Effect of encapsulated edible halophyte with different biopolymers on the inhibition of sodium absorption in mouse

Abstract The purpose of this study was to investigate the effects of edible halophyte Salicornia herbacea encapsulated with biopolymers on inhibition of sodium absorption in mouse. Salicornia herbacea encapsulated with four biopolymers (pectin, chitosan, cellulose and dextrin) were fed to mice for 48 hr, and inhibiting sodium absorption was measured. In primary in vitro condition, fresh Salicornia herbacea encapsulated with 1% cellulose had 40% binding rate. Juice residue Salicornia herbacea encapsulated with 1% chitosan had the highest sodium binding rate by 50%. In mouse model, fresh, juice, and juice residue of Salicornia herbacea encapsulated with 4% chitosan had the highest sodium absorption inhibitory rate by 19%. These results indicate that biopolymer‐encapsulated Salicornia herbacea could be combined with sodium under in vitro condition, and Salicornia herbacea encapsulated with biopolymers reduced sodium absorption in a mouse model. Chitosan and cellulose had the highest sodium absorption inhibitory effects compared with the other biopolymers.


| INTRODUC TI ON
Sodium is the principal cation in extracellular fluid in the body and is an essential nutrient necessary for maintenance of plasma volume, acid-base balance, transmission of nerve impulses and normal cell function (Organization, 2012). In healthy individuals, nearly 100% of ingested sodium is absorbed during digestion, and urinary excretion is the primary mechanisms for maintaining sodium balance. However, a high intake of sodium increases blood pressure and the risk of cardiovascular disease (Wang & Labarthe, 2011); thus, reduced dietary sodium intake has been recommended for at least a half century (Alderman & Cohen, 2012). Data from around the world suggest that the population average sodium consumption is well above the minimal physiological needs, and in many countries is above the value recommended by the 2002 Joint World Health Organization/Food and Agriculture Organization of the United Nations (WHO/FAO) Expert Consultation of 2 g sodium/day (equivalent to 5 g salt/day) (Brown et al., 2009;Organization, 2003). Therefore, reduction in sodium intake is beneficial to reduce the risk of cardiovascular disorders related to the high sodium intake. For that reasons, reduction in sodium intake has been extensively studied in food industries around the world. For instance, sodium replacer or saltiness enhancement such as SaltWise®, sub4salt®, Premier TM Light Salt 50/50 Blend, SodiumSense TM System V1 ~ V3, Salt Rite TM , AlgySalt®, Na-K TM , Kasomel TM , Sodium gluconate, PELL TM K Low Sodium Baking Powder, Reducit® 0402/20-MG-L, FlakeSelect TM Flour Sea Salt, or monosodium glutamate has been developed around the world. In the mean times, Cermak et al. (2002) reported that ammonia inhibits sodium absorption in the proximal colon of rats and the effect is independent of the inhibition of the Na + -H + exchanger isoforms NHE 2 (sodiumproton-exchanger subtype) and NHE 3 and requires the presence of chloride ions (Cermak et al., 2002). Tenapanor is an inhibitor of the sodium proton (Na + -H + ) exchanger NHE3, which inhibited sodium uptake in the gastrointestinal tract (Spencer et al., 2014). Linz et al. (2016 reported that reduction in sodium absorption by NHE3 inhibition in the gut lowered high blood pressure (Linz et al., 2016).
Although many sodium replacers and sodium enhancement have developed, no one can absolutely replace the natural functions of sodium (or salt) such as saltiness, flavor or antimicrobial activity. Therefore, we have developed a different approach to reduce sodium intake without reduction in natural sodium functions in foods, that is, inhibition of sodium absorption without sodium replacement or sodium reduction in foods after ingestion. Our several preliminary studies found that biopolymer encapsulation with halophyte decreased sodium absorption and increased sodium excretion without reduction in natural sodium function after their ingestion. Therefore, the aim of this study was to investigate the effects of edible halophyte Salicornia herbacea encapsulated with biopolymers on inhibition of sodium absorption in mouse, and to determine the binding rate of halophyte and various biopolymers under the in vitro condition.
2. Powder sample: Fresh Salicornia herbacea was dried at 70℃ for 48h and ground using blender for 1min.
3. Juice sample: Fresh Salicornia herbacea was squeezed using a juice extractor, and juice was collected.
4. Juice residue sample: Fresh Salicornia herbacea was squeezed, and residue of juice was collected.
Four Salicornia herbacea samples were mixed with DW in 1:10 ratio for 1hr using a magnetic stirrer ( Figure 1).

| Preparation of four biopolymer solutions
Four biopolymer (pectin, chitosan, cellulose, and dextrin) solution was prepared as follows: F I G U R E 1 A schematic flow diagram of the four Salicornia herbacea solutions 1. For initial biopolymer solution preparation, four biopolymers were mixed with DW (deionized water) and final volume of four biopolymer solutions was 1 to 5% v/w, respectively.
2. Solutions were stirred for 12 hr using a magnetic stirrer to mixing.

| Encapsulation of four Salicornia herbacea with four biopolymers
Four Salicornia herbacea (fresh, powdered, juice, and juice residue) solutions were mixed with four biopolymer solution using a magnetic stirrer for 30 min.

| Sodium binding rate of Salicornia herbacea encapsulated with four biopolymers in an in vitro condition
The sodium binding rate of four Salicornia herbacea solutions with four biopolymer solutions was measured using digital salimeter (DX223, Metelo-Toledo). Briefly, a probe of salimeter was placed into fifty mL of Salicornia herbacea encapsulated with biopolymer solution, and then, sodium concentration was analyzed.

| Preparation of animal diet
The dietary groups were divided into 20 (4 experimental group × 5 treatments) and diet was prepared as follows: First group (Figure 2) was divided into 5 treatments as follows: To make Salicornia herbacea encapsulated with biopolymers, four Salicornia herbacea solutions (1:10 ratio with DW) and two NaCl were mixed with different biopolymer (pectin, chitosan, cellulose, and dextrin) solutions for 30min. b Salicornia herbacea samples were prepared as follows: fresh was ground using blender; powdered was dried at 70℃ for 48h and ground; juice was squeezed using a juice extractor, and residue of juice was collected. c Biopolymers (pectin, chitosan, cellulose, and dextrin) made solution to a final volume of 1-5% v/w.

| Analysis of inhibition of sodium absorption in the mouse model
Inhibition of sodium absorption rate (%) in mouse models was determined with different sodium concentrations between consumed sodium in diet and sodium concentration in feces and urine of mice.
To determine inhibition of sodium absorption rate, animal diet was mixed with DW in 1:2 ratio and feces and urine of mice were mixed with DW in 1:2 ratio, and then, sodium concentration was measured using digital salimeter (DX223, Metelo-Toledo, Switzerland), respectively. The measurement was replicated five times.

| Statistical analysis
Statistical analyses were performed for three independent batches of samples, and a total of one hundred mice were used for analysis with three time repetitions. The data for each batch regarding inhibition of sodium absorption were analyzed using ANOVA with the SAS software (SAS Inst. Inc.). Significant differences (p < .05) between the mean values for different groups were determined for inhibition of sodium absorption (n = 5). were fed for 48hr in mouse models, and then, the concentration of sodium in consumed diets and feces and urine of mice was analyzed. reported that chitosan is effective in the uptake of transition metal since the amino groups on chitosan chains serve as coordination sites (Muzzarelli, 1983). The adhesive properties of chitosan in a swollen state have shown to persist well during repeated contacts of chitosan and the substrate, which implied that, in addition to the adhesion by hydration, many other mechanisms, such as hydrogen bonding and ionic interactions, might also have been involved (Lehr et al., 1992).

The inhibition of sodium absorption of fresh
In general, three steps are involved in the encapsulation of bioactive agents: 1) the formation on the wall around the materials to be encapsulated; 2) ensuring that undesired leakage does not occur; and 3) ensuring that undesired materials are kept out (Gibbs, 1999;Mozafari et al., 2008). Biopolymers are capable of trapping Na + in the gastrointestinal tract, and gel matrix formed by biopolymer is excreted in the feces. This is why biopolymer encapsulation reduced sodium absorption in mouse models. Second possible mechanism would be resulted in increased solution viscosity. In general, an increase in solution viscosity is responsible for a decreased transit time and digestion rate of the ingested food in the gastrointestinal tract .
Our previous study found that dietary biopolymers increased the viscosity and decreased digestion of the contents of the small intestine . In this study, biopolymer-encapsulated Salicornia herbacea increased viscosity and decreased diffusion of sodium ion in the gastrointestinal tract of mice and subsequently decreased sodium absorption in mice. Third possible mechanism would be electrostatic interaction of biopolymers. Chitosan has been insisted that chitosan entraps lipids in the intestine, because of its cationic nature (Hur et al., 2009;Kanauchi et al., 1995). Several studies reported that the positive charge on chitosan has been found for its enhanced bioadhesion to negatively charged cell membranes. This enables the site-specific applications in controlled delivery systems (Aksungur et al., 2004;He et al., 1998;Şenel et al., 2000). The authors assume that the positive charged chitosan under the acidic condition and negative charged chitosan under the alkali condition could be combined with Na + although ionic crosslink of chitosan is depending on pH and degree of acetylation. Chitosan has been shown to form complexes with a large number of different polyanions, such as DNA, alginates, pectins, xanthan, glucosaminoglycans, and carboxymethyl cellulose (Nilsen-Nygaard et al., 2015). Several studies also found that cellulose is attractive sorbent in terms of Na + ion binding. Deshpande et al. (2008) reported that the interaction between the positively charged Na ion and the partially negatively charged OH groups on cellulose is found to be purely ionic (Deshpande et al., 2008). Na + ions can combine with cellulose because hydrated alkali ions (Na + ) present in aqueous systems swell the cellulose by penetration into it and also an exchange of hydrated shell with OH groups of cellulose can occur. Electrostatic interaction between Na + ions and cellulose can occur in the absence of ligand in the solution. Therefore, edible dietary fibers in Salicornia herbacea and Na + ion could be combined with electrical charged chitosan and cellulose in this study. As result of this study, the authors assume that possible mechanism for inhibition of sodium absorption by Salicornia herbacea encapsulated with biopolymers is believed to originated from electrostatic attractive of chitosan or cellulose in the gastrointestinal tract of mice.

| CON CLUS IONS
This study determined the effect of edible Salicornia herbacea encapsulation with biopolymers on the sodium binding under in vitro condition and inhibition of sodium absorption in mouse models. As a result of this study, the authors found that biopolymerencapsulated Salicornia herbacea could be combined with sodium under in vitro condition, and Salicornia herbacea encapsulated with biopolymers reduced sodium absorption in the mouse model. In particular, chitosan and cellulose had greater effect of inhibition of sodium absorption both on in vitro condition and mouse model.
Although data are not shown, our preliminary study found that inhibition of sodium absorption effect of biopolymers was higher in Salicornia herbacea-encapsulated samples than pure sodium chloride encapsulated with biopolymer samples. Therefore, this approach, that is, Salicornia herbacea encapsulation with biopolymer, can be applied to one of the sodium reduction technologies.
However, sodium not only plays an important role for saltiness and flavor in food stuff but also affects the control of microbial growths in foods during storage. Therefore, further studies are required to understand how to improve the effect of inhibition of sodium absorption without negative effect of sodium reduction in terms of food flavor and microbial growth.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.