Document Type : Original Research Article
Subjects
Introduction
Carboxymethylcellulose (CMC) is an anionic natural polysaccharide. Carboxymethyl cellulose (CMC) possesses numerous compelling attributes as a polymer. It has high chemical stability, good solubility, and is non-toxic [1-4]. Furthermore, it is a biodegradable and cost-effective polymer suitable for biocompatibility and film production [5-7]. The pharmaceutical, food, and packaging sectors utilize it widely due to its safety and nontoxicity [8]. Conversely, carboxymethyl cellulose (CMC) is readily amenable to chemical modification and is utilized in various applications due to its abundant hydroxyl and carboxyl groups, as well as its environmental compatibility [9-13]. Pure CMC films readily dissolve in distilled water, necessitating the incorporation of other ingredients for various uses [14,15]. When CMC is combined with other consumable substances (such as CS) [16-19]. Chitosan is a natural polymer derived from the alkaline deacetylation of chitin, a structural component of the exoskeletons of crustaceans such as crabs and prawns [20,21]. Chitosan is the second most abundant organic substance in nature, following cellulose. Chitosan is a weak base that is insoluble in water [22]. This phenomenon is likely attributable to the existence of robust intermolecular hydrogen bonds generated between the molecular chains of chitosan [23]. However, it can dissolve in weak acidic water with a pH below 7, where it changes glucosamine units (-NH₂) into a form that can dissolve better (NH₃⁺). The solubility of chitosan is contingent upon its natural origin, molecular weight, and degree of deacetylation [24]. Chitosan is a robust polymer. because its molecules have hydrogen bonds. Chitosan is special because it is plentiful, can be modified on its surface, and has unique properties such as being biodegradable, biocompatible, non-toxic, antibacterial, hydrophilic, mucoadhesive, and having anticholesterolemic actions [25,26]. Chitosan is a very useful molecule that may be used in many different ways, such as in medicine, farming, food preservation, biocatalysis, and the environment [27]. Because it has amino and hydroxyl groups, chitosan does not have anionic characteristics. Biopolymer-based nanocomposites have rapidly emerged in recent years [28]. Researchers are currently looking at how to combine nanomaterials (NMs) with biodegradable polymers. Polysaccharides are safe for living things, break down quickly, and are quite common [29,30]. Polysaccharides are widely used to reduce and stabilize metal nanoparticles (MNPs) because they are good for the environment and easy to convert into different types of hydrogels [31]. Nanoparticles (NPs) make biopolymers better at blocking heat, mechanical stress, biological activity, and other things, which makes them more useful [32]. The present study to synthesize new series of imine compounds then modified carboxymethyl cellulose with imine prepared compounds and blending with chitosan , nanoparticles with screened of antibacterial , antioxidant and anticancer activities. Based on these preliminary theoretical and experimental results, these novel species have potential as drug candidates, and future work will involve in vivo studies.
AuNPs or MgONPs or AgNPs were supplied by US, Research Nanomaterials, Inc. Chemicals have been provided from BDH and CDH
With a range of 400 cm⁻¹ to 4000 cm⁻¹, the Shimadzu FT-IR-8400S was used to record the FT-IR spectra. Ultra Shield 400 MHz, manufactured by Bruker, University of Tehran, Iran, was used to perform the ¹H-NMR spectra. As an internal standard, TMS has been used using DMSO as the solvent. The University of Tehran in Iran was the site of the field emission scanning electron microscopy (FESEM) study. To visualize ligands, proteins, hydrogen bonding interactions, short contacts, and bond length measurements, molecular docking was carried out using the CCDC Hermes visualizer program (version 1.10.3). Biological activity took place at the University of Baghdad’s Central Environmental Laboratory, College of Science. Perform an acute toxicity test in the lab of the Centre for Cancer Research and Medical Genetics.
Synthesis of 2-amino-5-mercapato-1,3,4-thiadiazole[33]
This compound was synthesized in accordance with the literature (Ali H.Samir et.al. 2014). The M.P. (229-231) Co and the FTIR spectrum were in agreement with the literatures
Synthesis of compounds [I-VI][34]
2- amino-5- mercapto -1,3,4 - thiadiazole (1.33 gm.,0.01mol) or 4-amino benzene thiol (1.25gm.,0.01mol) mixed with( 0.01mol) of trichloro propanal or 2,4-dichloro benzaldehyde or syringaldehyde in 20ml of absolute ethanol with three drops of glacial acetic acid, the mixture was refluxing at(75-80) C° for 14h.The reaction mixture was cooled, the precipitate filtration and recrystallized from ethyl acetate to give:(Pale yellow, (248-250 (Co, yield65% ) ,(Very light brown, (137-138)Co, 72%),(Yellow,254-256Co, 96%), (Dark yellow, 229-230 Co, 70%), (Yellow green,158-160Co,81%),(Yellow green, 240-242Co, 54%) respectively. Scheme 2. shows the formation steps of compounds [I-VI]
Synthesis of compounds [VII-XII] [35]
Aliquot (0.01mol) one of the compounds [I-VI] mixed with (0.02 mol) anhydrous sodium carbonate in (15ml) distilled water then (0.01mol) of chloroacetic acid was added. The solution refluxed for 6h. then added conc. hydrochloric acid to reached out pH= 2. Filtered the product and washed with distilled water and recrystallized from absolute ethanol to produce: (Yellow, 187-189Co, 97%), (very light brown,228-230Co,78%), (Yellow,179-180Co, 82%), (White,193-195Co,78%), (White,164-165Co,75%), (Orange, 250-251Co, 90%) respectively. Scheme 2 shows the formation steps of compounds[VII-XII]
Synthesis of compounds [XIII-XVIII] [36] (0.01mol) of one of the compounds [VII-XII] mixed with SOCl2 (0.01mol) in 15ml dry benzene was refluxed for (8 h.) The excess of thionyl chloride and benzene were outlying under vacuum. (Dark brown,160-162Co, 93%) (Dark orange,239-241Co,96%), (Dark brown, 118-120Co ,94%), (Dark indigo,183-185Co,69%), (Red, >300 Co, 63%), (Brown,210-212Co, 92%) respectively. Scheme 2. shows the formation steps of compounds[XIII-XVIII]
Synthesis of Modified CMC [XIX-XXX] [37]
Modified CMC [XIX-XXX] has been prepared by the reaction (0.1gm.) of one compounds [XIII-XVIII] with 0.1 gm. of CMC in 20 ml of DMF, the mixture was refluxed for 8 h. The solution was poured on a Petri dish and left to dry at room temperature. shown in Scheme 3 and Scheme 4
Synthesis of Blend Polymers [XXXI-XLII] [38]
Solvent casting was employed to create polymer blends. One of modified CMC [XIX-XXX] was dissolved in water and stirred for 1 hour at room temperature to create the modified CMC [XIX-XXX] solutions, 1 gm of chitosan dissolved in (49 mL of 2% percent aqueous acetic acid solution) and stirring for 1 hour at room temperature to create the chitosan solutions. Two polymer solutions (CMC solutions and Chitosan solutions) were mixed to prepare homogenous solution by using hotplate stirrer for 60 min after the two polymers’ solutions had been combined. The modified CMC/CS blends were created by combining Modified CMC: CS (5:5) in one ratio. shown in Scheme 5.
Synthesis of Nanocomposites (Modified CMC/CS / Nanoparticles)[XLIII-LX] [39]
In order to bond the gold, or magnesium or silver nanometals in the blend matrix, 100mg of the dried modified CMC/CS [XXII-XXVII] was added to 50mL of AuNPs or MgONPs or AgNPs solution of a 250 mg/L concentration. This was done with the use of a hotplate stirrer for three hours. shown in Scheme 6
FTIR and 1HNMR of Synthesis Compounds
Scheme (1) illustrates the process of synthesizing new compounds from Schiff bases. The compounds [I-VI] are synthesized by combining 2-amino-5-mercapto-1,3,4-thiadiazole or 4-amino benzene thiol with various aldehydes in ethanol and heating the mixture for 14 hours The FTIR spectra of compound [VI] exhibited peaks at 2325, 1640cm⁻¹. these peaks are linked to the SH group and C=N, respectively, Table 1. Show FT-IR data of compounds [I-VI] . 1H-NMR of compound [VI] showed a singlet at δ3.36 ppm for the proton of SH , doublets at 4.59–4.76 ppm for 2(OCH₃), a singlet at δ5.06 ppm for the proton of OH, an extra signal at δ9.82 ppm for the proton of CH-N, and several peaks at δ6.50–7.73 ppm for aromatic protons. The reaction of compounds [I-VI] with chloroacetic acid in distilled water produced compounds [VII-XII] in a basic environment. The FTIR. spectra of compound [XII] show peaks for the hydroxyl group at 3400–2400 cm⁻¹ and for the carboxylic group at 1680 cm⁻¹, Table 2. shows FT-IR of compounds[VII-XII] . 1H-NMR of [XII] showed a singlet signal at a chemical shift of δ 13.21 ppm, which was caused by carboxylic proton. There was a signal at δ 9.78 ppm that had to do with the proton of CH-N. There were several peaks in the region of δ 7.12–7.77 ppm for aromatic protons, as well as a single signal at δ 3.85 that corresponded to two protons of S-CH₂. There were doublet indications at 3.76–3.77 ppm for 2(OCH₃). When the synthesized chemicals [VII-XII] were mixed with SOCl2 in dry benzene, they made many derivatives[XIII-XVIII]. The FTIR analysis of the compound[XVIII] shows disappearance band at 3400–2400 cm⁻¹, which is linked to the (OH) group of the carboxylic acid. Instead, there is a band at 1740 cm⁻¹, which indicates the acyl chloride, Table 3. FT-IR spectroscopy of compounds [XIII-XVIII].
CMC derivatives [XIX-XXX] were prepared through the reaction between [VII-XVIII] with the CMC in DMF as the solvent. The FT-IR spectrum of the polymer [XXIX] illustrated a large peak at 3275 cm⁻¹, which indicates the hydrogen bonding OH stretching region. The small hump at 29224cm and 2900 cm⁻¹ shows the attributable C-H stretching vibration. The sharp peak observed at 1731 cm⁻¹ confirms the presence of C=O, which is assigned to the stretching of the ester group, Table 4 FT-IR data of polymers[XIX-XXX]. 1H-NMR spectrum of modified CMC[XXIX], singlet signal with a chemical shift at δ 12.6 ppm as a result of the proton of carboxylic protons, additional signal at δ δ8.87 ppm due to the presence of protons for CH-N, multiple peaks appeared at δ δ7.01-8.00ppm for aromatic protons, and a singlet signal at δ 3.85 for two protons for the S-CH2, The characteristic peak of CH₂COOH on C₆ of CMC is near 4.27-4.64, signals at 2.10-2.26 (H₂, H₃, H₄, H₅) of CMC, signals at 4.66-4.81 due to H₁ of CMC, signals at 6.98 for OH of CMC, and signals at 12.6 due to CH₂COOH on C₆ of CMC . 1H-NMR spectrum of modified CMC[XXX], singlet signal with a chemical shift at δ 11.82 ppm as a result of the proton of carboxylic proton on C6 of CMC, additional signal at δ9.70 ppm due to the presence of proton for CH-N, multiple peaks appeared at δ7.23-8.27ppm for aromatic protons , a singlet signal at δ5.96 ppm due to presence of proton for OH singlet signal at δ3.85 for two protons for the S-CH2 , doublet signals at 2.98-3.07ppm at 2(OCH3) ,The characteristic peak of CH2COOH on C6 of CMC is near 4.27- 4.64, signals at 2.10-2.36 of (H2,H3,H4,H5) of CMC, signals at 4.66-4.81 due to H1 of CMC , signals at 5.96 for OH of CMC. Modified CMC Blended with CS to prepare a blend, polymer research of the characteristics of the obtained blends had shown a good level of miscibility between the CMC and CS that had been shown by FT-IR results of the blend polymer[XLII], the band broadening in the (2400-3600) cm⁻¹ region because of a strong intermolecular bonding of hydrogen that exists between amino groups of CS and CMC’s hydroxyl groups, and 1612 cm⁻¹ as a result of (C=N) and 1712 cm⁻¹ cm-1due to carbonyl of ester,Table 5.FT-IR data of polymers[XXXI-XLII].The preparation of nanocomposites[XLIII-LX] using blend polymers [XXXIV-XXXIX] with AuNPs, MgONPs, and AgNPs. FT-IR data of nanocomposites [XLIII] shows that the presence of peaks at 3288 cm⁻¹ reveals O-H stretching from the inter- and intra-molecular hydrogen bonds and shifting asymmetric and symmetric stretching vibrational of C-H from alkyl groups at (2861, 2920) and 400 to 800 cm⁻¹ of (Au, MgO, Ag) NPs bonding also supports the formation of particles.
Field Emission Scanning electron microscope studies (FESEM) [40,41]
The FESEM approach reveals that the surface morphology differs for the Modified CMC blend with CS [XXV] (Fig.1), Modified CMC/CS/AuNPs [XLIII] (Fig.2), Modified CMC/CS/MgONPs [XLIV] (Fig.3) and Modified CMC/CS/AgNPs [XLV] (Fig. 4). The incorporation of CS modifies the surface topography of the composite membrane and significantly influences cell spreading. The FESEM image indicates a homogeneous dispersion of nanoparticles (NPs) on the matrix surface. The average particle size of the modified CMC blend with CS [XXV] ranges from 428 to 532 nm in the presence of CS. The average particle size of modified CMC/CS/Ag ranges from 34 to 79 nm, while that of modified CMC/CS/MgO ranges from 46 to 80 nm. The average nanoparticle size for gold nanoparticles is between 24 and 33 nm, which exhibit a homogeneous distribution on the matrix surface. The particles in the nanocomposite film exhibited an almost spherical shape. Nevertheless, certain clusters of nanoparticles were observed, and the surface exhibited a degree of roughness. The nanoparticles displayed a uniform dispersion in CMC/CS and a robust attachment to the polymer. The nanoparticles did not exhibit any significant cytotoxic effects on human cells. The coating on the nanoparticle surface might help reduce harmful effects or completely prevent toxicity.
Transmission Electron Microscopy (TEM) [42,43]
The TEM picture of the modified CMC/CS suspension drop-cast with AuNPs or AgNPs reveals that the AuNPs or AgNPs are uniformly disseminated and exhibit a semi-spherical morphology. Despite the differing orientations of the particles and their closed nature, there are no indications of agglomeration. The AuNPs or AgNPs generated in the improved CMC/CS exhibit enhanced stability. The TEM picture revealed that the synthesized modified CMC/CS/AuNPs [XLIII] and modified CMC/CS/AgNPs [XLV] were semi-spherical, with dimensions ranging from 50 to 100 nm, as illustrated in Figs 5 and 6. The particles exhibited a spherical morphology, encased in a thin layer of gold or silver around modified CMC/CS.TEM micrographs revealed homogeneous layers of gold or magnesium nanostructures enveloping the modified CMC/CS. The altered CMC/CS seemed to be enveloped in a coating of gold or silver particles, confirming the formation of CMC/CS/AuNPs and CMC/CS/AgNPs.
Molecular docking Study [44,45]
Molecular docking Study Molecular docking for compounds [XII-XIV] is studied, where operations are used to predict the binding status of compounds with the enzyme and to calculate the free energy (ΔG) of the compounds prepared with the human topoisomerase II alpha, which was chosen to perform the molecular docking of the derivatives, as this enzyme plays an important role in cell replication and division, and its inhibition reduces or inhibits the process of non-programmed cell division (cancer cells). The enzyme with the symbol (PDB:ID:5GWK) was chosen; its dimensions were (40, 40, 40) (X, Y, Z), and its location coordinates were (36.578, -24.476, 39.415) (X, Y, Z), and the following figures 7-10 and Table 6 show the interaction between the protein and these compounds.
Molecular docking studies showed the interference of doxorubicin with the binding site of the protein at the docking point value -9.0 kcal/mol and at the conformational value (RMSD.i.b(3.031 RMSD.u.b(8.702)) with the presence of two hydrogen bonds between the amino acid residues SER:A:64, the OH group in the compound, and the hydrogen bond between the amino acid residues. SER: A:75 with the C=O group in the drug show Fig.7.
Fig.8 shows the interaction of compound [XII]at the docking score value equal to -9.5 Kcal/mol and at the conformational value RMSD.i.b(1.493), RMSD.u.b(1.911. The two and three-dimensional cross-sectional images showed the presence of a hydrogen bond between the amino acid residue B: SER:56 with the NH group. In addition to some other bonds, such as a pi-cation and pi-alkyl, as in Table 6.
Molecular docking studies showed compound [XIII] with the protein binding site at the docking point value -9.1 kcal\mol and at the conformational value (RMSD.i.b(5.647), RMSD.u.b(6.865) with the presence of four hydrogen bonds between the amino acid residues GLUB:87, ARG:B:111, GLY:B:163 with a number of OH groups in the compound and the fourth hydrogen bond between The amino acid residues ALA:B:192 are with the NH group, show Fig.9.
Molecular docking studies showed compound [XIV] with the protein binding site at the docking point value -9.8 kcal\mol and at the conformational value (RMSD.i.b(22.870) RMSD.u.b(6.865) with the presence of four hydrogen bonds between the amino acid residues GLUB:87, ARG:B:111, GLY:B:163 with a number of OH groups in the compound and the bond. The fourth hydrogen between the amino acid residues ALA:B:192 with the NH group, as shown in the fig.10 and table 6.
From the results obtained above, it is clear that compounds [XII], [XIII]and [XIV] have a better docking score than the drug doxorubicin used as a reference. It also has more hydrogen bonds than the reference, as compound [XII] was linked to three hydrogen bonds , compound [XIII]to four hydrogen bonds and [XIV] to six hydrogen bonds, while the reference drug was linked to two hydrogen bonds, and the RMSD value appeared lower than the reference drug, which indicates a better match with the binding site.
The biological activities of modified CMC [XXIII-XXVI] , modified CMC blended with CS [XXXV-XXXVIII] , polymer blends of CS / modified CMC with gold [XLVI, XLIX] or magnesium [XLVII] or silver [XLVIII] nanocomposites have been tested against two pathogenic bacteria types (G+) Staphylococcus aureus and E. coli (G-) and compared with two drugs (Amoxicillin ,Tetracycline) and DMSO. The results of antimicrobial activity have been represented in Table 7 and Figures 11-14. The ternary mix (modified CMC/CS) with Au or MgO or Ag nanocomposite exhibited good antimicrobial activities comparable with Amoxicillin , Tetracycline as standard antibiotics. The AuNPs have shown excellent antibacterial activities towards E. coli through the absorption of the light and converting it to heat. The AuNPs are taken up easily by the immune cells as a result of their excellent cell affinity that results in precise delivery to the infected area, which facilitates the inhibition and the damage to the microbial pathogens. MgONPs was observed that these particles had the capability to generate a notable concentration of active O₂⁻. Particularly in acidic or neutral environments, this generation was attributed to the production of hydroperoxyl radicals (•HO₂). Furthermore, Ag silver nanocomposite exhibits good antibacterial properties leading to biomedical applications. The antibacterial effect of silver depends on Ag⁺, as it binds tightly to electron donor groups in microbial cell walls such as sulphur, nitrogen, or oxygen. However, silver nanoparticles affect bacterial cells by attaching to and entering the bacterial cell wall, and by producing free radicals that can harm the cell and break its membrane.
Antioxidants serve as a countermeasure against oxidants. Antioxidants are natural or manmade agents that can inhibit or postpone cellular damage induced by oxidants (ROS, RNS, free radicals, and other unstable chemicals). Halliwell and Gutteridge characterized an antioxidant as any chemical that inhibits, stops, or eliminates oxidative damage to a target molecule. The quantity must be sufficiently high to deactivate the target molecule, necessitating a reaction with oxygen or nitrogen free radicals. No universal antioxidant exists, as distinct antioxidants interact with various reactive substances by diverse methods, at different sites and safeguard specific biochemical targets. The DPPH method was employed to assess the free radical scavenging activity of chemically produced nanocomposites [XLVI, XLVIII]. The DPPH radical scavenging capacity of nanocomposites was compared to that of ascorbic acid. Enhanced outcomes were noted for the synthesized nanocomposites. The nanocomposites demonstrate considerable antioxidant activity and have been utilized in the treatment of disorders induced by oxidative stress. The in vitro antioxidant efficacy of the chosen compounds was assessed by examining their DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ability in comparison to ascorbic acid, a standard antioxidant. The data presented in Table 8, Fig.15, indicate that the nanocomposites exhibit superior activity compared to standard ascorbic acid. The previously stated NH-C=S- nanocomposites exhibited enhanced activity. This is due to a complex group that may absorb and stabilize free radicals through consecutive systems, thereby scavenging them, which then becomes conjugated.
Preparation of cell lines for cytotoxicity assays [50] uses grown cells on a 96-well microtiter plate. Absorbance was measured at 620 nm using a microplate reader. The calculated cell growth inhibition rate is defined by the Equation [51]:
Inhibition rate =
× 100
Inhibition rate = (mean of control-mean of treatment)/(mean of control) × 100
The anticancer efficacy of different concentrations of modified CMC/CS [XXXV] and the nanocomposites (modified CMC/CS/AuNPs) [XLVI], (modified CMC/CS/MgONPs) [XLVII] was examined against A172 (human brain cells) and rat embryonic fibroblasts (REF), demonstrating significant activity without impacting the proliferation of normal rat embryonic fibroblasts. Tables 9-12, Fig.16-18 show Nanocomposites [XLVI], [XLVII] demonstrate superior inhibition compared to the polymer blend [XXXV]. It is essential to further investigate the mechanisms by which nano and heterocyclic units exert powerful cytotoxic effects, which may render chitosan derivatives promising candidates for anti-cancer medications. AuNPs, MgONPs demonstrate significant cytotoxicity. A dose-dependent increase in the activation of apoptosis and reactive oxygen species production. Bio-synthesized metal nanoparticles, particularly gold, magnesium, and their conjugates with biopolymers, possess significant potential across various scientific domains due to their extensive variety of applications, including biomedical uses.
This investigation was carried out at the laboratory of the Centre for Cancer Research and Medical Genetics to assess the acute toxicity of certain synthesized polymer nanocomposites, employing the Lorke method. The study involved (25) laboratory albino mice, aged three months, with average weights ranging from (20-26) grams, all of which were male. These animals were housed in plastic cages with metal lids, coated with fine sawdust, and provided with water via plastic bottles, along with food supplies. Mice were subjected to an 18-hour fasting period with unrestricted access to water and food before the test. The compounds were dissolved in distilled water and subsequently administered via injection at dosages of (5 g/kg and 10g/kg). The treatment group and the control group were evaluated based on injection dosages, revealing after (14) days: no mortality at (5 g/kg and 10g/kg) body weight doses, no significant differences in daily weight measurements between the control and treatment groups, no alterations in mouse behavior, and no reported symptoms of toxicity. Furthermore, several mice have been euthanized via cervical dislocation, and the liver, kidneys, heart, and lungs have been weighed. The visual assessment of murine organs exhibited a normal appearance. These results suggested that polymer nanocomposites have low toxicity towards both examined organisms.
Histological examination of the liver, kidney, and brain related to the treated group showed no changes but similarities to those of the control group.
In this study, the synthesis, characterization and study of molecular docking, antibacterial, antioxidant and anticancer activities of some new modified CMC containing chitosan and nanoparticles such as gold nanoparticles, magnesium oxide nanoparticles and silver nanoparticles. TEM and FESEM studies showed the changes in the surface morphology of the synthesized polymers due to the new bonds between chitosan and modified CMC and nanoparticles. Results have indicated that nanocomposites had a greater diameter of the growth inhibition zone compared to standard antibiotics (amoxicillin, tetracycline). Finally, the antioxidant activity of modified CMC/CS/NPs exhibited a high inhibition rate compared to standard ascorbic acid. MTT assay was used to estimate the cytotoxic effect of different concentrations of the created nanocomposites for the cancer cell line (A172) and compare with the normal cell line (REF); the nanocomposites exhibited a very excellent inhibition rate. Finally, toxicity tests and histological studies for these nanocomposites are conducted, where it showed the non-toxicity of these nanocomposites.
The authors thank the Dean of the College, the Head of the Department, and the professors working in the scientific laboratories for their continuous support throughout the research.
we affirm that our submitted manuscript adheres to the ethical guidelines outlined above regarding the use of animals in research.
Conflicts of Interest: None. We hereby confirm that all the Figures and Tables in the manuscript are ours. Furthermore, any Figures and images, that are not ours, have been included with the necessary permission for republication, which is attached to the manuscript. Ethical Clearance: The project was approved by the local ethical committee at University of Baghdad
Authors’ contribution statement
Study conception and design: Ruwaidah S.Saeed; data collection: Huda H.Saeed; analysis and interpretation of results: Huda H.Saeed , Ruwaidah S.Saeed and Lyazzat Bekbayeva; draft manuscript preparation: Lyazzat Bekbayeva. All authors reviewed the results and approved the final version of the manuscript.
