Antioxidant Activities, Anticancer Activity, Physico-Chemistry Characteristics, and Acute Toxicity of Alginate/Lignin Polymer

Alginate/lignin is a synthetic polymer rich in biological activity and is of great interest. Alginate is extracted from seaweed and lignin is extracted from corn stalks and leaves. In this paper, antioxidant activities of alginate/lignin were evaluated, such as total antioxidant activity, reducing power activity, DPPH free radical scavenging activity, and α-glucosidase inhibition activity. Anticancer activity was evaluated in three cell lines (Hep G2, MCF-7, and NCI H460) and fibroblast. Physico-chemistry characteristics of alginate/lignin were determined through FTIR, DSC, SEM_EDS, SEM_EDS mapping, XRD, XRF, and 1H-NMR. The acute toxicity of alginate/lignin was studied on Swiss albino mice. The results demonstrated that alginate/lignin possessed antioxidant activity, such as the total antioxidant activity, and reducing power activity, especially the α-glucosidase inhibition activity, and had no free radical scavenging activity. Alginate/lignin was not typical in cancer cell lines. Alginate/lignin existed in a thermally stable and regular spherical shape in the investigated thermal region. Six metals, three non-metals, and nineteen oxides were detected in alginate/lignin. Some specific functional groups of alginate and lignin did not exist in alginate/lignin crystal. Elements, such as C, O, Na, and S were popular in the alginate/lignin structure. LD0 and LD100 of alginate/lignin in mice were 3.91 g/kg and 9.77 g/kg, respectively. Alginate/lignin has potential for applications in pharmaceutical materials, functional foods, and supporting diabetes treatment.


Introduction
Alginate is a bioactive polymer extracted from brown algae (Phaeophyceae), such as Sargassum, Laminaria, Tubinaria, Macrocystis, and Ascophyllum grown in tropical and subtropical coasts [1][2][3]. Alginates exist in seaweed cell walls and play a remarkable role in other fields, such as food, functional food, and pharmaceutical, as well as in different industries in the economy [4,5]. Alginate is composed of basic units of (1→4) α-L-guluronic acid (G) and (1→4) β-D-mannuronic acid (M) [6][7][8]. The arrangement of M and G in alginate leads to the different structures and activities of alginate, and this depends on the species of seaweed, the season, and the geographical location of the algae. Alginate possesses different bioactivities, such as antioxidant [9,10], anti-tumor, anti-fungal, neuroprotective, anticancer, and immuno-stimulant bioactivities [11][12][13]. In brown algae, known as Sargassum in Vietnam, the alginate content is about 16 to 36 (%wt) of dry algae, and the yield of sargassum seaweed is about 10,000 kg dry per year.
Six metals were detected in alginate/lignin composed of Sr, Ta, Fe, Mg, Na, and K. Three non-metals in alginate/lignin were P, Si, and S. Content of six metals achieved a value from 0.1 to 54.9%, corresponding to Ta and Na, respectively. The content of six metals was 1 to 38.7%, corresponding to P and S, respectively. Nineteen oxides of alginate/lignin were shown including to SiO 2 , P 2 O 5 , SO 3 Figure 5) with DMSO solvent for running spectrum. The solvent peak was in the signal at 2.5 ppm ( Figure 5). when analysis of them was via XRF spectrum. CKa, OKa, NaKa, SKa, and SKb of alginate/lignin occurred in the energy range under 03 keV (Figure 4b). The results of SEM_EDS mapping presented the mass percentage of elements C, O, Na, and S of alginate/lignin corresponding to 22.16 ± 0.08, 41.55 ± 0.13, 27.62 ± 0.12, 8.66 ± 0.08 (%), respectively. The atom percentage of elements C, O, Na, and S of alginate/lignin were 31.2 ± 0.11, 43.91 ± 0.13, 20.32 ± 0.09, and 4.57 ± 0.04 (%), respectively (Table S1).             The 1 H-NMR spectrum of alginate/lignin gave signals at 0.852, 1.068, 1.158, 1.238, 1.361, and 1.535 ppm ( Figure 5) with DMSO solvent for running spectrum. The solvent peak was in the signal at 2.5 ppm ( Figure 5).

Discussion
The total antioxidant activity of alginate/lignin was higher than alginate in the previous notice [9,10] and polyphenol of sweet rowanberry cultivars [31]. Sodium alginate achieved a total antioxidant activity of 23.62 ± 17.52 and 188.54 mg ascorbic acid equivalent/g DW, corresponding to brown algae Sargassum duplicatum [9] and Sargassum polycystum [10], respectively. The reducing power activity of alginate/lignin was higher than that of alginate of brow algae Cystoseira schiffneri [32]. The total antioxidant activity of alginate/lignin demonstrated that alginate/lignin possessed antioxidants (218.73 ± 10.45 mg ascorbic acid equivalent/g DW), and that the assay is a pioneer test for further antioxidant assay. The total antioxidant activity of alginate extracted from brown algae Sargassum polycytum corresponded to 188.54 mg ascorbic acid equivalent/g DW [10]. Thus, the particles of alginate/lignin could lead to a synergism. Alginates play a role in the absorbent substance for metal, so their reducing power activity is usually high. Alginate/lignin showed the capacity of methylene blue movement [33] and metal in the industry [34]. Reducing

Discussion
The total antioxidant activity of alginate/lignin was higher than alginate in the previous notice [9,10] and polyphenol of sweet rowanberry cultivars [31]. Sodium alginate achieved a total antioxidant activity of 23.62 ± 17.52 and 188.54 mg ascorbic acid equivalent/g DW, corresponding to brown algae Sargassum duplicatum [9] and Sargassum polycystum [10], respectively. The reducing power activity of alginate/lignin was higher than that of alginate of brow algae Cystoseira schiffneri [32]. The total antioxidant activity of alginate/lignin demonstrated that alginate/lignin possessed antioxidants (218.73 ± 10.45 mg ascorbic acid equivalent/g DW), and that the assay is a pioneer test for further antioxidant assay. The total antioxidant activity of alginate extracted from brown algae Sargassum polycytum corresponded to 188.54 mg ascorbic acid equivalent/g DW [10]. Thus, the particles of alginate/lignin could lead to a synergism. Alginates play a role in the absorbent substance for metal, so their reducing power activity is usually high. Alginate/lignin showed the capacity of methylene blue movement [33] and metal in the industry [34]. Reducing power activity is necessary for numerous applications of alginate/lignin. The current study showed that the DPPH free radical scavenging activity of alginate/lignin was higher than one in the notice (EC 50 = 1.15 mg/mL) [35], and its highest value only corresponded to Trolox at 22.5 µg/mL (Table 2). DPPH free radical scavenging efficiency of alginate/lignin is only 1% of that of Trolox. α-glucosidase inhibition activity of alginate/lignin was ten times more than acarbose. The results demonstrated the potential of alginate/lignin in the application of diabetes medicine, functional food, or pharmaceutical raw materials. The results of α-glucosidase inhibition activity in the current study was higher than previous notices on benzoylphloroglucinols from Garcinia schomburgakiana [36] and flavonoids [37]. The former publications did not indicate any antioxidant activities of alginate/lignin.
The results on the anticancer activity of alginate/lignin showed that alginate/lignin was non-selective on tested cancer cells because the inhibition percentage of cancer cells is lower than that of fibroblast cells. Alginate/lignin was not toxic to cancer cells. Alginate/lignin did not have potential in the research and development of K treatment products. The results showed that alginate/lignin is non-toxic.  Figure S1). FTIR spectrum did not show the 1738 cm −1 peak that exists in the alginate structure ( Figure 1). Some typical peaks of lignin disappeared, such as the typical band for the symmetric and asymmetrical vibrations of the CH 3 group at 2920 and 2850 cm −1 , respectively, and the characterized peak for the C=C oscillation in the aromatic ring at about 1510 and 1460 cm −1 [39]. The band's disappearance showed the interaction of the two materials to form a stable composite system. DSC spectrum of alginate/lignin showed that the melting temperature was similar to lignin in the notice [40,41]. SEM_EDS, SEM_EDS mapping, XRD, and XRF have shown the morphology, elemental distribution, and content of metal and oxide. Some flattened flakes occurred, which may be the materials that were decomposed/torn and not attached to the particles alginate/lignin at the beginning. Synthetic alginate/lignin particles have a perfect spherical structure. The spheres are unequal in size, intertwined, and stick together. The XRD spectrum of the alginate/lignin system shows the formation of a crystalline phase with sharp peaks on the amorphous background. In previous studies, the XRD spectra of alginate and lignin often showed broad peaks of the carbon system, which are typical for the amorphous or poor crystalline structure of the materials [42,43]. Alginate and lignin formed sphere particles with the enhancement of the crystalline phase on an amorphous substrate. These results demonstrate the applicability of alginate/lignin in pharmaceutical materials and functional foods. The structural properties of alginate/lignin are necessarily analyzed via other solvent systems. In this study, 13 C NMR spectroscopy did not work because a suitable solvent was not found to conduct the 1 H NMR spectroscopy for liquid samples. The solvents DMSO, D 2 O, methanol, and acetone could be used for performing NMR spectroscopy of the alginate/lignin samples. The NMR spectrum and conditions for alginate/lignin derived from seaweed and corn by-products have not been noticed. The solvent of D 2 O and 01% CD 3 COOD with an internal standard of DSS and water reduction measurement technique was used for the 13 C NMR spectroscopy of sodium alginate [10] ( Figure S2). The 1 H NMR analysis of maize stalks lignin was conducted with C 2 D 6 OS solvent and tetramethylsilane as a standard for calibrating the peaks shift [44]. According to Mtibe et al., the shift from 1.5 to 2.4 ppm shows the presence of the group of aromatics [44] ( Figure S3). LD 0 and LD 100 are the highest and the lowest doses that cause 0% and 100% death of alginate/lignin-used mice, respectively. The mice were tested over three dose levels with six to eight mice (50% male, and 50% female) per dose, and the lethal dose was of 50% (LD 50 ), see Table 5. After drinking alginate/lignin for 30-45 min, the activity of the mice decreased, they laid still, and had diarrhea. Some mice had severe diarrhea and weak breathing and died within 2-4 h. The number of mice with diarrhea was proportional to the numbers of mice administered the oral dose ( Table 6). The surviving mice recovered, and diarrhea stopped after 8-24 h of drinking the sample. These mice ate bran pellets and drank water normally. Their stool, urine, and weight were not unusual after recovery. Abnormalities in circulatory function, digestive, sensory reflexes, excretory status, and hair of mice were absent. Mice lived within the first 72 h of observation and for 14 days of follow-up. The recovery time was proportional to the test dose level. Dead mice during the observation period were operated on for further studies. For example, macroscopic examination showed that the internal organs and organs (heart, lungs, liver, stomach, and intestines) were not abnormal. The alginate/lignin material exhibited acute oral toxicity in mice with an LD 50 value of 8.15 ± 0.32 g/kg, corresponding to an oral dose of 55 kg average adult of 36.443 ± 1.43 g/day (the dose conversion factor between adults and mice is 12.3). After taking the alginate/lignin material, some mice showed diarrhea and decreased activity after 30-45 min; no abnormalities in circulation and sensory reflexes were found; then the mouse died within 2-4 h. Brown seaweed Sargassum polycystum was selected, cleaned, and stored below 10 • C during transportation to the laboratory. Salt and impurities of the seaweed were removed with fresh water in the laboratory and then the seaweed was dried until its moisture content of 19 ± 1%. After that, the seaweed was ground into a fine powder and stored at 10 • C for further studies. Phlorotannin was separated from brown algae using 96% ethanol in the ratio of 1/10 (w/v). Fucoidan was separated via H 2 SO 4 solution (pH 2) for two hours at 80 • C. After fucoidan extraction, the liquid-separated residue was kept for four hours at 60 • C with the ratio Na 2 CO 3 (pH 9)/dried seaweed (40/1, v/w) for extracting alginate. Alginate was extracted from the residue. After that, the mixture was hot-filtered to collect the filtrate and added to 10% CaCl 2 solution (the ratio of CaCl 2 to alginate was 2.0/1.0) to form a calcium alginate precipitate. Calcium alginate was washed with distilled water and decolorized with 20-30 mL of chlorine solution (1% chlorine solution/100 g calcium alginate antioxidant) for 30 min, and the residual chlorine was removed with fresh water. Bleached calcium alginate was converted to alginic acid by the solution (pH 2.0). Next, the alginic acid was converted to sodium alginate by dissolving the alginic acid in a Na 2 CO 3 solution with a Na 2 CO 3 /alginic acid ratio of 0.35/1 (w/w). The mixture was filtered to remove the insoluble fraction of Na 2 CO 3 solution and then sodium alginate was precipitated in 40% ethanol. The sodium alginate precipitate was filtered and dried at 50 • C under a vacuum to obtain the sodium alginate dry powder.

Extraction of Alkaline Lignin
Corn by-products (corn stalks and leaves) were extracted from polyphenols and chlorophyll by 96% ethanol and were dried in the aerated shade until the moisture was below 20 ± 1% for ground and were stored in breathable solid bags for further studies. Samples were soaked in 4N NaOH at 80 • C for 150 min and filtered. The filtrate was adjusted to pH 5 to form a cellulose precipitate, and then the filtrate was collected. The filtrate was adjusted to 70% ethanol concentration using 96% ethanol to obtain a hemicellulose precipitate. After the removal of hemicellulose, the filtrate was adjusted to pH 2 to collect the precipitated lignin. After centrifugation, the lignin precipitate was washed with clean water and dried at 50 • C under vacuum conditions to constant weight.

Preparation of Alginate/Lignin Particles
Alginate/lignin powder was prepared via a spray-drying method. Sodium alginate in Section 4.1.2 (5% w/v) was dissolved in de-ionized water at 80 • C for 30 min with stirring at 500 rpm, followed by lignin addition in Section 4.1.3 (2% w/v, in 0.1 N NaOH) according to the alginate-to-lignin ratio of 90:10 and assimilated at 500 rpm. Alginate/lignin (90/10) spray drying resulted in the best recovery rate, compared with other spray drying conditions. Tween 80 and tripolyphosphate were, in turn, added to the ultrasound-assisted mixture at the rate of 0.1%, compared with the total content of alginate and lignin. The mixture was continuously homogenized during spray drying. The spray drying conditions were as follows: a pump speed of 1500 mL/h, pressure in the chamber of 0.2 atm, air heating temperature of 180 • C, and outlet temperature of 90 • C. Alginate/lignin powder was sifted and preserved in a sealed aluminum bag for further evaluation of the antioxidant activity, anticancer activity, physico-chemistry characteristics, and acute toxicity.

Total Antioxidant Activity
Briefly, 100 µL sample was diluted ten times with distilled water and mixed into solution A (0.6 M H 2 SO 4 , 28 mM sodium phosphate, and 4 mM ammonium molybdate) for 5 min. Then, the mixture was incubated at 95 • C for 90 min, and the mixture absorbance at the wavelength of 695 nm was measured. Ascorbic acid was the standard substance [45].

Reducing Power Activity
The determination of reducing power activity was according to the description of Zhu et al., (2002) [46]. Briefly, 500 µL sample was mixed in 0.5 mL of phosphate buffer (pH 7.2), 0.2 mL of 1% K 3 [Fe(CN) 6 ], and kept for 20 min at 50 • C. Then, the mixture was added to 500 µL of 10% CCl 3 COOH, 300 µL distilled water, and 80 µL of 0.1% FeCl 3 for vortexing for 5 min. The absorbance measurement of the compound was at 655 nm with FeSO 4 used as the standard.

DPPH Free Radical Scavenging Activity
DPPH in 80% methanol was diluted to form 150 µM of DPPH solution and was used immediately. Each well on a 96-well plate was then added to 200 µL of 150 µM DPPH and 25 µL of the sample at different concentrations. The mixture measured the optical density (OD) at 517 nm for 30 min with a jump of 5 min/time. The positive control was Trolox [47].
The percentage of DPPH free radical scavenging activity was calculated using the following formula: OD t and OD c were the optical density of the test sample and the control, respectively, and these OD values have been subtracted from the OD value of solution wells of non-containing DPPH. SC 50 value (test concentration of 50% DPPH free radical scavenger) was determined based on a standard curve of optical density values of samples at different concentrations (using Prism software with multi-parameter nonlinear regression and R 2 > 0.9).

α-Glucosidase Inhibition Activity
Add 120 µL of sample and 20 µL of α-glucosidase (1 unit/mL) to each well on a 96-well plate to incubate the mixture at 37 • C for 15 min. Then, add 20 µL of 5 mM pnitrophenyl-α-D glucopyranoside solution/well and keep for 15 min at 37 • C. Finish the reaction by adding 80 µL of 0.2 M Na 2 CO 3 solution/well. The absorbance measurement of the mixture was at the wavelength of 405 nm, and the positive control was acarbose [48].
The percentage of α-glucosidase inhibition was calculated using the formula: OD t and OD c were the optical density of the test and control samples, respectively. The OD value of these samples did not include blank OD value (without α-glucosidase). IC50 value (concentration of 50% α-glucosidase inhibitor test substance) was determined based on the standard curve of optical density values of samples at different concentrations (using Prism software with R 2 > 0.9). After obtaining the optical density values at 492 nm and 620 nm (denoted OD 492 and OD 620 ): Calculate the value of OD TS = OD 492 − OD 620 , Calculate OD 492 (or OD 620 ) = OD av − OD blank , Calculate the percentage (%) of cytotoxicity according to the following formula: where, OD av : average OD value of the cells well, OD blank : OD value of blank well (no cells), OD TS : the OD value of the test sample calculated from Formulas (1) and (2), OD C : OD value of the control specimen calculated from Formulas (1) and (2). IC 50 was determined using Prism software with regression multi-parameter non-linearity and R 2 > 0.9 [49,50].

Functional Groups
The sample was measured at a wavelength range from 4000 to 500 cm −1 on an IRAffinity-1S of Shimadzu.

Surface Morphology and Elemental Composition
Surface morphology and elemental composition of alginate/lignin were measured via scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) (SEM_EDS), respectively.

Thermal Analysis
Thermal analysis of alginate/lignin was conducted using the differential scanning calorimetry (DSC) method on a NETZSCH DSC 204F1 Phoenix. The surveyed temperature was 30 • C/10.0 (K/min)/400 • C and the atmosphere of N 2 was 40.0 mL/min/N 2 , 60.0 mL/min.

NMR Spectra
NMR spectra were measured on a Bruker AVANCE Neo 600 MHz instrument at 70 • C, using DMSO as solvent and DSS as an internal standard with a water-reduced measurement technique.

Investigation of Acute Oral Toxicity
Ten mice were kept in fasting for at least 12 h before giving them the maximum possible oral dose of the test sample with a volume of 50 mL/kg [51]. Mice were kept in the same condition, fed the same volume of the test sample, and their general movement, behavior, hair state, eating, urination, and death within 72 h were recorded. If the mouse shows no abnormality or dies after 72 h, continue monitoring for 14 days. There are three possible cases: -Case 1: After the mice drank the test sample, the mice did not die, continue determining the highest possible dose of the test sample through the needle without causing the mouse to die (D max ). -Case 2: After giving the test sample to mice, the mortality rate is 100%, then try 1 /2 dose of the first dose until a minimum amount is lethal to 100% of mice (LD 100 ) and a maximum amount that is not lethal to rats (LD 0 ). Conduct testing to determine LD 50 . Divide mice into four lots, each batch of 6 mice. Divide the four doses by an exponential interval from LD 0 to LD 100 . At doses close to LD 50 , the number of mice was increased for more accurate measurements and monitored for 72 h to record the movements of mice, and the number of dead mice in each batch, and to calculate the mortality fraction to find LD 50 . -Case 3: After giving the test sample, the death rate is lower than 100%, the dose of LD 100 cannot be determined, and the LD 50 cannot be determined. In this case, it is only possible to determine the maximum dose which is not lethal to the mice, called sub-lethal dose (LD 0 ). The LD 50 value determined via the Behrens method based on two doses close to the LD 50 lethal dose: in which: D 1 is the lethal dose a% of test animals (dose close to 50%). D 2 is the deadly dose b% of test animals (the upper dose is nearly 50%). d = D 2 − D 1 is the dose step between 2 doses near LD 50 .

Data Analysis
Each experiment was triplicated (n = 3) and the results were represented as mean ± SD. ANOVA and statistical analyses were conducted on the software MS Excel 2010. IC50 determination of cell toxicity activity, DPPH free radical scavenging activity, and α-glucosidase inhibition activity were analyzed using GraphPad 7.0 Prism software.

Conclusions
Alginate/lignin is a potential antioxidant material for pharmaceutical materials, functional foods, and for supporting diabetes treatment. Alginate/lignin exhibited high antioxidant as evident from its total antioxidant activity and reducing power activity, especially its α-glucosidase inhibition activity. Alginate/lignin showed insignificant DPPH free radical scavenging activity, and as well insignificant effect in three cancer cell lines (Hep G2, MCF-7, and NCI H460) and fibroblast cells. Alginate/lignin was in a thermally stable regular spherical shape containing six metals, three non-metals, and nineteen oxides. Typical elements of the alginate/lignin structure were C, O, Na, and S. When alginate was combined with lignin to form a unified crystal, some functional groups of alginate and lignin did not exist in the new alginate/lignin structure. The acute toxicity test of alginate/lignin in mice presented an LD 0 of (3.91 g/kg) and LD 100 of (9.77 g/kg). The current study showed potential applications of active alginate/lignin formed by combining alginate from Sargassum polycystum and lignin from corn by-products in treating anti-aging, diabetes, and digestive system diseases.