Isolation And Characterization of Biopolymers From Chenopodium Quinoa And Their Immunostimulatory Activity

Non-starch polysaccharides derived from natural resources play a signicant role in the eld of food science and human health due to their extensive distribution in nature and less toxicity. In this order, the immunostimulatory activity of a non-starch polysaccharide (CQNP) from Chenopodium quinoa was examined before and after and deproteination in murine macrophage RAW264.7 cells. The chemical composition of CQNP and deproteinated- CQNP (D-CQNP) were spectrometrically analysed that revealed the presence of carbohydrate (22.7 ± 0.8% and 39.5± 0.8%), protein (41.4± 0.8% and 20.8± 0.5%) and uronic acid (8.7±0.8% and 6.7± 0.2%). The monosaccharide composition results exposed that CQNP possesses a high amount of arabinose (34.5±0.3) followed by galactose (26.5±0.2), glucose (21.9±0.3), rhamnose (7.0±0.1), mannose (6.0±0.1) and xylose (4.2±0.2). However, after deproteination, a difference was found in the order of the monosaccharide components, with galactose (41.1±0.5) as a major unit followed by arabinose (34.7±0.5), rhamnose (10.9±0.2), glucose (6.6±0.2), mannose (3.4±0.2) and xylose (3.2±0.2). Further, D-CQNP potentially stimulate the RAW 264.7 cells through the production of nitric oxide (NO), upregulating inducible nitric oxide synthase (iNOS) and various pro-inammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α). Moreover, stimulation of RAW 264.7 cells by D-CQNP takes place along the NF-κB and the MAPKs signaling pathways through the expression of CD40.


Introduction
Quinoa (Chenopodium quinoa Willd.) is one of the ancient Pseudo-cereal crops belonging to the family of Chenopodiaceae. It is a broadleaf non-grasses plant originated from South America and their seeds have been included in cereal-based foods. It is largely produced by small farms and associations in the Andean region of South America. Nevertheless, it is currently cultivated in more than 70 countries, including Kenya, India, United States and many European countries. It considers as a starch-rich component that is high in carbohydrates and also contains starch and small amounts of sugars. Quinoa starch content varies from 51 to 61% and its granule diameter is 3 m [1]. Currently, quinoa is gaining more attention in the nutrition and pharmaceutical industries because it is rich in nutrients, especially the dietary ber content (1.1 % to 16.3%) is higher than other cereals such as rice (0.4%), wheat (2.7%) and corn (1.7%). Also, there is no gluten compared to other grains [2]. Quinoa is important for patients with celiac disease because it is believed that gluten-free ber de ciency can be prevented by adding quinoa seeds to the diet [2].
Previous studies on quinoa proteins have shown that it contains a balanced essential amino acid composition and high levels of essential amino acids. Its value is higher than that of common grains [3].
The lipid content of quinoa is two to three times higher than that of general grains and contains high levels of unsaturated fatty acids, which contribute an important role in nutrition [2]. In addition, it contains some important micronutrients such as minerals and vitamins, as well as signi cant amounts of bioactive components such as polyphenols, avonoids that exhibit various biological activities (Alvarez-Jubete, Wijngaard, et al., 2010). It has been reported that quinoa can help reduce the risk of complications such as diabetes, heart disease, obesity, anemia and dyslipidemia [6].
Non-starch polysaccharides are the important components of dietary ber that are formed by the binding of many monosaccharide units by glycosidic bonds, which play an important role in regulating cellular growth and development. Several studies are reported that non-starch polysaccharides of cereals have shown countless biological activities including immunogenic [7], anti-oxidants [8], antiatherosclerogenic [9], anti-cancer [10] and anti-diabetics [11]. Of these, immunostimulatory activities are directly connected to health bene ts. Promoting innate immunity in a controllable way will enhance the host defence activities [12]. Polysaccharides from various natural sources such as plants [13], algae [14], fungi [15] stimulated the innate and cellular community via interactions with T cells, monocytes, macrophages, and polymorphonuclear lymphocytes [16]. When stimulated, the macrophage can destroy the pathogens directly by phagocytosis and indirectly through the secretions of NO and various cytokines TNF-α, IL-1β, and IL-6 ( L. Zhang & Wang, 2014). Usually, non-starch polysaccharide stimulate the macrophages by binding to pattern recognition receptors (PRRs) such as toll-like receptors (TLRs), Dectin-1, and complement receptor type 3 (CR3), and trigger the signal transduction pathways including phosphoinositide-3-kinase (PI3K)/Akt, mitogen-activated protein kinases (MAPKs), as well as transcription factors such as nuclear factor (NF)-κB and activator protein (AP) [7].
In this study, we reported the extraction of non-starch polysaccharides (CQNP) and deproteinated CQNP from C. quinoa and their structural characterization and immunomodulatory properties.
Material And Method

Isolation of non-starch polysaccharide
A non-starch polysaccharide (CQNP) was isolated from C. quinoa using the α-amylase treatment. The C. quinoa seeds were collected from the local market of XiNing city, QingHai province, China. The seeds thoroughly cleaned with distilled water, dried at 45°C, and were milled into a ne powder. For CQNP extraction, 20 g of powdered sample was extracted with 200 mL of distilled water at 65°C. The collected extract was centrifuged at 4000 × g for 15 min. Afterwards, the extract was mixed with an equal volume of phosphate-buffered saline (PBS, pH 6.0) and incubated with 4 mg of α-amylase (Sigma-Aldrich, St. Louis, MO, USA) for 16 h at 55°C. The reaction was arrested by raising the temperature to 100°C for 10-15 min. Finally, the reaction mixture was ltered through a 110 nm size of the membrane, dialyzed against distilled water and freeze-dried.

Deproteination of CQNP
About, 1mg of CQNP was dissolved in 0.1 M sodium phosphate buffer and then incubated with 10-20% of proteinase K (w/v) in a water bath at 58°C for 24 h. The reaction was stopped by keep the sample at 100°C for 10 min. Afterwards, the reaction mixture was centrifuged (10000 rpm for 10 min) and the supernatant was dialyzed against distilled water and freeze-dried [21]. The deproteinated CQNP was named as D-CQNP.

Chemical and Monosaccharide composition analysis
The presence of carbohydrate, protein and uronic acid were examined by phenol-sulfuric acid (Dubois et al., 1956), folin-phenol reagent (Lowry et al., 1951) and m-hydroxydiphenyl reaction [24], respectively. For the monosaccharide composition analysis, three milligrams of the sample was hydrolyzed with 0.5 mL of 4 M tri uoroacetic acid (TFA) at 100°C for 6 h. The hydrolyzed product was reduced by NaBD 4 and then acetylated using acetic anhydride. The monosaccharide composition analysis was carried out by a gas chromatography mass-spectrometry analysis (GC-MS, 6890 N/MSD 5973, Agilent Technologies, Santa Clara, CA, USA) equipped with the HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm; Agilent Technologies).

Molecular weight analysis
The average Mw and Rg values were estimated by following the method of Tabarsa et al . Brie y, the sample was solubilized in distilled water (2 mg/mL) and heated for 30 S using a microwave bomb (#4872; Parr Instrument Co., Moline, IL, USA). Then, the samples were ltered through a cellulose acetate membrane (3.0 µm pore size; Whatman International) and injected into a TSK G5000

Cell proliferation and NO production
In this study, the immunostimulatory activities of CQNP and D-CQNP were tested on RAW 264.7 macrophage cells (ATCC; Manassas, VA, USA and ATCC, Rockville, MD, USA). RAW 264.7 cells were grown in RPMI-1640 medium in the presence of 10% fetal bovine serum (FBS) under 5% CO 2 atmosphere condition. The cell proliferation activity was tested using WST-1 assay. RAW 264.7 cells were cultured in a 96-well microplate (1×10 6 cells/mL) with different concentration of CQNP or D-CQNP (12, 25 and 50 µg/mL) for 24 h at 37°C. Add 100 µL of 10% WST-1 solution into the wells and extend the incubation for 1 h. The absorbance was read at 450 nm and the proliferation activity (%) was calculated using the following equation. Similarly, the cells were treated with CQNP or D-CQNP and the NO production was measured using Griess reaction (Green et al., 1982).
Cell proliferation (%) = Absorbance of sample/Absorbance of control x 100 2.8 Real-time PCR analysis RAW 264.7 cells (1×10 6 cells/mL) were incubated with polysaccharide or LPs (1 µg/mL) at the concentration of 50 µg/mL in a 96-well microplate under 5% CO 2 atmosphere for 18 h at 37°C. The total RNA content was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was constructed using oligo-(dT) 20 primer and Superscript III RT (Invitrogen) by following the steps provided in the kit. PCR ampli cation was performed by a Real-Time PCR system using Fast Start DNA Master TB Green II kit (Takara Bio Inc., Shiga, Japan) using the speci c primers (Table. 1)  Table 1 Sequences of the primers used in real-time PCR analysis

Gene
Sequences of the primers

Statistical analysis
All experiments were performed in triplicate (n = 3), and the value was expressed as the average and standard deviation (SD). The statistical analysis was carried out using SPSS software (Version 16; SPSS Inc., Chicago, IL, USA). The differences between groups were analyzed using one-way analysis of variance (ANOVA) and Duncan's multiple-range test, and a p-value of < 0.05 was considered statistically signi cant.
Result And Discussion

Proximate composition of CQNP and D-CQNP
The non-starch polysaccharide from C. quinoa was extracted by hot water extraction and α-amylase treatment. The yield and chemical composition of CQNP was displayed in Table. 1. The amount of extracted CQNP was 1.7% of the starting raw material. Primarily, the CQNP constituted by carbohydrate (22.7 ± 0.8%), protein (41.4 ± 0.5%) and uronic acid (8.7 ± 0.3%). Of these result, CQNP has a high protein content because the protein is not hydrolyzed before the extraction of the non-starch polysaccharides [27]. Further, the existence of free protein in the polysaccharide maybe disturbs the structural and pharmaceutical properties. Hence, the protein content was removed from CQNP by proteinase K that showed notable changes in protein content. Totally, 20.4% of D-CQNP was obtained, which consisted of 39.5 ± 0.8% carbohydrate, 20.8 ± 0.5% protein, and 6.7 ± 0.2% uronic acid.

Molecular weight analysis
The molecular weight of polysaccharide is an important factor to be associated with various biological activities [29]. The average molecular weight of CQNP and D-CQNP were examined using a multi angle laser light scattering technique (MALLS) through a high-performance size exclusion column (HPSEC). As shown in Figure.2 and Table. 1, CQNP exhibited two different peak at the elution time between 11.6 and 14.7 min (Peak I) and 15.4 and 17.9 min (Peak II). The average molecular weight of CQNP calculated using the MALLS system, which was 148.5 ± 36.6 kDa and 24.9 ± 1.9 kDa, respectively. Next, the D-CQNP emerged from the HBSEC as three distinct peaks between 11 and 13.7 min (peak I), 13.8 and 15.4 min (peak II), and 15.5 and 17.8 min (peak III) indicating that the molecular weight of 774.3 ± 13.5 kDa and 90.9 ± 7.7 kDa, and 77.2 ± 6.8 kDa, respectively. Similarly, the non-starch polysaccharide has a molecular weight of 15-150 kDa when isolated from green gram using hot water extraction [30]. Further, Rg values were calculated from the spectrum, which showed approximate sizes of the molecules ranging from 60.3-74.6 nm for CQNP and 57.5-62.3 for D-CQNP.

Cell proliferation and nitric oxide production
Generally, macrophages or monocytes play a signi cant role in both immunities, which considered being important immunocytes to protect the host from the pathogen, including cancer (Kohchi et al., 2004). In this study, the immunostimulatory effect of CQNP and D-CQNP were tested on murine RAW 264.7 macrophage cells. Initially, the RAW 264.7 cells were incubated with different concentration of CQNP and D-CQNP for 24 h and assess the cell proliferation activity using WST-1 assay. The cell proliferation results were compared with LPS treatment (1µg/mL). As shown in Figure. 3a, RAW 264.7 cells showed an enhanced level of cell proliferation than LPs treatment when incubating with CQNP and D-CQNP. While increasing the concentration the cell proliferation activities were also increased. Further, the extracted non-starch polysaccharides did not show any toxic effects to the RAW 264.7 cells at the tested concentration. Subsequently, the NO production that could occur during treatment with different concentrations of CQNP and D-CQNP was measured in RAW 264.7 cells. NO is one of the primary molecule produced by stimulated macrophages that shows potent cytotoxic effects on pathogen and cancer cells. It is also performed as an intracellular messenger in the regulation of various physiological process [33]. In this study, RAW 264.7 cells treated with D-CQNP exhibited higher NO production than CQNP, demonstrating that the deproteinated non-starch polysaccharide has a strong immunostimulatory function ( Figure. 3b).

Expression of iNOS and cytokines
iNOS is one of the major enzyme involved in the NO production in stimulated macrophages [34]. Therefore, the experiments are being conducted to examine whether the increase in NO production is associated with increased iNOS activity and/or gene expression. Here, the gene expression of iNOS and various cytokines such as IL-1β, IL-6, IL-10 and TNF-α were studied by Real-Time PCR analysis. As shown in Fig. 4, the RAW 264.7 cells treated with 50 µg/mL of D-CQNP revealed signi cantly higher iNOS expression than those treated with CQNP. These ndings are consistent with the result of NO production. Several studies reported that the increase in NO production in RAW 264.7 cells is associated with an increase in iNOS expression [35][36][37]. Similarly, the mRNA expression of various cytokines including IL-1β, IL-6, IL-10 and TNF-α was signi cantly increased (p 0.05) when RAW 264.7 cells were treated with D-CQNP (50 µg/mL). These results con rmed that the RAW 264.7 cells are stimulated by D-CQNP by enhance the NO production through the mRNA expression of iNOS and various cytokines.

Western blot analysis
In addition, the underlying mechanisms for the activation of RAW 264.7 cells were explored by evaluating the protein expressions of NF-κ B and MAPK (p38, ERK, and JNK) signaling pathways using Western blot analysis. NF-κ B is one of the major transcriptional factors that controls pro-in ammatory gene expression. The treatment of RAW 264.7 cells with CQNP and D-CQNP promote the phosphorylation of NF-κB (Fig. 5). In a previous study in support of this result, it was reported that the translocated NF-κB induce the pro-in ammatory mediators expression including iNOS and cytokines ( Liu et al., 2017). Similarly, the pathways of MAPKs also play a major role in regulating cellular functions and regulating pro-in ammatory responses (Kim & Choi, 2010). In particular, p38 MAPKs play an important role in responding to cellular processes [42]. The phosphorylation of MAPKs, p38, ERK, and JNK has remarkably induced in RAW 264.7 cells by the treatment of CQNP and D-CQNP. Of these, the phosphorylation of ERK and JNK was markedly higher than others. Furthermore, D-CQNP showed an expression equivalent to a positive control (LPS). Hence, these results demonstrated that the immunostimulating activity of CQNP and D-CQNP was related to the activation of NF-κB and the MAPKs signaling pathways.

Flow cytometry analysis
Flow cytometry analysis was used to evaluate the surface biomarkers on RAW 264.7 cells treated with CQNP or D-CQNP (50 µg/mL) for 24 h. In this study, two biomarkers, CD11b and CD40 were used; CD11b is an indicator of activated macrophage that can be detected from the surface of granulocytes, monocytes, macrophages and natural killer cells and subsets of B-and T-cells. However, CD40 is a costimulatory molecule that induces the IL-2 and TNF-α through T-cell activation [43]. When treating RAW 264.7 cells with CQNP or D-CQNP at 50 µg/mL the expression of CD11b and CD40 was 23.82% and 27.90% for CQNP and 80.25% 81.33% for D-CQNP, respectively ( Figure. 6). Moreover, the activity of D-CQNP more or less similar to the positive control (LPS). This result was agreed in previous studies, where CD11b and CD40 expression were found to be higher when macrophages were activated by micro bers [44]. Furthermore, the expression of CD40 was higher than CD11b, which demonstrates the stimulation of RAW 264.7 cells by non-starch polysaccharide of C. quinoa through the NF-κB and MAPK signaling pathways by the production of pro-in ammatory cytokines and NO production via CD40 expression.

Figure 6
Evaluation of CD11b and CD40 expression in CQNP and D-CQNP treated RAW 264.7 cells. Cells treated with LPS and the medium alone (RPMI) served as a positive and negative control. The letters a and b indicate signi cant differences (p < 0.05) between the polysaccharide treatments. CD11b, cluster of