Spectroscopic, density functional theory, cytotoxicity and antioxidant activities of sulfasalazine and naproxen drugs combination

2.1 Materials

Sulfasalazine and naproxen drugs were purchased from Sigma-Aldrich at 99% purity. DPPH and BHT (butylated hydroxytoluene) compounds were procured from Sigma-Aldrich Ltd. (Germany). Also, methanol, 99% (CAS Number: 67-56-1), and acetone (CAS Number: 67–64-1), H₂SO₄ (CAS Number: 7664-93-9), NaH₂PO₄•H₂O (CAS Number: 10049–21-5), and (NH₄)₆Mo₇O₂₄ * 4H₂O (CAS Number: 12054-85-2) have been purchased from Merck. Doubly distilled water was used as solvent within the experiments.

2.2 Preparation of Sulfoxen nanocomplex

Sulfoxen nanoparticles were prepared by mixing the SSZ (50 mg in 20 mL methanol) and NAP (50 mg dissolved in 20 mL methanol) in 1:1 wt ratio with constant stirring (Fig. 1). The mixture was stirred at 40 ˚C for 12 h to obtain an orange color solution. Afterwards, the mixture was sonicated at 37 kHz, 80 W and 40 ˚C for 10 min. The mixture was centrifuged at 10000 rpm for 10 min. Finally, the orange color precipitate was filtered and carefully washed with cold methanol and dried at 50 ˚C in the oven for 24 h.

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Fig. 1

Schematic diagram of the interaction between NAP and SSZ. The dashed lines between the COOH groups of both drugs represent the assumed H-bonding by the M06-2XD functional in methanol phase.

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2.3 DPPH radical scavenging activity

A solution of 4.3 mg of DPPH in 3.3 mL of methanol was prepared in a test tube and protected from light by covering with aluminum foil (Tepe and Sokmen, 2007). A 150 μL of the solution was then taken and diluted up to 3 mL with methanol and then the absorbance was recorded at 517 nm by using UV–Visible spectrophotometer. This absorbance was assigned as the control. For the test and standard (std), the different concentrations of drugs were provided. For the assay, 150 μL of DPPH solution was added to 150 μL of the test or std solution and diluted up to 3 mL in methanol, the absorbance of the reaction mixture was determined after 30 min at 517 nm by UV–Vis spectrophotometer (methanol as blank). The percentage (%) scavenging activity was calculated by using the following equation: $$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\%=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$$

2.4 Total antioxidant capacity

The total antioxidant capacity (TAC) is based on the decrement of Mo (VI) to Mo (V) by the compound and the subsequent formation of a green phosphate/Mo (V) complex at acidic pH (Prieto et al., 1999). An aliquot of 0.1 mL of sample solution and 1 mL of reagent solution (H₂SO₄ (0.6 M), NaH₂PO₄•H₂O (28 mM), and (NH₄)₆Mo₇O₂₄*4H₂O (4 mM)) was mixed in methanol and incubated at 95 ˚C for 90 min. After the reaction mixture cooled for 15 min at room temperature, the absorbance was determined at 695 nm. A typical blank solution included 1 mL of reagent solution and the convenient volume of the same solvent was prepared for the compound and it was incubated under the same conditions as the rest of the compounds.

2.5 FRAP assay

The FRAP (ferric reducing antioxidant power) assay of different parts was determined by the method of Yildirim et al. (Yildirim et al., 2001). The dried compound (125–1000 μg) in 1 mL of the corresponding solvent was blended with 2.5 mL of K₃Fe (CN)₆ [potassium ferricyanide; 10 g/L] and 2.5 mL of phosphate buffer (0.2 M), then the reaction mixture was allowed to react at 50 ˚C for 30 min. Then, 2.5 mL of TCA (trichloroacetic acid; 100 g l⁻¹) was added to the mixture and centrifuged at 1650 rpm for 10 min. Finally, 2.5 mL of the supernatant solution was blended with 2.5 mL of distilled water and 0.5 mL of FeCl₃ (1 μg/L) and the absorbance was determined at 700 nm. High absorbance indicates high reducing power.

2.6 Fish preparing

Herein, zebrafish (Danio rerio) was selected as an experimental model. For acute toxicity bioassay 147 healthy, live specimens (∼0.8 gr weight) of both sexes were used in this study. Fish were prepared from a breeding center, transferred to a workshop and adapted to the environment for one week. Fish were maintained in a stable environmental condition (Water temperature: 27.5 ± 0.5; hardness: 430 ± 25; pH: 7.5) during the adaptation. They were fed on a commercial diet twice daily. Feeding was stopped 24 h before experimenting.

2.7 Range finding test

A range finding test was performed to characterize the range of the substance according to OECD 203. The highest concentration of test substance to use in the range-finding test was 12.5 mM and the lowest one was 2 mM. Fish in the test and control chambers were monitored periodically to remove possible mortality.

2.8 UV–Vis analysis

UV–Vis spectra of SSZ, NAP and Sulfoxen samples in methanol were recorded in an (UV–Vis machine name) with a spectral range from 200 nm to 700 nm and spectral resolution of 1 nm. Quartz cuvettes were filled with 3 mL of each sample and a quartz cuvette with 3 mL of methanol was used as blank sample. Each spectrum was recorded in triplicate.

2.9 Physicochemical characterization

FT-IR spectroscopy (JASCO680 plus FT-IR spectrophotometer, Perkin-Elmer) and Raman (SENTERRA, BRUKER, Germany) were utilized for evaluating the combination. The rate of decomposition of free SSZ and NAP and their combined complex were evaluated using TGA (thermal gravimetric analysis) (Q50, Thermal Analysis -TA) with the experimental conditions of scanning from 25 to 700 °C under argon flow at a heating rate of 20˚C/min. Sample was also scanned from 25 to 700 °C at 20 °C/min in The LINSEIS Differential Scanning Calorimeters (DSC) to detect for thermal induced phase transition or decomposition of sample. The crystallinity of the complex was evaluated using XRPD analysis. Particle size of nanocomplex was also analyzed using FE-SEM (DSM-960A, Zeiss Company).

2.10 Cell toxicity assay

Cell toxicity MTT assay was accomplished with a tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) provided by a Bio-IDEA MTT assay Kit. The MCF-7 and KYSE30 cell lines were cultured as a monolayer in a 25-cm² flask containing DMEM enriched with a mixture of 100 U/mL penicillin and 100 µg/mL streptomycin, and 10% FBS. The cells were preserved in a 95% humidity and 5% CO₂ at 37 °C incubator. Then, the cell morphology was determined by an inverted microscope. Ultrasonic dispersion and a vigorous vortex of the sulfasalazine-naproxen combination colloidal stock was done, serial dilutions of treatments were provided ranging from 5 to 250 µM, and their cytotoxic effects were tested on standard cell lines using MTT assay. Untreated cells served as the control. The cells were exposed to MTT (0.5 mg/mL in phosphate buffered saline) for four hours at 37 °C, the medium was eliminated. The absorbance measurement was computed at 490 nm, and then the cell viability percentage was compared with untreated control cells.

2.11 Theoretical method

All calculations were performed via the GAMESS package (Schmidt et al., 1993) at the density functional theory (DFT) level using the hybrid M06-2X exchange functional augmented with an empirical dispersion term (M06-2X-D) (Ghasemi et al., 2019). The optimized molecules were further subjected to the calculations for harmonic vibrational frequencies at the M06-2X/6-311+G** level of theory. The excited states are determined with TDDFT (time-dependent density functional theory). The functional used in TDDFT calculation is M06-2X with 6-311+G** basis set. Vibrational frequencies were also evaluated by the M06-2X/6-311+G** level of theory to determine the reliability of the relaxed structures, in addition to study assign experimental data.

3.1 Characterization of complex

3.1.1

3.1.1 UV–Vis spectra and interaction energies

As displayed in Fig. 2, the ultraviolet–visible absorption spectra (UV–visible) of the SSZ, NAP and Sulfoxen compounds in methanol were measured. NAP shows broad band at 332 nm due to π-π* transition of naphthalene ring and 244 and 272 nm possibly owing to the n-π* transitions of carboxyl and ether groups in the presence of polar solvent Perez et al., 2015. SSZ also shows two peaks at 253 and 359 nm which are respectively attributed to n-π* transition of carboxylic group and π-π* transition of benzene ring (Fig. 2) (Ghashim, 2012). Sulfoxen compound shows a slight hypsochromic shift in the UV–Vis band of carboxyl group at 240 nm. Formation of Sulfoxen involves hydrogen bonding between the two drugs at carboxyl group which prevents the conjugation of carboxylate ions causing the conjugation system to increase in energy gap. Absorbance band of Sulfoxen between 300 nm and 500 nm is approximately equivalent with the sum of NAP and SSZ absorbance due to aromatic rings with 1:1 wt ratio. Based on theoretical modeling of UV–Vis absorption, Sulfoxen compound represents a strong absorption peak at 220 nm with oscillator strength of 0.911 and a strong absorption peak at 312 nm with oscillator strength of 1.02 in methanol phase which are in corroboration with the UV–Vis spectral data. In UV–Vis absorption spectra of pure SSZ demonstrates the main band maximum is observed at 226 and 369 nm while the NPX drug shows the main band maximum is observed at 221 and 286 nm.

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Fig. 2

UV–visible spectra of NAP, SSZ and coamorphous (1:1) Sulfoxen.

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We also evaluated the interaction energy between NAP and SSZ through carboxyl (–COOH) groups by the M06-2XD functional. Fig. 1 represents the formation of hydrogen bonding between carboxyl groups of both drugs in methanol phase. The estimated value of the interaction energy is −14.89 kcal/mol. The most substantial geometrical variations in the interaction process are inconsiderable increase in C-O bond length of SSZ from 1.224 Å to 1.244 Å and little increment of C-O bond length of NAP from 1.209 Å to 1.229 Å.

3.1.2

3.1.2 FT-IR and Raman spectra

FT-IR spectra of the free SSZ, NAP, and its combination complex (Sulfoxen) were recorded in the range of 400–4000 cm⁻¹. The FT-IR spectra of free SSZ and NAP drugs have a strong intensity band at 1638 and 1629 cm⁻¹ range due to ʋ(C = C). Whilst the Sulfoxen drug shows a weak IR mode at 1460 cm⁻¹. The phenolic C-O stretching mode from 1167 and 1141 cm⁻¹ in the free SSZ and NAP drugs shifted to 1111 cm⁻¹ in the combination drug M.G. Abd EL-WAHED, M.S. REFAT, S.M. EL-MEGHARBEL, , 2009; Bhise et al., 2008. The theoretical result of the combination drug indicates that the phenolic C-O stretching mode is shifted to lower wavenumber (1108 cm⁻¹). Presence of the OH group showed a shift for the δ(OH) in-plane bending from 1394 and 1362 cm⁻¹ in the free SSZ and NAP drug to 1354 cm⁻¹ in the Sulfoxen compound.

FT-IR spectrum of naproxen (data shown in Table 1) displays fairly sharp bands at 1236, 1362, 3188, and 1677 cm⁻¹ in turn due to C-O stretching vibration, CH₃ bending, OH stretching band, ν(C = O) stretching mode. Meanwhile, the modes at 2926–3140 cm⁻¹ illustrate C-H aromatic and aliphatic stretching vibrations (Srivastava et al., 2018). The free SSZ and NAP drugs indicate respectively the OH stretching vibrations at 3315 and 3188 cm⁻¹. In the combination drug, this characteristic band shifts to 3454 cm⁻¹ with a reduced intensity which is assigned to the formation of intermolecular hydrogen bonding. The result of the calculated theoretical harmonic frequency shows abroad band in the region of 3540 cm⁻¹ which can attribute to hydroxyl groups (OH) in the combination drug, which is close to the experimental result obtained. Abd El-Wahed and co-workers reported the IR spectrum of sulfasalazine that indicated a medium broadband at 3439 cm⁻¹, assigning the OH stretching vibration of the phenolic and carboxylic OH groups M.G. Abd EL-WAHED, M.S. REFAT, S.M. EL-MEGHARBEL, , 2009.

Table 1 FT-IR data for the SSZ and NAP and its combination complex.

ν(OH)	ν(C = C)	νsym.(C-C)	ν(N = N)	δ(OH)	ν(C = O)	νasym.(SO2)	ν(C-O)	Compound
3454	1607	1458	1577	1354	1702–1761	1364	1111	Sulfoxen
3315	1638	1487	1587	1394	1675	1360	1167	SSZ
3188	1629	1481	–	1362	1677	–	1141	NAP

FT-IR spectrum indicates that the SO₂ group in the combination drug leads to emerging strong bands at 1364 cm⁻¹ stemming from the symmetric vibration, whereas this group observed at 1360 cm⁻¹ in the free SSZ drug. Based on the FT-IR spectrum in the combination drug, the medium band at 1607 cm⁻¹ is related to the aromatic ring skeleton stretching vibration of benzene ring: ʋ(C = C), which is comparable to theoretical calculations (1632–1663 cm⁻¹). In addition, the theoretical result on the combination drug represents that the ʋ(C = O) vibration observed in region between 1694 cm⁻¹ and 1807 cm⁻¹, which is comparable to experimental data (1702–1761 cm⁻¹). The vibration bands at 2864 and 2922 cm⁻¹ are assigned to C-H aromatic ring and asymmetric stretching vibration of the methyl group: ʋ_asym(CH₃), respectively.

We studied the Raman analysis for the combination of sulfasalazine with naproxen in the range of 2500–400 cm⁻¹. The Raman spectrum of naproxen shows sharp peaks at 400, 560, 700, 750, 860, 1170, 1400, 1420, 1480, 1580 and 1620 cm⁻¹ whereas sulfasalazine shows sharp peaks at 630, 800, 1110, 1150, 1250, 1300, 1400, 1450, 1490 and 1580 cm⁻¹. The major peaks in the combined drug are located at 1149, 1396, 1444, 1589, and 1742 cm⁻¹ (Fig. 3). Several peaks in the absorbance range of 400–1000 cm⁻¹ were significantly reduced in intensity which indicates the reduction in vibration energies associated with C-C and C-O bonds, ring deformation and skeletal vibration.

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Fig. 3

Raman spectrum of the Sulfoxen compound.

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3.1.3

3.1.3 Thermal analysis

The blue curve in Fig. 4 is the DSC curve which measures temperature differences while the green curve is TG curve which measures weight loss of sample. To avoid confusion on the blue curve as DTG curve, the caption of Fig. 4 was updated for further distinction on TG and DSC curves. We found that the naproxen drug completely decomposes (99%) within the temperature range of 150–480 °C with a main mass loss at 266.93 °C based on TGA curve. Zayed et al. proposed that the thermal decomposition of naproxen occurs due to three fragmentations: carboxyl at 150–170 °C, ethyl at 210–280 °C and methoxyl at 400–480 °C (Zayed et al., 2017). The differential scanning calorimetry (DSC) analysis of the naproxen also represents endothermic and exothermic mass loses involved in the decomposition of this drug. Moreover, the sulfasalazine drug decomposes at 279 °C resulting in the mass losses of C₄H₆N₄SO₃ (47.20%,) and C₂H₈O₂ (16.50%) as the organic moieties which are endothermic mass losses (Refat et al., 2011). Combination drug which is comprised of those two drugs shows a similar behavior to the precursors with some negative shifts in the temperature values. The thermal analysis of the combination drug was performed in a temperature range of 0–600 °C using a Universal V4.5A TA Instrument.

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Fig. 4

Thermal analysis of Sulfoxen compound: TG curve (Green) and DSC curve (Blue).

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The TGA curve stands for couple of steps in the mass loss with the main mass loss of 63.63% placed between 200 and 400 °C (Soliman, 2006). The DSC curve also represents an exothermic (at 154.5 °C) and three endothermic (211, 236 and 318 °C) processes occurring within thermal decomposition of the combination drug (Fig. 4). These results illustrate that thermal decomposition of the combination drug occur in four consecutive steps. The DSC curve further confirmed that the mass loss of 63.73% (3.285 mg) is occurred at 200–400 ˚C. DSC profile represents three endothermic peaks, the first at 352 K corresponding to the melting of the combined drug, the second at 600 K assigns to the dehydration and decomposition of the sulfoxen compound and the third board endothermic peak related to the final decomposition of the sulfoxen.

3.1.4

3.1.4 XRPD analysis

XRPD analysis was done in order to obtain further evidence about the structure and lattice dynamics of the combination drug. The effect of temperature on drugs can be investigated by observing the alteration of lattice parameters and the reactions of drug Shen et al., (2018). The melting point of USP Naproxen Reference Standard was 154.4–156.2 °C [lit. 155.3 °C 3)], and that of USP Sulfasalazine Reference Standard was 260–265 °C [lit. 262.5 °C (12)]. The XRPD pattern of the pure SSZ is consistent with those reported in the literature da Costa et al., 2015; Rajesh et al., 2011 and exhibits well-defined crystalline structure with characteristic signals at 11.9°, 15.1°, 23.1°, and 27.6°. The XRPD pattern of the pure NAP have the distinct peaks at 2θ˚: 6.5˚, 12.4˚, 16.6◦, 19◦, 20◦, 22.5◦, 24◦ and 28.5◦ (Javadzadeh et al., 2010). As displayed in Fig. 5, the XRPD pattern obtained for the Sulfoxen indicates high crystallinity of the combination drug. The XRPD analysis of the combination drug presents reflecting peaks at 27.50, 31.90, 45.50, 53.95, 56.50 and 66.32˚ attributing the (1 1 1), (2 0 0), (2 2 0), (3 1 1), (2 2 2), and (4 0 0) crystal planes respectively. Shown in Fig. 5, the last four XRPD peaks were attributed to the heterodimer structure of Sulfoxen with the coformers via strong hydrogen bonds between NAP and SSZ. The presence of heterodimer in the crystal lattice resulted in substantial discrepancy in the crystal structure of Sulfoxen have profound impact on drug features.

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Fig. 5

XRPD patterns of the SSZ, NAP and Sulfoxen complex.

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3.1.5

3.1.5 FESEM analysis

The morphology, size, shape and elemental composition of the combination drug (Sulfoxen) are displayed in Fig. 6. FESEM image of the sulfoxen nanoparticles demonstrated that the nanoparticles have a solid dense cubical or cuboidal structure and not aggregated. As can be seen in Fig. 6, Sulfoxen nanoparticles represent the almost spherical shape with the mean particle size of 50–100 nm. EDX spectrum of combination drug nanoparticles is shown in Fig. 6. The EDX spectrum represents the attendance of C (56.36%), O (17.26%), S (4.61%), and N (5.62%) in the combination drug. Elemental maps are presented in Fig. 7 and indicated that the entire particle surface consists of C, N, and O.

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Fig. 6

FESEM image and EDX spectrum of Sulfoxen compound.

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Fig. 7

EDX elemental map for different elements of the Sulfoxen compound.

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3.1.6

3.1.6 AFM and DLS analyze

In Fig. 8(a), AFM analysis indicated that the size of the the nano combination products ranging from 50 to 150 nm with near cubical or cuboidal shapes. AFM image of combination products presents the height of nanoparticles to be 20 nm as shown in Fig. 8. Dynamic light scattering (DLS) assay performed to define the physical features of combination products (Fig. 6(b)). The mean size of Sulfoxen nanoparticles was analyzed with a value of 157.7 ± 45.7 nm.

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Fig. 8

AFM (a) and DLS (b) images of sulfoxen compound.

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3.2 Anti-oxidizing activity

The TAC and FRAP (Abdolahi et al., 2018) of the tested compounds, containing SSZ, NPX, and sulfoxen nanoparticles have been determined by DPPH assay. The obtained results in Table 2 represent the effect of each of these molecules on the decrement of oxidation, which is presented by the concentration index required for 50% inhibitory IC₅₀. The highest antioxidant activity of the Sulfoxen was found to be 73.0% inhibition at concentration of 100 mg/mL, while highest antioxidant activity for the pure sulfasalazine and naproxen are found to be 23% and 1%, respectively. The result represents the sulfoxen has a higher antioxidant capacity than pure samples of SSZ, and NPX. Sulfoxen has IC₅₀ values way below the average of IC₅₀ values of sulfasalazine and naproxen, indicating that the drug combination does provide synergistic effect instead of additive property. See Table 3

For pure drug samples, NAP has lower reducing power than SSZ as the former has naphthalene and carboxyl group as the stronger conjugated system to provide better redox resistance. With the drug combination, conjugation of carboxyl groups in both NAP and SSZ were both prevented due to intermolecular hydrogen bonding between carboxyl groups. This resulted in the weakened conjugated system of the overall structure thus significantly improve the reducing power of sulfoxen. The results demonstrated that the best oxidation inhibitory effect between the three test specimens with all three methods is to evaluate the composition of naproxen-sulfasalazine, which has the lowest concentration used for inhibiting the specific percentage (IC₅₀). This amount of inhibition is generally lower than the synthetic antioxidant effect of BHT.

3.3 Cell toxicity studies

We evaluated cytotoxic effects of Sulfoxen nanoparticle on KYSE30 and MCF-7 cell lines in compared to NPX and SSZ alone. For MCF-7 cell line, IC₅₀ values of free naproxen and sulfasalazine were 2300 and 775 µM. For KYSE30 cell line, IC₅₀ values of free naproxen and sulfasalazine were 1900 and 500 µM Deb et al., 2014; Narang et al., 2007. We did not observe a significant toxicity for Sulfoxen nanoparticles until 300 µM concentration. We previously demonstrated that ST8MNV nanoformulation (Squalene (S) and Tween 80 (T8) Micellar/Niosomal Vesicles) significantly improved solubility of naproxen which leads more efficient anticancer property of naproxen Erfani-Moghadam et al., (2020). This formulation also improves the bioavailability of sulfasalazine and promotes its effectiveness through the induction of cancer cells apoptosis (Aghaei et al., 2021).

3.4 In vivo toxicity against zebra fish

We explored the use of zebrafish as an alternative model for toxicity testing of sulfoxen compared to the free sulfasalazine and naproxen drugs in order to provide an in vivo assessment of combined effects of drugs at an earlier stage in drug discovery. In the course of resistance test, Sulfoxen compound showed no toxicity towards zebrafish at concentrations between 1 millimolar (mM) to 12.5 mM after 120 h of treatment. Therefore, the results of the test showed that zebrafish is not affected during the administration of this substance in our determined doses range, suggesting their safety for potential clinical use.