for their thermal decomposition [46]. The TGA results depicted the degradation of Ca(II) complex in three steps. The mass (7.34%) loss for water molecules was found in first step of degradation at T max 190°C. In the second step of degradation, 48.91% mass was lost due to ethene and nitrous oxides of the organic ligand at T max 261, 288°C. The third step of degradation was recorded at T max 566°C owing to the decomposition of second ligand moiety, and CaCO 3 was the final decomposition product with some residual carbon about 0.024 g (Fig. 6). The activation energy (E α 323.84 kJ/mol) value for Ca(II) complex was calculated by Coats Redfern equation, which shows a high thermal stability of the complex [23].

Antioxidant Assays
The free radical scavenging, metal chelating and reducing abilities of meloxicam and its synthesized Ca(II) complex determined by using five different in vitro assays as more than one method are used to evaluate the antioxidant capacity/activity of a desired sample [47]. The radical scavenging, metal chelating and reducing capabilities of compounds were summarized in the form of IC 50 values (Table 1). DPPH and ABTS radical scavenging activity has been widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors and thus to evaluate the antioxidant activity [48]. The ABTS assay is better than DPPH as ABTS is soluble in water and organic solvents, and it reacts relatively rapidly with the tested compounds compared to DPPH [49]. The tested compounds interact with the stable free radical DPPH which shows their radical scavenging ability. This interaction was found to be concentration and time dependent, but the IC 50 values of meloxicam and its Ca(II) complex were higher; > 2000 μM, while in ABTS radical scavenging assay, Ca(II) complex showed prominent radical scavenging potential in a concentration-dependent manner as compared to meloxicam and standard ascorbic acid. The IC 50 values for ABTS radical scavenging of Ca(II) complex, meloxicam and standard ascorbic acid showed decreasing trend with respect to time, and lowest IC 50 values were recorded at 120 min: 44.8, 82.22 and 104.12 μM, respectively. Meloxicam itself has anti-radical properties and has been reported to reduce the formation of reactive oxidants [50], but complexation with calcium metal enhanced its ABTS radical scavenging properties.
Iron chelating activity is based on the absorbance measurement of iron (II)-phenanthroline complex. This complex produced a red chromophore with a maximum absorbance at 512 nm [51]. The synthesized compounds act as chelating agents and capture the ferrous ion (Fe 2+ ) before phenanthroline. This method is used to determine the extent of Fe 2+ + chelation by meloxicam and its Ca(II) complex. It was found that IC 50 values of meloxicam were much lower as compared to its Ca(II) complex showing its greater Fe 2+ chelating abil- ity. Higher IC 50 value (> 2000 μM at 120 min) of Ca(II) complex corresponds to its lower chelating ability, which might be due to complexation as meloxicam is already in complexed form with calcium. It might be possible that the moieties of meloxicam responsible for chelating Fe 2+ are now involved in complexation with calcium metal and therefore exhibited lower iron chelating activity.
In FeCl 3 reducing power assay, meloxicam and its Ca(II) complex reduced potassium ferricyanide (Fe 3+ ) to potassium ferrocyanide (Fe 2+ ) which then reacted with ferric chloride to form ferric ferrous complex [52]. In this assay, IC 50 of Ca(II) complex was found to be lower than un-coordinated meloxicam at all time intervals. Lower IC 50 values indicate a higher antioxidant power of Ca(II) complex to reduce Fe 3+ to Fe 2+ . So, it is observed that complexation of meloxicam with cal-cium enhanced its iron reducing capabilities but lowers iron chelating capacity. Similarly, phospho-molybdenum (PM) activity is based on the reduction of Mo (VI) to Mo (V) by the test compounds, giving a direct approximation of reducing capacity of the compounds [53]. Similar results were observed in PM activity, and IC 50 of Ca(II) complex was found to be lower than meloxicam and ascorbic acid, indicating higher reducing activity of meloxicam in complex form. From the overall results, it is concluded that the Ca(II) complex of meloxicam exhibited potent radical scavenging and reducing abilities in ABTS, FeCl 3 reducing power and phospho-molybdenum antioxidant assays than uncoordinated meloxicam. The Ca(II) complex of meloxicam did not show promising iron chelating ability which is the indication of complexation between meloxicam and calcium.

In Vitro Cyclooxygenase Inhibitor Assay
The COX-1/2 inhibitory activities of the synthesized Ca(II) complex were evaluated and compared with free meloxicam using the enzyme immunoassay (EIA) method against ovine COX-1 and human recombinant COX-2. The half maximal inhibitory concentration IC 50 values calculated from experimental data are shown in Table 2. The selectivity index was calculated as the ratio IC 50 COX-1/IC 50 COX-2. The results obtained for the standard NSAIDs meloxicam are similar to those described by the previous studies [26,54].
The tested Ca(II) complex showed half maximal inhibitory concentration for COX-1 by concentrations of 211.95 μM, while for COX-2 half inhibitory concentration is achieved by concentrations as low as 4.84 μM. The Ca(II) complex and meloxicam both have a potent COX-2 inhibitory effect with IC 50 < 30 μM. When considering the selectivity index as well as the inhibitory potency, the Ca(II) complex was proved to be more selective toward COX-2, as compared to meloxicam. According to Warner et al., selectivity indices ranging from 5 to 50 are associated with compounds that markedly inhibit more the COX-2 isoform than COX-1 and represent the ideal range for the development of safe and selective COX-2 inhibitors [55]. This Ca(II) complex can be considered as a selective COX-2 inhibitor as it showed 43.77 selectivity index (SI) toward COX-2, while meloxicam with 10.39 selectivity is considered as preferential COX-2 inhibitor [56].

Acute Toxicity of Ca(II) Complex of Meloxicam
Before the in vivo evaluation, we evaluated the toxicity of Ca(II) complex to demonstrate safety and their safe dose [27]. The results suggested that the oral LD 50 of Ca(II) complex is 1000 mg/Kg (Table 3, rats 10). The toxicity of Ca(II) complex appeared to be much lower than the toxicity of the reference drug meloxicam. The LD 50 threshold for oral meloxicam is 470 mg/Kg [57,58]. According to OECD guidelines for acute oral toxicity, an LD 50 dose of > 300-2000   is categorized as category 4 and hence the drug is found to be safe. The extended safety index of Ca (II) complex of meloxicam is probably related to the complexation of Calcium with meloxicam through amide oxygen and thiazolyl ring nitrogen.

In Vivo Anti-Inflammatory Activity
To evaluate the in vivo anti-inflammatory potential of synthesized Ca(II) complex at different doses (5, 10, 20 mg/Kg), the carrageenan-induced paw edema method was employed (Fig. 7). It was found that meloxicam and its Ca (II) complex showed a significant anti-inflammatory effect by reducing the paw volume after 2 h of activity, while the paw volume of carrageenan group rats was at peak after 3 h. The Ca (II) complex at 10 mg/Kg showed significant reduction in paw volume (*P < 0.05) followed by its 20 mg/Kg dosage and standard meloxicam, while Ca (II) complex at 5 mg/Kg exhibited the least response. The development of edema induced by carrageenan is biphasic: The first phase occurs within 1 h of carrageenan inflammation and is attributed to the release of the neurotransmitter molecules histamine and serotonin. The second phase (over 1 h) is mediated by an increased release of prostaglandins in the inflammatory area, and the continuity between the two phases is provided by kinins [59]. After 3 h of activity, carrageenan group rats exhibited maximum swelling and redness in inflamed paw as compared to other treated groups which was scored from 0-4 on the basis of severity (Fig. 8). In scoring, 0 was assigned to normal; 1 represented minimal inflammation and edema of injected paw; 2 was denoted to mild paw inflammation and edema; 3 was allocated to moderate inflammation and edema; and 4 was assigned to severe inflammation and redness of paw.

Histamine-Induced Paw Edema
Histamine-induced inflammation has been well established as a valid model to study paw edema because histamine evokes the release of neuropeptides and prostaglandins from endothelial cells leading to hyperalgesia [60,61]. Rats of histamine-induced paw edema group showed rise in paw volumes after 1, 2 and 3 h of sub-plantar injection of histamine  (Fig. 9a). Rats pretreated with oral Ca(II) complex and meloxicam showed slight decrease in paw volumes after 2 h. There was no significant difference between paw volumes among the treated groups, but Ca(II) complex at 10 mg/Kg showed prominent response at 2, 3 and 4 h (Fig. 9a). After 2 h of activity, histamine group rats exhibited maximum swelling and redness in the inflamed paw as compared to other treated groups (Fig. 9b). Inflammation likely consists of three stages; an increase in vascular permeability; leukocyte migration; and proliferation of connective tissue. Swelling is the first stage in the inflammatory process. Although the second stage of inflammation is not dependent on the first stage, the therapeutic effects of agents acting on the second stage may be influenced by the first stage [61].

PGE 2 -Induced Paw Edema
PGE 2 is a very important mediator of all types of inflammation and is responsible for increased prostaglandin production in inflamed tissue [62]. Rats of PGE 2 -induced paw edema group showed rise in paw volumes after 1 and 2 h of sub-plantar injection of PGE 2 compared with the paw volume of rats of the untreated control group (Fig. 10a). Rats pretreated with oral Ca(II) complex at 10 mg/Kg showed significant decrease in paw volumes after 1 h of activity (P < 0.05). All the treated group showed decrease in paw volume, but Ca(II) complex at 10 mg/Kg showed significant response at 1, 2, 3 and 4 h (Fig. 10a). After 2 h of activity, PGE 2 group rats exhibited maximum swelling and redness in the inflamed paw as compared to other treated groups (Fig. 10b). Our results indicated that Ca(II) complex was more potently inhibited PGE 2 -induced paw edema than meloxicam at similar doses (10 mg/Kg). In the prostaglandin (PGE 2 ) biosynthesis pathway, (COX-2) is the key enzyme that catalyzes the conversion of arachidonic acid to PGE 2 [63] and this finding was confirmed by our observation in the in vitro inhibition of COX-2 by Ca(II) complex. These results indicate that the inhibitory effect of Ca(II) complex on carrageenan edema is probably due to PGE 2 reduction  since its effect on carrageenan edema was more pronounced than that one produced by histamine.

Acetic Acid-Induced Analgesic Potential
Acetic acid-induced writhing test was used to evaluate the analgesic potential of meloxicam and its Ca(II) complex at 10 mg/Kg. Acetic acid-induced writhing test is a model of peripheral pain that is useful for anti-nociceptive drug development [64]. The Ca(II) complex showed more significant response (*P < 0.05) and reduced the number of characteristic writhing in rats as compared to meloxicam (Fig. 11). Consequently, highest percentage (85.26%) inhibition of writhing response was produced by Ca(II) complex at 10 mg/Kg, while meloxicam showed 67.89% inhibition (Table 4). NSAIDs act by the reduction of sensitization of pain receptors caused by prostaglandins at the inflammation site [65].

In Silico Molecular Docking Study
The molecular docking study was performed in order to investigate the binding interaction of Ca(II) complex at the binding/active site of COX-1 and COX-2 protein (PDB code: 1CQE and 6COX, respectively) [66,67]. The software Deepsite-PlayMolecule helped to assess the binding sites in the three-dimensional structure of enzymes (Fig. 12). The molecular docking was performed according to the given literature for binding pocket of COX enzymes containing THR 118 , ARG 120 , GLN 192 , VAL 349 , TYR 355 , GLU 364 , PHE 518 , VAL 523 (ILE 523 in COX-1), ALA 527 , MET 535 . Structural differences among the binding sites of COX-1 and COX-2 provided valuable guidelines for the design of selective COX-2 inhibitors [68,69]. The main difference consists in the existence of a second pocket inside of COX binding site, which is more accessible in COX-2 because of the replacement of ILE 523 in COX-1 with a smaller side chain residue VAL 523 , linked with conformational changes at TYR 355 , which opens the hydrophobic chain of the additional pocket including LEU 352 , SER 353 , TYR 355 , PHE 518 and VAL 523 [70]. Meloxicam showed close interaction with VAL 116 , LEU 359 , LEU 352 , SER 353 , GLU 524 , TRP 387 , LEU 384 , TYR 385 , GLY 526 , ALA 527 , respectively. The two residues LEU 352 and SER 353 of hydrophobic chain showed interactions with meloxicam and thus account for the selectivity of meloxicam for COX-2. The docking score, approximate area of interface and ACE of meloxicam were found as 5150, 643.2 and − 281.06 kJ/mol for COX-2 and 4350, 565.1 and − 280.03 kJ/mol for COX-1, respectively. Diclofenac sodium showed the parameters as 5054, 621.10 and − 121.24 kJ/mol for COX-2 while 4336, 474.7 and − 113.5 kJ/mol for COX-1, respectively. The Ca(II) complex showed close hydrophobic interaction with LEU 80, LEU 81 , LEU 82 , LYS 83 , VAL 89 , ARG 120 , TRY 122 , SER 471 , LEU 472 , LYS 473 , SER 119 , VAL 523 and GLU 524 with docking score, approximate area of interface and ACE as 6438, 857.6 and − 289.87 kJ/mol for COX-2 ( Fig. 13a and b) while 5732, 767.2 and − 193.77 kJ/mol for COX-1, respectively ( Fig. 13c and  d). The highly negative ACE values depict greater potential of formation of the enzyme-inhibitor complex due to exothermic energy change. The Ca(II) complex fitted well into COX-2 binding site occupying a similar but greater region in the binding site as meloxicam. This pose might benefit from additional interaction energy due to the relative proximity of the VAL 116 , which can generate an additive effect determining the selectivity for COX-2 [71].
The docking of Ca(II) complex revealed intricate interactions with COX-2 channel, including hydrogen bonds and hydrophobic interactions having highest dock/binding score compared to meloxicam. Furthermore, it was observed previously that the compounds having higher selectivity index (SI) for COX-2 than COX-1 in the in vitro experiments also showed higher binding interactions (dock score) with COX-2 than COX-1 in molecular docking studies [26,32,[72][73][74][75]. On this basis, the compounds with higher docking score are recognized as selective COX-2 inhibitors.

Conclusion
A new Ca(II) complex of meloxicam was synthesized and investigated by various spectroscopic and biological techniques. The spectral studies envisaged that the meloxicam ligand is bidentate in nature, which coordinate with the Ca(II) metal ion through oxygen of amide group and nitrogen of thiazolyl ring. The correlation of the experimental data allows in assigning an octahedral geometry for the Ca(II) complex. SEM and XRD analysis showed crystalline morphology of complex and is in good agreement with each other. TGA data revealed that the complex decomposes into three steps resulting in a metal carbonate as final decomposition product. The new Ca(II) complex showed prominent in vitro antioxidant activities and higher selectivity toward COX-2 than uncoordinated meloxicam. It showed lower toxicity with LD 50 1000 mg/Kg and acts as a potent anti-inflammatory and analgesic agent. It inhibits PGE 2 -induced inflammation more strongly than histamine which justifies the anti-inflammatory and analgesic action of complex. Molecular docking data provide new insights about COX-2 inhibitions by the new Ca(II) complex, having higher binding score as compared to COX-1, and may be considered as a potent COX-2 inhibitor. Moreover, Ca(II) meloxicam complex can be further used to explore its beneficial impacts at molecular level.