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Introduction
In nations with emerging economies, the issue of antimicrobial resistance remains a concern because it makes it harder to treat infectious diseases, which are responsible for a substantial portion of disease and death1,2. For a long time, diseases from microorganisms have been the second most common cause of death3-5. Heterocyclic compounds, widely studied because of their diverse biological, medicinal, industrial, antimicrobial, and therapeutic applications, are a focal and increasingly studied area within organic chemistry6,9. Schiff base compounds are precious in medicine and play a vital role in combatting various organisms10. Hugo Schiff first reported imine synthesis in the 19th century (1864) and since then, multiple methods for producing imines have been recognized11. Schiff bases are chemical compounds but they contain an imine or azomethine group in place of the carbonyl group. Schiff bases exhibit a diverse range of biological characteristics, suggesting their potential utility as agents with antioxidant, antifungal, anti-inflammatory, antitumor, antibacterial as well as anticancer activities12–18. Furthermore, heterocyclic thiazole compounds, which contain both sulfur and nitrogen atoms, have relevance to diverse research fields19-21. Several synthetic as well as naturally occurring substances that incorporate the thiazole unit have exhibited physiological potential as anti-microbial, free radical scavenging, anti-fungal, anti-cancer, anti-candida and cytotoxicity, anti-inflammatory agents22–27. Reactive oxygen species (ROS) are essential contributors to the development of several significant diseases including cancer28, heart diseases29, diabetes30, arteriosclerosis31 and cataracts32 causing biological harm through oxidative stress induced by free radicals33. Oxidative strain occurs when there is a disparity, with the production of oxidants exceeding their degradation rate34. Antioxidants, neutralizing free radicals, offer protection against severe and potentially severe diseases35. An antioxidant is a compound with the ability to hinder the oxidation process of other molecules36-38. Due to ongoing microbial resilience against antibiotics, scholars are constantly exploring substitutes for traditional medications, recognizing essential role played by thiazoles in the biological diversity of various drugs.
To explore the combined effectiveness of a novel pharmacophore, we aim to generate a set of innovative compounds by synthesizing Schiff bases that include thiazole-nitro hybrids by changing substituents at various positions into the ring of thiazole. This will facilitate advancement in novel and more potent drugs. In current scientific studies, understanding the electronic structure of chemical compounds is essential for unraveling the mechanisms behind pharmacological activities39. Theoretical predictions play a crucial role in identifying potent biologically active compounds by generating results such as Molecular orbitals (LUMO, HOMO) and other electronic characteristics that closely align with experimental data40, while the research involves evaluating computational toxicity, adherence to drug characteristics, and pharmacokinetic scoring for the synthesized compounds and includes studies on molecular docking to understand how proteins interact with the synthesized analogs.
Materials and Methods
Chemicals
Reagents purchased from Sigma-Aldrich and Merck were used as received, without additional purifications.
Experimental
Measurements
Infrared spectra were recorded by Shimadzu FTIR spectrophotometer (Model FTIR-IR Affinity-1) and melting points with a Fisher JOHN m.p. apparatus (Model 1A 9000). The Proton and Carbon-13 NMR spectra (DMSO-d6, δ, ppm, J: Hz), including DEPT-135, COSY, HSQC, and HMBC were recorded by Bruker HD spectrometer (operated at 400 and 100 MHz). Structure of synthesized compounds was drawn in ChemDraw Ultra 12.0 software, In Silico Molecular Docking was performed using Gaussian 09, PyMol (version 2.4), AutoDock Vina in PyRx 08 and Discovery Studio 4.1 soft wares.
Synthesis of Thiosemicarbazone 1a
Synthesis of hydrazinecarbothioamide (1a)
Substituted 3-nitroacetophenone (5mmol) and thiosemicarbazide (5mmol) were taken in two-neck flask with 15.0 mL of ethanol followed by refluxed at 80°C with stirring. After completion (checked by TLC), it was cooled and separated by filtration. The product was recrystallized from ethyl acetate, affording the compound in an excellent yield (77%).
Synthesis of Thiazole (1b-4b)
Procedure for the synthesis of thiazolederivatives (1b-4b)
Thiosemicarbazone derivatives (g, mmol) and substituted phenacyl bromide (5 mmol) was refluxed in acetone (15.0mL) at approximately 60°C with stirring. After completion (checked by TLC), it was allowed to cool down and subsequently filtered to isolate the final desired products. The solid was further purified by recrystallization from acetone, affording the final compounds in excellent yields (72–78%).
Antibacterial and antifungal screening
In vitro antimicrobial studies of the compounds (1b-4b) were evaluated by using the agar disc diffusion41 method. Potato dextrose agar (PDA) and mueller–hinton agar (MHA) were employed for the cultivation of fungal and bacterial strains. Both PDA and MHA plates were incubated for 24 hours to ensure sterility and confirm the absence of contamination. Following incubation, sterile cotton swabs were employed to uniformly inoculate the test microorganisms onto the respective media. Discs loaded with 25μL of a DMSO solution containing 300μg of each synthesized compound were placed on the plate`s surfaces. Then, it was incubated aerobically for 24 h at 37°C for antibacterial studies and 48 h at 26°C for antifungal evaluation. For comparison, discs containing equivalent volumes of ceftriaxone (for antibacterial assays) and amphotericin B (for antifungal assays) dissolved in DMSO were used as standard drugs. Finally, the zones of inhibition were determined.
Antioxidant activity
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method42-44 was used to assess the antioxidant properties. DPPH (EtOH) solution (6μg/mL) was prepared and kept in stirring for 24 hours. Sample solutions were prepared in ethanol at concentrations 500μg/mL to 31.25μg/mL. For each test, 100μL of the sample solution was mixed to 4.0 mL of the DPPH followed by incubation in an ice bath under dark conditions. Ascorbic acid, dissolved in ethanol at corresponding concentrations, was used as the standard. After brief centrifugation (10 seconds), all samples were kept in the dark for an additional 15 minutes. The absorbance (Asample) of each sample was then recorded at 517 nm and compared with the control (Acontrol). Antioxidant activity was determined by the equation:
Molecular Docking
In silico docking studies were undertaken to clarify how the proposed inhibitors interact with the binding site of the target protein. In this purpose, the crystal structure corresponding to PDB ID: 5JBO45 (taken from protein data bank), representing the co-crystal of T. harzianum. Protein was prepared using PyMol (version 2.4) by removing crystallographic H2O molecules, ligands and attached heteroatoms. Energy minimization of the processed protein was subsequently conducted using Swiss-PdbViewer.
Frontier Molecular Orbital Analysis
The energies of the HOMO and LUMO orbitals are key indicators for interpreting the electronic behavior, charge-transfer capability, and overall chemical reactivity of a molecule, which together contribute to its biological activity46. Various biological activity descriptors of the compounds were computed based on the frontier molecular orbital values using the standard quantum chemical equations.
In silico ADMET prediction
Assessment of absorption, distribution, metabolism, and excretion (ADME) characteristics for the synthesized analogs was carried out using the online versions of Swiss ADME and Molinspiration tools. The absorption (% ABS) for each compound was estimated by the equation:
Table 1: Synthesis of the compounds with yield (%) and time (hour)
Compounds Colors and Textures m.p. oC Reaction Time (h) Yield 1a Yellow amorphous solid 210-212 9 77% 1b Brown amorphous solid 228-230 6 74% 2b Light yellow amorphous solid 178-180 8 78% 3b Brown amorphous solid 182-184 6 72% 4b White amorphous solid 195-197 6 75%Results and Discussion
Chemistry
Four novel thiazole derivatives (1b–4b) were successfully synthesized, and their molecular structures were confirmed through IR, Proton and Carbon-13 NMR, DEPT-135, COSY, HSQC, and HMBC spectroscopic analyses.
Characterization of Compounds
2-(1-(3ʹ-nitrobenzylidene)ethylidene)hydrazinecarbothioamide (1a)
Amorphous solid, m.p. 210~212°C. IR: ῡmax. (KBr, cm-1): 3402 and 3199 (N-H) and 1598 (C=N). 1H-NMR: 8.61 (1H, s, H2′), 8 .22 (1H, d, J = 8.0,H4′ ), 7.67 (1H, t, J = 8.0, H5′), 8.41 (1H, d, J = 8.0, H6′) 8.61 (1H, s, NH), 10.39 (2H, s, NH2) and 2.37 (3H, s, CH3); 13C-NMR: δ C1ꞌ(139.9), C2ꞌ(121.4), C3ꞌ(148.6), C4ꞌ(124.0), C5ꞌ(130.1), C6ꞌ(133.4), C=N(146.0), C=S(179.6) and CH3(14.5).
4ʹʹ-Phenyl-2ʹʹ-(2-(1-(3ʹ-nitrobenzylidene) ethylidene)hydrazinyl)thiazole (1b)
Brown amorphous solid, m.p. 228~230°C.IR: ῡmax. (KBr, cm-1):. 3130 and 3115(N-H) and 1610 (C=N). 1H-NMR : δ 8.57 (1H, s, H2′), 7.73 (1H, t, J = 7.2, H4′), 7.42 (1H, t, J = 8.4, H5′), 8.22 (1H, t, J = 8.4, H6′) 7.37 (1H, s, H5′′), 7.87 (1H, d, J = 7.6, H2′′′), 7.33 (1H, t, J = 7.2, H3′′′),7.31 (1H, t, J = 7.2, H4′′′), 8.22 (1H, t, J = 8.4, H5′′′),7.87 (1H, d, J = 7.6, H6′′′), 8.57(1H, s, NH) and 2.39 (3H, s, CH3); 13C-NMR: δ C1ꞌ(140.0), C2ꞌ(120.3), C3ꞌ(148.5), C4ꞌ(123.4), C5ꞌ(130.5), C6ꞌ(132.3), C=N(144.7), C2ꞌꞌ(169.9), C4ꞌꞌ(134.9), C5ꞌꞌ(104.9), C1ꞌꞌꞌ(134.9), C2ꞌꞌꞌ(129.1), C3ꞌꞌꞌ(126.0), C4ꞌꞌꞌ(128.0), C5ꞌꞌꞌ(126.0), C6ꞌꞌꞌ(129.1) and CH3(14.3).
4ʹʹ-(4ʹʹʹ-Chlorophenyl)-2ʹʹ-(2-(1-(3 nitrobenzylidene)ethylidene)hydrazinyl)thiazole (2b)
Light yellow amorphous solid, m.p. 178~180°C, IR: ῡmax. (KBr, cm-1): 3066 and 3026 (N-H) and 1606 (C=N). 1H-NMR: δ 8.56 (1H, s, H2′),7.72 (1H, d, J = 7.2, H4′), 7.46 (1H, t, J= 8.4, H5′), 8.22 (1H, d, J = 8.4, H6′)7.44 (1H, s, H5′′), 7.91 (1H, d, J = 7.6, H2′′′), 7.49 (1H, d, J = 7.2, H3′′′),7.49 (1H, d, J = 7.2, H5′′′), 7.91 (1H, d, J = 7.6, H6′′′), 8.18 (1H, s, NH) and 2.37 (3H, s, CH3) ;13C-NMR: δ C1ꞌ(139.9), C2ꞌ(120.3), C3ꞌ(148.5), C4ꞌ(123.4), C5ꞌ(130.5), C6ꞌ(132.3), C=N(144.7), C2ꞌꞌ(149.7), C4ꞌꞌ(133.9), C5ꞌꞌ(105.7), C1ꞌꞌꞌ(132.4), C2ꞌꞌꞌ(129.1), C3ꞌꞌꞌ(127.7), C4ꞌꞌꞌ(144.7), C5ꞌꞌꞌ(127.7), C6ꞌꞌꞌ(129.1) and CH3(14.3).
4ʹʹ-(4ʹʹʹ-Methoxyphenyl)-2ʹʹ-(2-(1-(3 nitrobenzylidene)ethylidene)hydrazinyl)thiazole (3b)
Brown amorphous solid, m.p. 182~184°C.IR: ῡmax. (KBr, cm-1): 3118 and 3080 (N-H) and 1608 (C=N). 1H-NMR: δ 8.57 (1H, s, H2′), 8.22 (1H, t, J = 7.2, H-4′), 7.73 (1H, t, J= 8.4, H5′), 8.22 (1H, t, J = 8.4, H6′)7.19 (1H, s, H5′′), 6.99 (1H, d, J = 7.6, H2′′′), 7.82 (1H, d, J = 7.2, H3′′′),7.80 (1H, d, J = 7.2, H5′′′) and 6.97 (1H, d, J = 7.6, H6′′′), 8.18 (1H, s, NH), 3.79 (3H, s, OCH3) and 2.39 (3H, s, CH3); 13C-NMR: δC1ꞌ(140.0), C2ꞌ(120.3), C3ꞌ(148.5), C4ꞌ(123.4), C5ꞌ(130.5), C6ꞌ(132.3), C=N(144.7), C2ꞌꞌ(169.8), C4ꞌꞌ(140.0), C5ꞌꞌ(102.7), C1ꞌꞌꞌ(132.3), C2ꞌꞌꞌ(114.4), C3ꞌꞌꞌ(127.7), C4ꞌꞌꞌ(159.3), 5ꞌꞌꞌ(127.3), C6ꞌꞌꞌ(114.4), CH3(14.3) and OCH3(55.5).
4ʹʹ-(Chloromethyl)-2ʹʹ-(2-(1-(3 nitrobenzylidene)ethylidene)hydrazinyl)thiazole (4b)
White amorphous solid, m.p. 195~197°C .IR: ῡmax. (KBr, cm-1): 3417 and 3126 (N-H) and 1598 (C=N). 1H-NMR: δ 8.57 (1H, s, H2′), 7.73 (1H, t, J = 7.2, H-4′), 7.42 (1H, t, J= 8.4, H5′), 8.22 (1H, t, J = 8.4, H6′), 7.37 (1H, s, H5′′), 8.54 (1H, s, NH), 2.37 (3H, s, CH3) and 4.66 (2H, s, CH2Cl); 13C-NMR: δ C1ꞌ(139.8), C2ꞌ(120.3), C3ꞌ(148.5), C4ꞌ(123.5), C5ꞌ(130.5), C6ꞌ(132.4), C=N(147.3), C2ꞌꞌ(170.1), C4ꞌꞌ(145.4), C5ꞌꞌ(109.5), CH3(14.3) and CH2Cl(41.8).
Antimicrobial Studies
In vitro antimicrobial investigation of the synthesized derivatives (1b-3b) was assessed by bacterial and fungal species. The zones of inhibition (mm ± SD) are summarized in Table 2. Amphotericin B (Amp B) and Ceftriaxone (Cef) were used as reference standards. Among the tested compounds, 3b exhibited the highest antibacterial activity, showing 13.7 ± 0.6 mm against Salmonella typhimurium. All compounds demonstrated significant antifungal activity, particularly against Aspergillus niger.
Table 2: Antibacterial activities of compounds (1b-3b)
Compounds Gram (+)ve bacteria Gram (-)ve bacteria Fungal Strains S. aureus B. subtilis S. typhimurium E. Coli T. harzianum A. niger 1b 11.3 ± 0.6 – 6.3 ± 0.6 7.0 ± 1.0 21.0 ± 1.0 26.3 ± 1.5 2b 5.3 ± 0.6 6.3 ± 0.6 4.3 ± 0.6 6.0 ± 1.0 10.3 ± 0.6 26.0 ± 1.0 3b 10.3 ± 1.5 12.0 ± 2.0 13.7 ± 0.6 12.0 ± 1.0 10.7 ± 1.2 36.3 ± 1.5 DMSO – – – – – – Cef 40.3 ± 0.6 50.0 ± 1.0 42.7 ± 1.5 37.3 ± 1.2 – – Amp B – – – – 17.7 ± 0.6 15.3 ± 0.6Diameter ± SD (standard deviation), – Represents no activity.
Antioxidant Activity Assay
The DPPH test was employed to examine the antioxidant capacity of the samples at varying concentrations. The compounds exhibited IC50 values ranging from 31.82 to 60.42 µg/mL. Among them, compound 3b demonstrated the highest activity (IC50 of 31.82 µg/mL) comparable to that of ascorbic acid (27.34 µg/mL). In particular, the para-positioned methoxy group in 3b enhances its radical scavenging ability through electron-donating effects, whereas compound 2b displayed the lowest activity in presence of halogen (chlorine). Due to limitations in laboratory facilities, compound 4b was not subjected to antimicrobial evaluation.
Table 3: Antioxidant activities of (1b-3b)
Compounds IC50 (µg/mL) 1b 44.07 ± 2.920 2b 60.42 ± 3.90 3b 31.82 ± 3.80 Ascorbic acid (AA) 27.34 ± 1.86Docking Analysis
Compounds (1b–4b) were subjected to docking studies against the co-crystal structure of 5JBO (PDB ID) to evaluate their antifungal potential. All compounds exhibited favorable binding affinities (–8.4 to –9.5 kcal/mol). Among them, 1b demonstrated the highest affinity, whereas compound 4b showed comparatively moderate binding (–8.4 kcal/mol) relative to the other synthesized analogs.
Table 4: Docking Results of compounds (1b-4b)
Compounds Protein (PDB ID) Binding Affinity (Kcal/mol) Residue in Contact Types of Interactions Bond Distance (Å) 1b 5JBO -9.5 GLU172 AC 4.85981 ASP243 AC 5.56333 TYR245 CHB 2.91468 GLU172 CHB 2.9389 ARG262 CHB 2.89845 ARG262 CHB 3.1789 HIS266 CHB 2.87799 ASP243 PA 3.59632 ASP444 PA 3.89965 HIS266 PDH 3.40432 TRP126 PS 5.32234 TRP126 PPT 5.4008 ILE175 Pi-Al 4.91473 2b 5JBO -9.4 GLU172 AC 4.75713 ASP243 AC 5.33181 GLU172 CHB 2.83221 ARG262 CHB 2.89863 ARG262 CHB 3.1345 HIS266 CHB 2.82444 ASP243 PA 3.36468 ASP444 PA 3.86893 HIS266 PDH 3.36524 TRP126 PS 5.54955 TRP126 PPT 5.47412 ILE175 Pi-Al 4.89729 3b 5JBO -9.3 ASP243 AC 4.48688 TYR316 CHB 2.85983 ASP243 CHB 2.89236 TRP357 Pi-C 4.03109 GLU172 PA 3.68864 ASP243 PA 3.49978 TRP434 PDH 4.19158 GLN319 PDH 4.09525 HIS266 PS 5.95345 ILE175 Pi-Al 5.3695 PHE356 Pi-Al 5.1782 4b 5JBO -8.4 GLU172 AC 3.90228 GLU172 CHB 2.55379 GLU172 CHB 2.3281 TYR179 CHB 2.42071 TRP357 Pi-C 4.7087 GLU384 PA 3.86456 TRP357 PS 4.82807 TYR316 PPS 4.52261 TRP357 PPS 3.78312 TRP357 PPS 4.34145 TRP434 PPS 5.3004 TRP434 Pi-Al 4.1378 TRP434 Pi-Al 3.92438 TRP442 Pi-Al 5.27478CHB = Conventional Hydrogen Bond, PPS = Pi-Pi Stacked, AC= Attractive Charge, PA = Pi-Anion, PDH = Pi-Donor Hydrogen Bond, Pi-Al = Pi-Alkyl, , PS = Pi-Sulfur, PPT = Pi-Pi T-shaped, Pi-C = Pi-Cation.
In silico ADMET Prediction
Pharmacokinetic properties are commonly evaluated using Veber’s and Lipinski’s Rule of Five, provide insight into oral bioavailability. Key descriptors considered include molecular weight (MW)a, no. of H-bond acceptors (HBA)b, no. of H-bond donors (HBD)c, lipophilicity (clogP)d, no. of rotatable bonds (NROTB)e, topological polar surface area (TPSA)f, solubility (logS)g, and percentage absorption (% ABS)h. TPSA is particularly important in drug design, as values above 140 often suggest limited oral bioavailability. The pharmacokinetic profiles of compounds 1b–4b, along with reference standards ascorbic acid (AA), ciprofloxacin (Cip), and miconazole (Mic), are summarized in Table 5.
Table 5: ADME Prediction’s properties of Compounds (1b-4b)
Comp. Lipinski’sViolations
Lipinski’s rule Veber’s rule logSg % ABSh MWa(≤500) HBAb(≤10) HBDc(≤5) clogPd(≤5) NROTBe(≤10) TPSAf (140 Å2) 1b 0 338.39 6 1 5.05 5 111.3 -5.48 70.5 2b 0 372.84 6 1 5.67 5 111.3 -6.22 70.5 3b 0 368.42 7 1 4.99 6 120.5 -5.50 67.32 4b 0 310.76 4 1 3.90 5 111.3 -4.84 70.48 Cip 1 331 5 2 -1.53 3 72.88 -3.32 83.85 AA 0 176 6 4 -2.46 2 107.2 -0.35 71.91 Mic 1 416 3 0 4.85 6 27.5 -5.08 99.67Frontier Molecular Orbital Analysis:
The energy difference (EHOMO − ELUMO) is a significant descriptor that reflects a molecule’s chemical hardness or softness, optical polarizability, kinetic stability, and electron transport capability. A smaller HOMO–LUMO gap indicates a softer and more chemically reactive molecule, whereas a larger gap is associated with greater hardness and lower reactivity. The most antioxidant-active compound is 3b (IC50= 31.82 ± 3.80 μg/mL) and the least active compound, is 2b (IC50 = 60.42 ± 3.90 μg/mL). Compound 3b exhibited a narrower energy gap (ΔE = 3.97 eV) compared to 2b (ΔE = 4.22 eV), indicating higher reactivity. The corresponding HOMO–LUMO values are summarized in Table 6. Furthermore, compound 2b showed a greater hardness value (η = 2.1106 eV) relative to 3b (η = 1.935 eV), suggesting lower reactivity and enhanced stability, along with greater resistance to electron density distortion. Electrophilicity (ψ) analysis also indicated that compound 2b possesses the highest predicted toxicity among the studied compounds.
Table 6: Energies of HOMO–LUMO and other parameters of compounds (1b-4b)
Chemical reactivity indices (eV) 1b 2b 3b 4b EHOMO -7.7384 -7.9620 -7.5059 -7.07 ELUMO -3.7067 -3.7408 -3.6354 -3.28 Energy Difference, ΔE 4.0317 4.2212 3.9705 3.79 Hardness, η 2.01585 2.1106 1.93525 1.895 Softness, ζ 0.248 0.2369 0.2584 0.2638 Ionization potential, I = – EHOMO 7.7384 7.9620 7.5059 7.07 Electron affinity, A = – ELUMO 3.7067 3.7408 3.6354 3.28 Chemical potential, μ -5.72255 -5.8514 -5.570 -5.175 Electrophilicity, ψ 8.12252 8.111 8.0174 7.066 Electronegativity, χ= – μ 5.72255 5.8514 5.570 5.175
Toxicity And Drug – Score Properties
In Silico evaluation of toxicity risks as well as Drug-Likeness Properties for Compounds (1b-4b), Ascorbic Acid, Ciprofloxacin and Miconazole. Reproductive (R), Irritating (I), Tumorigenic (T) and Mutagenic (M) are the four categories used to represent toxicity effects.
Table 7: In Silico toxicity properties of Compounds (1b-4b).
Compound Toxicity Effects Drug-Likeness Drug-Score R I T M 1b L L H L -2.04 0.16 2b L L H L -1.29 0.14 3b L L H H -1.90 0.10 4b H L H H -2.39 0.08 AA H L H H 0.02 0.16 Cip. L L L L 2.07 0.63 Mico. L L L L 7.64 0.18L=Low, H= High, AA = Ascorbic Acid
Conclusions
A new series of thiazole derivatives (1b–4b) were synthesized and characterized to evaluate their antimicrobial and antioxidant activities against selected bacterial and fungal strains associated with severe or fatal infections. Compound 3b showed potential antimicrobial and antioxidant activities. On the other hand, compounds (1b-3b) showed excellent antifungal activities. In addition, docking studies, ADMET predictions were performed to analyze the potentiality of the compounds as drug candidates. Docking studies showed that all four compounds exhibited favorable binding affinities with the 5JBO protein along with multiple strong interactions at the binding sites of the receptor. In silico studies were consistent with the observed antimicrobial activity. Overall, the synthesized thiazole derivatives (1b–4b) demonstrated promising drug-like properties, suggesting their potential for further development as potent therapeutic agents.
Acknowledgement
We are grateful to the authority of Chittagong University of Engineering and Technology for providing necessary research fund to complete this work.
Funding Sources
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Conflict of Interest
The author(s) do not have any conflict of interest.
Data Availability Statement
This statement does not apply to this article.
Ethics Statement
This research did not involve human participants, animal subjects, or any material that requires ethical approval.
Authors’ Contributions
Ranajit Kumar Sutradhar: Paper writing, Supervision,
Md. Abu Bakkar Siddiki: Methodology, Investigation,
Md. Mohiuddin Emon: Formal analysis,
Md. Din Islam: Formal analysis,
Md. Aminul Haque: Biological Test and Analysis,
Mohammad Mostafizur Rahman: Biological Test and Analysis
References
Groome, M. J., Albrich, W. C., Wadula, J., Khoosal, M. & Madhi, S. A., Paediatr. Int. Child Health, 2012, 32, 140–146.
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