Influence of Reduction with NaBH4 and HCl in Obtaining Amino Derivatives of Cashew Gum and Cytotoxic Profile

Tree-exuded gums are natural polymers that represent an abundant raw material in the food and pharmaceutical industries. The cashew gum can be obtained by exudation of trees of the genus Anacardium, a native species of the Brazilian northeast; its polymer consists of monosaccharide units propitious to the action of chemical reactions that tend to improve their intrinsic characteristics among them, as the degree of hydro-solubility. The objective of this work was to modify the exudate gum of Anacardium occidentale (cashew gum (CG)) through an amine reaction. The modification was confirmed by Nuclear Magnetic Resonance (1H NMR), infrared spectroscopy (FTIR), gel permeation chromatography (GPC), zeta potential, and thermogravimetric analysis (TG). In addition, the chemical modification altered the molar mass and surface charge of the CG, and the amino group binding to the CG polymers was confirmed by FTIR spectra. In addition, cytotoxicity tests were performed where cell viability was estimated by an MTT assay on RAW 264.7 macrophages. Through these tests, it was found that the amine caused an increase in the thermal stability of the amino compounds and did not present cytotoxic potential at concentrations below 50.0 mg/L.


Introduction
Natural gums can be divided into vegetable exudates gums, plant seed gums, seaweed gums, and gums produced by microorganisms. The interest in gums obtained from plant exudates has grown considerably due to their structural properties and their functions in food, pharmaceutical, cosmetic, textile, and biomedical products [1].
Water-soluble gums, also known as hydrocolloids, are used for various applications such as dietary fibers, texture modifiers, gelling agents, thickeners, stabilizers, emulsifiers, coatings, films, encapsulating agents, and drying aids [2,3]. There is a strong tendency to replace synthetic materials with natural gums due to non-toxicity, low cost, safety, and availability [4]. The most industrially used gums are Starch, Cellulose, Guar gum, Arabic technological and functional properties [20]. The long-term strategy to promote the use of GC in industry is therefore to understand and explore the physicochemical properties of gum, in its original or chemically modified state, isolated or mixed with other polymers.
Thus, in this study, cashew gum was modified by reductive amine reaction in two separate routes and with a different reducing agent for each route in order to identify the amino derivative with the highest degree of substitution. The derivatives were characterized, and the biological profile was tested in different cell lines.
The specimen Anacardium occidentale L. has already registered in the National System of Genetic Heritage Management and Associated Traditional Knowledge, with the number AAA024E.

Purification of the Cashew Gum
To obtain the gum, a method adapted from [28] was used. This used 10.0 g of crushed exudate immersed in 100.0 mL of deionized water, being under constant agitation for 12 h for the solubilization of the polymer. After solubilization, it was filtered in an analytical funnel for retention of impurities that may be adhered to the exudate. The pH was adjusted to 7 with NaOH at 0.1 mol.L −1 and 2.0 g of NaCl was added, leaving the solution in agitation for another hour. Subsequently, the polymer was recovered by precipitating it in ethanol at a 4:1 ratio (ethanol/gum). Sediment was expected, and filtration was carried out in a sintered plate funnel. The precipitate was washed two more times with 99.0% ethanol and once with acetone. The drying was performed with the aid of a mortar and pistil and dried under the continuous flow of hot air until obtaining powder.

Modification Reaction
Two routes were used to obtain the modification reaction of Cashew Gum (CG) to compare their effectiveness. On route 1 it was performed and adapted as described by [22] by the addition of ethylenediamine (C 2 H 8 N 2 ) as an amine agent by the substitution of the -OH group of the CG by -NHCH 2 CH 2 NH 2 step 1. In addition, the -NHCH 2 CH 2 NH 2 group was reduced to -NH 2 by the addition of NaBH4 as shown in Figure 1 with [24]. In a 100.0 mL beaker (bottle 1), 1.0 g of CG was added and dissolved in 10.0 mL of distilled water, and stirred for 30 min to obtain a homogeneous solution. An amount of 12.0 mL of ethylenediamine was added, which was placed to react under agitation for 24 h at room temperature. Overtime, 5.0 mL of 5% NaBH 4 solution was added to the reaction mixture contained in the flask. The CG modification route 2 (CG) was performed and adapted as described by [25], and using ethylenediamine (C 2 H 8 N 2 ) as an amine agent by replacing the -OH group of the GC by -NHCH 2 CH 2 NH 2 step 2 of Figure 1.
In a 100.0 mL beaker, 1.0 g of CG was added which was dissolved in 10.0 mL of distilled water, stirred for 30 min to obtain a homogeneous solution,12.0 mL of ethylenediamine. The homogeneous system was placed to react under agitation for 24 h at room temperature. After passing the time, we added 5.0 mL of HCl solution 1 mol.L −1 , and the flask was subjected to vigorous agitation for a period of 3 h. After finishing the samples were precipitated, washed with 99% ethyl alcohol and acetone, and precipitated dried in a warm air stream, and then sprayed to uniform the particles. temperature. Overtime, 5.0 mL of 5% NaBH4 solution was added to the reaction mixture contained in the flask. The CG modification route 2 (CG) was performed and adapted as described by [25], and using ethylenediamine (C2H8N2) as an amine agent by replacing the -OH group of the GC by -NHCH2CH2NH2 step 2 of Figure 1. In a 100.0 mL beaker, 1.0 g of CG was added which was dissolved in 10.0 mL of distilled water, stirred for 30 min to obtain a homogeneous solution,12.0 mL of ethylenediamine. The homogeneous system was placed to react under agitation for 24 h at room temperature. After passing the time, we added 5.0 mL of HCl solution 1 mol.L −1 , and the flask was subjected to vigorous agitation for a period of 3 h. After finishing the samples were precipitated, washed with 99% ethyl alcohol and acetone, and precipitated dried in a warm air stream, and then sprayed to uniform the particles.
After drying, a yield of 67% for ACG1 and 65% for ACG2 was obtained.

Fourier Transform Infrared Spectroscopy (FTIR)
The infrared spectroscopy was performed in an infrared spectrometer by Fourier transform (Nicolet iS5-iD7 ATR-Thermo Fisher Scientific Mississauga, Mississauga, ON, Canada) using the technique of total attenuated reflection, with a spectrum ranging from 4500 to 400 cm −1 .

Thermogravimetric Analysis (TGA)
The thermograms were obtained through the thermogravimetric analyzer (TGA51 Thermogravimetric Analyzer SHIMADZU), in a nitrogen atmosphere with a flow of 50 mL/min, heating rate of 10 °C/min, and range of analysis from 25° to 600 °C. The sample was packed at platinum sample ports and a mass of 4.0 0.1 mg. After drying, a yield of 67% for ACG1 and 65% for ACG2 was obtained. The infrared spectroscopy was performed in an infrared spectrometer by Fourier transform (Nicolet iS5-iD7 ATR-Thermo Fisher Scientific Mississauga, Mississauga, ON, Canada) using the technique of total attenuated reflection, with a spectrum ranging from 4500 to 400 cm −1 .

Thermogravimetric Analysis (TGA)
The thermograms were obtained through the thermogravimetric analyzer (TGA51 Thermogravimetric Analyzer SHIMADZU), in a nitrogen atmosphere with a flow of 50 mL/min, heating rate of 10 • C/min, and range of analysis from 25 • to 600 • C. The sample was packed at platinum sample ports and a mass of 4.0 0.1 mg.

Elemental Analysis(C, H, N)
For the determination of the percentage of carbon, nitrogen and hydrogen, the samples were processed in duplicates in an Elemental Analyzer-Perkin Elmer model PE 2400 in CHNS mode with thermal conductivity detector.

Degree of Substitution
The degree of substitution (DS) of the CG derivatives was determined based on that of the % N (nitrogen percentage) obtained from the elemental analysis (minus the insignificant amount present in CG), and calculated according to the following Equation(1) [29]. DS was defined as the number of hydroxyl groups replaced per unit of sugar CG.

Zeta Potential
Zeta potential measurements were analyzed in a Malvern Zetasizer Nano Modelo ZS90 for each sample at a concentration of 1.0 mg.mL −1 . The Zeta potential (mV) was determined from the measurements the samples were diluted in Milli-Q water (1:10; v/v)as suggested by [30]. The parameters were estimated by the average of three measures.

Gel Permeation Chromatography (GPC)
The molar mass distribution was determined by gel permeation chromatography (GPC) in the Shimadzu LC-20AD equipment coupled to a refractive index detector (RID-10A). For the analysis, a column PolySep-GFC-P Linear (300 × 7.8 mm) and NaNO 3 (a) (0.1 mmol·L −1 ) were used as eluent. The measurement was made at 30 • C, with a flow of 1.0 mL/min, and the injected volume of the sample was 50.0 µL. Molar mass was calculated using a pullulan curve (Equation (2)) or (Equation (3)).
Note: Equation (2) was used only for the samples GCA1 and GCA2.

Nuclear Magnetic Resonance Spectroscopy ( 1 H NMR)
To obtain the NMR spectra, a sample of CG and ACG (20.0 mg) each was dissolved in deuterium oxide (D 2 O) 1 H spectra obtained from the NMR spectrometer Agilent 400 MHz at 40 • C.

MTT Assay
The cells were plated into a 96-well multiplate at a density according to the doubling time of each lineage (8 × 10 4 cells/mL of MDA-MB-231; 6 × 10 4 cells/mL of the HCT-116; 10 × 10 4 of L929; 10 × 10 4 cells/mL of RAW macrophages 264.7). After 4 h of incubation at 37 • C and 5% CO 2 for cell adhesion, three washes with sterile PBS were performed. Then, 100.0 µL of supplemented DMEM medium containing different concentrations of substances (50.0 to 1.56 mg/mL) was added and incubated for a period of 72 h. At the end of the period 10.0 µL of MTT (5.0 mg/mL) was applied and incubated for more than 4 h. Then the supernatant was removed and 100.0 µL of DMSO was added in all wells. After 30 min of agitation, the reading was performed at 540 nm in a plate reader. Supplemented DMEM medium containing 0.5% DMSO was used as negative control and considered as 100% macrophage viability.

Fourier Transform Infrared Spectroscopy
The chemical characterization was performed to confirm the chemical modification of cashew gum by the insertion of amino on the polysaccharide surface. In the spectra, it is possible to observe characteristic bands of O-H (3290 cm −1 ) and the emergence of a shoulder (3345 cm −1 ) corresponding to the replacement of OH groups by NH 2 groups on the surface of CG [8].We can also identify bands at 1132 cm −1 , 1078 cm −1 ,and 1013 cm −1 attributed to C-O-C angular deformations of glycosidic bonds and O-H bending of alcohols and that are also present in ACG2.
The most striking difference between CG and its amine derivatives was the new absorption bands at 1250-1300 cm −1 and 700-800 cm −1 .It is possible to observe a weak band at 1572 cm −1 , which corresponds to the angular deformation of NH 2 and the band at 1456 cm −1 due to angular deformation of primary amine N-H bond confirming the chemical modification of CG [31,32]. Bands are observed in the regions of 1366 cm −1 and 1323 cm −1 referring to the angular deformation of germinal CH 3 appearing as doublet and evident only in ACG1 [17].
The ACG2 sample presented a spectrum similar to that of the CG with some displacements, with no significant changes and/or emergence of new bands as shown in Figure 2.

Fourier Transform Infrared Spectroscopy
The chemical characterization was performed to confirm the chemical modification of cashew gum by the insertion of amino on the polysaccharide surface. In the spectra, it is possible to observe characteristic bands of O-H (3290 cm −1 ) and the emergence of a shoulder (3345 cm −1 ) corresponding to the replacement of OH groups by NH2 groups on the surface of CG [8].We can also identify bands at 1132 cm −1 , 1078 cm −1 ,and 1013 cm −1 attributed to C-O-C angular deformations of glycosidic bonds and O-H bending of alcohols and that are also present in ACG2.
The most striking difference between CG and its amine derivatives was the new absorption bands at 1250-1300 cm −1 and 700-800 cm −1 .It is possible to observe a weak band at 1572 cm −1 , which corresponds to the angular deformation of NH2 and the band at 1456 cm −1 due to angular deformation of primary amine N-H bond confirming the chemical modification of CG [31,32]. Bands are observed in the regions of 1366 cm −1 and 1323 cm −1 referring to the angular deformation of germinal CH3 appearing as doublet and evident only in ACG1. [17] The ACG2 sample presented a spectrum similar to that of the CG with some displacements, with no significant changes and/or emergence of new bands as shown in Figure 2.  Changes in the chemical structure of polysaccharide gums can lead not only to changes, but also to adding new properties. Modification of the amine of polysaccharide gums can create amphiphilicity properties by integrating the alkyl chains of the amine or creating a positive charge after the introduction of a primary amine into the structure.

Thermogravimetric Analysis (TG)
The thermogravimetric curves of cashew gum and amine derivatives show weight loss events in four, three, and two stages for GC, CGA1, and GCA2, respectively, shown in Figure 3 and the parameters obtained from the curves in Table 1. The initial decomposition temperature (To) is above 200 • C, as verified for some other polysaccharides [33]. The comparison of the curves reveals that the thermal decomposition processes of the samples are different and that the first event occurs at (Tmax. = 60 • C) related to water loss and is present in all curves.
The CGA1 and CGA2 gums show a new event at Tmax = 531 • C that is not present in the original gum TG. Similar results were obtained for carboxymethylated cashew gum [34]. This event is probably related to the thermal fission of carbohydrate segments.
However, the Tmax of the second event (309 • C) is similar to that previously reported for cashew gum [35]. The maximum degradation temperature in the range of 270-320 • C was determined for other polysaccharides: gum Arabic (316 • C) [33] and chitosan (300 • C) [36]. The decomposition of derivatives of some polysaccharides, such as guar gum [37] and galactoxyloglucan [38], also begins at a lower temperature than that of the original polymer. Below 200 • C, the amine derivatives had less weight loss than GC. Subsequently, weight loss increases sharply with temperature. However, above 330 • C, ACG1 and ACG2 had a lower weight loss than CG, an important factor regarding the thermal stability of CG after the amine reaction, corroborating with the results found by [24].
changes, but also to adding new properties. Modification of the amine of polysaccharide gums can create amphiphilicity properties by integrating the alkyl chains of the amine or creating a positive charge after the introduction of a primary amine into the structure.

Thermogravimetric Analysis (TG)
The thermogravimetric curves of cashew gum and amine derivatives show weight loss events in four, three, and two stages for GC, CGA1, and GCA2, respectively, shown in Figure 3 and the parameters obtained from the curves in Table 1. The initial decomposition temperature (To) is above 200 °C, as verified for some other polysaccharides [33]. The comparison of the curves reveals that the thermal decomposition processes of the samples are different and that the first event occurs at (Tmax. = 60 °C) related to water loss and is present in all curves.  The CGA1 and CGA2 gums show a new event at Tmax = 531 °C that is not present in the original gum TG. Similar results were obtained for carboxymethylated cashew gum [34]. This event is probably related to the thermal fission of carbohydrate segments.
However, the Tmax of the second event (309 °C) is similar to that previously reported for cashew gum [35]. The maximum degradation temperature in the range of 270-320 °C was determined for other polysaccharides: gum Arabic (316 °C) [33] and chitosan (300 °C) [36]. The decomposition of derivatives of some polysaccharides, such as

Elementary Analysis (C, H, N)
The elemental analysis of CG and ACG samples indicated the presence of 36.03% and 32.12% carbon, 5.68% and 6.01% hydrogen, 0.76% and 8.73% nitrogen, respectively to CG and ACG2 ( Table 2). The increase in the percentage of nitrogen in the product confirms that the amino group was incorporated into the polymer skeleton. The results clearly indicate the presence of the NH 2 group on both routes and confirm the modification of the CG. The small amount of nitrogen in CG reflects the presence of protein traces (0.15%). The ACG1 (route with NaBH 4 ) presented a greater DS compared to that observed fromACG2 (route with HCl). Therefore, the process of route 1 was more effective than route 2; it is expected that the amended results can create ionic imbalances in the biopolymer due to the formation of NH 2 [26].

Zeta Potential
The surface charge is an important parameter to be investigated in the characterization of modified gums because this factor can influence its properties involving electrostatic interactions. The zeta potential measurement reflects the effective charge of the particle and refers to the electrostatic repulsion between them. The results indicate that the CGmodification process promoted a change in the polymer surface charge. The CG surface charge is −26.5 mV, and this negative value is justified by the presence of glucuronic acids (13.5%) in the CG composition. After modification, lower anionic characters of −3.6 mV for ACG2 and +0.16 mV for ACG1 were observed, probably due to the insertion of positively loaded amino groups, previously proven by FTIR, in the CG polymeric chain.

Gel Permeation Chromatography
Natural gums are composed of polysaccharides of multiple sugar units linked together to form large molecules; they have a high molecular weight. However, chemical modification can cause changes in the molecular weight of CG, which can influence physicochemical properties of CG. Thus, the GPC analysis was performed to investigate possible changes in the molecular mass of the CG after chemical modification by amination. The amines and the original gum Figure 4 and ( Table 2) presents physicochemical and mass distribution data, where we can conclude that they are polydispersed macromolecules, with polydispersity values (Mw/Mn) of 6.1 and 3.6 for ACG1 and ACG2, respectively. We can still observe that the molecules of ACG1 and ACG2 present bimodal distribution indicating the formation of amino products similar results found in [17] These results corroborate with the results obtained by [24], who performed this same synthesis pathway to obtain the xyloglucan gum amine, where this reaction also promoted reduction in the molecular mass of the modified polysaccharide.  Table 2) presents physicochemical and mass distribution data, where we can conclude that they are polydispersed macromolecules, with polydispersity values (Mw/Mn) of 6.1 and 3.6 for ACG1 and ACG2, respectively. We can still observe that the molecules of ACG1 and ACG2 present bimodal distribution indicating the formation of amino products similar results found in [17] These results corroborate with the results obtained by [24], who performed this same synthesis pathway to obtain the xyloglucan gum amine, where this reaction also promoted reduction in the molecular mass of the modified polysaccharide. 3.6. Hydrogen Magnetic Resonance (NMR 1 H) Figure 5 Shows the 1 H NMR spectra of CG(A), ACG1(B), and ACG2(C), respectively. The NMR spectrum for cashew gum without modification in Figure 5A showed characteristic anomeric protons in the region of 4.4 to 5.0 ppm using D2O as solvent [23]. Signals  Figure 5 Shows the 1 H NMR spectra of CG(A), ACG1(B), and ACG2(C), respectively. The NMR spectrum for cashew gum without modification in Figure 5A showed characteristic anomeric protons in the region of 4.4 to 5.0 ppm using D 2 O as solvent [23]. Signals in this region are reported as a duet α-D-glucose (4.95 ppm), α-L-rhamnose (4.81 ppm), β-Dgalactose (1→3) (4.69 ppm and 4.43 ppm), and glycuronic acid (4.51 ppm). The H-2 to H-5 Figure 5B,C signals are superimposed in the regions of 3.4 ppm to 4.3 ppm, and a quartet signal in the region of 1.26 ppm is due to the methyl protons of rhamnose [39]. Signals in the region of 1.5 to 2 ppm are due to protons of NH 2 ( Figure 5B,C). Comparing to native GC ( Figure 5A), ACG1, and ACG 2 ( Figure 5B,C, respectively) exhibited an additional peak close to 3.1 ppm, which was attributed to methyl groups attached to primary amines [40]. The existence of this signal is a result of the hydrogen atom of the substituent that was introduced into the chain. In Figure 5C it is more intense, and thus, we can conclude based on the results of the zeta potential and elemental analysis that the sample ACG 2 presents a lower degree of substitution.

Cytotoxicity
Cytotoxicity analysis is a prerequisite for assessing the biocompatibility of biomaterials. In this context, cell viability was estimated by the MTT assay on RAW 264.7 macrophages, and the results are expressed as the percentage of the sample group in relation to the blank control. The results demonstrated a CC50 of 48.3 (mg/mL) for ACG1 and 40.02 (mg/mL) for ACG2. These results point to low cytotoxicity in RAW 264.7 macrophages at concentrations up to 25.0 (mg/mL), which showed viability above 65% for both ACG1 and ACG2 samples, indicating their good cell compatibility. In contrast, the highest tested concentration of 50.0 (mg/mL) showed a decrease in viability. These results corroborate those found by [41] who report low cytotoxicity for RAW 264.7 macrophages with polysaccharides. Our results indicate that the tested samples do not present cytotoxic effects at concentrations up to 25.0 (mg/mL) for this immunological cell line, and are, therefore, a valid and safe alternative for biomedical applications.

Cytotoxicity
Cytotoxicity analysis is a prerequisite for assessing the biocompatibility of biomaterials. In this context, cell viability was estimated by the MTT assay on RAW 264.7 macrophages, and the results are expressed as the percentage of the sample group in relation to the blank control. The results demonstrated a CC50 of 48.3 (mg/mL) for ACG1 and 40.02 (mg/mL) for ACG2. These results point to low cytotoxicity in RAW 264.7 macrophages at concentrations up to 25.0 (mg/mL), which showed viability above 65% for both ACG1 and ACG2 samples, indicating their good cell compatibility. In contrast, the highest tested concentration of 50.0 (mg/mL) showed a decrease in viability. These results corroborate those found by [41] who report low cytotoxicity for RAW 264.7 macrophages with polysaccharides. Our results indicate that the tested samples do not present cytotoxic effects at concentrations up to 25.0 (mg/mL) for this immunological cell line, and are, therefore, a valid and safe alternative for biomedical applications.
Cytotoxicity analysis was also performed on mouse fibroblast cell lines (L929), human colorectal carcinoma (HCT-116), and human mammary adenocarcinoma (MDA-MB-231). Figure 6, together with the IC50 values shown in Table 3, shows the low selectivity of the molecules tested, with no significant differences between tumor and non-tumor cells. The cytotoxicity profile of the cashew tree gum modifications in a 72 h treatment shows that ACG1 and ACG2 showed a significant reduction (p < 0.05) compared to the control, for all strains tested, especially at the highest concentrations ( Figure 5). These data indicate that the effect of cytotoxicity was dose-dependent, so that, with the increase in its concentration, cell viability was reduced.
Polymers 2023, 15, x FOR PEER REVIEW 11 of 14   5). These data indicate that the effect of cytotoxicity was dose-dependent, so that, with the increase in its concentration, cell viability was reduced.  Some polysaccharides may have in vitro and in vivo biological activities against tumor cells, but most of them have only a considerable effect at high doses. Previous work using cashew gum against cancerous and non-cancerous strains did not observe a cytotoxic effect, and this is possibly due to the low concentrations used [42], for example, did not observe cytotoxicity when using concentrations < 0.1 (mg/mL) in the HCT-116  Some polysaccharides may have in vitro and in vivo biological activities against tumor cells, but most of them have only a considerable effect at high doses. Previous work using cashew gum against cancerous and non-cancerous strains did not observe a cytotoxic effect, and this is possibly due to the low concentrations used [42], for example, did not observe cytotoxicity when using concentrations < 0.1 (mg/mL) in the HCT-116 strain, in their work on the antitumor potential of cashew tree gum. In their study, [43] managed to observe a moderate effect of this same strain, with an IC50 value > 0.5 (mg/mL) in their study of the cytotoxic profile of cashew tree gum. By using concentrations between 0.16 and 2.5 (mg/mL) of a polysaccharide obtained from Juniperus convallium, [41] found a significant antiproliferative effect on the cell lines used in their study, including the MDA-MB-231 line age.
The IC50 values are presented in mg/meal and were calculated from nonlinear regression using GraphPad Prism Software version 8.

Conclusions
In this study, the amination of cashew gum was performed using an easy and simple synthetic strategy. The modification of the polysaccharide improved the thermal properties of the CG; it is observed that the route 1 is more effective in the amination synthesis of the cashew gum.
Modified gums showed low invitro cytotoxicity at concentrations lower than 50 mg/L. The invivo cytotoxicity profile of cashew gum modifications in a 72 h treatment shows that ACG1 and ACG2 showed a significant reduction (p < 0.05) control, for all strains tested, especially in the highest concentrations. These data indicate that the effect of cytotoxicity was dose-dependent, so that, with the increase inits concentration, cell viability was reduced. These properties can be attractive for safe applications to biomedicine.

Data Availability Statement:
The data presented in this study are available only by the requesting them from the corresponding author due to the further work in progress.