**1. Introduction**

Oncological immunotherapy, also known as immuno-oncology, is a modern method of cancer treatment consisting of administering non-toxic preparations, modulating the immune system's response by increasing and/or improving its ability to prevent, control, and fight cancer. Immuno-oncology can educate the immune system to better recognize, more effectively attack, and successfully eliminate cancer cells. At the beginning of the 21st century, the American Society of Clinical Oncology announced that oncological immunotherapy will dominate the treatment of neoplastic diseases and, after 2020, it could be used in the treatment of even every second type of cancer. In the beginning, immunooncology mainly focused on immunostimulatory cytokine treatments. Recently, natural killer cell-based immunotherapy has emerged as the most promising therapeutic approach for both hematological malignancies and solid tumors [1]. The natural killer (NK) cells were first identified as unique large granular lymphocytes able to detect and rapidly kill abnormal cells without prior sensitization; thus, they are the first line of defense for the immune system against cells infected with bacteria and viruses, as well as malignant cells [2]. The importance of NK cells for cancer treatment has been confirmed in epidemiological studies, which revealed the negative correlation between NK cells activity and the risk of cancer development and metastasis [1]. The main considerations relating to NK cell-based immunotherapy are the NK cell source and the method of enhancement of

**Citation:** Lemieszek, M.K.; Komaniecka, I.; Chojnacki, M.; Choma, A.; Rzeski, W. Immunomodulatory Properties of Polysaccharide-Rich Young Green Barley (*Hordeum vulgare*) Extract and Its Structural Characterization. *Molecules* **2022**, *27*, 1742. https:// doi.org/10.3390/molecules27051742

Academic Editors: Sokcheon Pak and Soo Liang Ooi

Received: 14 February 2022 Accepted: 4 March 2022 Published: 7 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

NK cell anticancer activity. The presented studies address the second issue, in particular, being focused on barley (*Hordeum vulgare*), which is the fourth most important grain crop in the world and the richest source of dietary fiber among the cereals. For many years, barley was valued solely for its nutritional properties; recently, more and more scientific reports have indicated its health benefits. In the early 1990s, Japanese scientists showed that the most valuable source of both nutrients and bioactive substances is young green barley, defined as seedlings up to 200 h after sprouting, with a height of between 20 and 30 cm [3–5]. More recent studies have also indicated barley grass as a functional food with many health-promoting effects (e.g., anti-aging, antioxidant, anti-inflammatory, immunity enhancement, detoxification, hypolipidemic effects, improvement of gastrointestinal activity, blood pressure regulation, anti-hypoxia, antifatigue) as well as a potential agent against several human chronic diseases, including cancer, cardiovascular disease, obesity, diabetes, and stroke [6]. The health benefits of young green barley are associated with many of its different elements; however, immunomodulatory properties are typically considered to be due to providing a large amount of dietary fiber, especially in the form of polysaccharides [6–8]. The immunoenhancement effects of polysaccharides isolated from young barley leaves have been demonstrated by Kim et al. and Han et al., in both in vitro and in vivo studies [9,10]. Kim et al. have reported the stimulation of proliferation and the production of cytokines (IL-6, GM-CSF) in cells derived from Peyer's patches, in response to arabinoxylan- and rhamnogalacturonan I-rich polysaccharides that were isolated from barley grass (BLE0) [9]. Furthermore, the intestinal immunostimulatory activity observed in vitro has been also proved in mice, wherein the oral administration of BLE0 augmented IgA production and increased the levels of IgA-related cytokines (IL-10, TGF-β) [9]. Further studies on the abovementioned crude barley polysaccharides (BLE0) confirmed their immunostimulatory effects. They have been shown to elicit an increase in the proliferation and secretion of cytokines (IL-2, IL-4, IL-10, IFN-γ) in CD3/CD28-activated mice splenocytes. Additionally, oral administration of barley polysaccharides enhanced the immune system of mice with cyclophosphamide-induced immunosuppression, via the stimulation of splenocyte proliferation and acceleration of NK cell cytotoxic activity [10]. Han et al. have shown that water-soluble barley polysaccharide (BP-1) significantly improved the immune ability of immunosuppressive mice in several different ways, including: increasing the number of bone marrow cells as well as white blood cells in peripheral blood; promoting the proliferation of spleen cells, NK cells and macrophages; stimulating NK cell cytotoxic activity; improving the phagocytosis activity of macrophages; increasing the level of IL-2, TNF-α and IFN-γ in serum and the spleen; and enhancing the production of IgG and IgM in the spleen [11]. Ryu et al. have shown that water-soluble barley β-glucan suppressed the proliferation of mice splenocytes, accelerated by concanavalin A, IL-2, or alloantigen. Additionally, the tested β-glucan suppressed the death-induced activity of allogenic lymphocytes Tc and allogenic lymphokine-activated killer cells [12]. These immunomodulatory properties were also reported in the case of commercially available barley β-glucan. Misra et al. demonstrated that β-glucan derived from barley improved immune system activity in fish through an increase in leukocyte number, complemented activation, and stimulated phagocytic activity as well as bactericidal activity [13]. Aoe et al., who investigated the impact of the high-fructan barley product named BARLEYmax (BM; containing fructan 9.0%, β-glucan 6.3%, and resistant starch 3%) on the growth of microbiota that are present in the rat gut, revealed a beneficial effect of the investigated carbohydrates on health-promoting species belonging to the *Bifidobacterium*, *Oscillospira*, *Parabacteroides* and *Sutterella* species, which may prevent colonic inflammation and enhance intestinal immunological functions [14]. Furthermore, Aoe et al. have shown that BM possesses stronger prebiotic properties than the high-β-glucan barley line (BG; containing β-glucan 9.0%, fructan, 2.2%, and resistant starch, 0.5%) [14]. The beneficial effect of BARLEYmax on the gut microbiome was also proven in clinical studies, wherein 4 weeks' consumption at 12 g/day of this high-fructan barley product increased the abundance of *Bacteroides* and decreased the abundance of *Clostridium subcluster* [15]. Similarly, Sasaki et al. revealed in an

in vitro model of the human colonic microbiota that polysaccharide-rich young green barley leaf extract (YBL; containing carbohydrates at 46.5%, including fructan at 9.4%, proteins at 29.0%, and minerals at 17.5%) increased the abundance of bacteria related to the genus *Bifidobacterium* and stimulated the growth of bacteria related to the genera *Faecalibacterium, Lachnospira*, *Roseburia,* and unclassified *Ruminococcaceae*. The discovered bifidogenic and butyrogenic effects of YBL have been associated with health-promoting properties as a result of gut immune system stimulation, as well as via inflammatory inhibition [16].

Despite the fact that several scientific reports have revealed the immunoenhancement properties of polysaccharides derived from barley, due to the high variability of polysaccharides and the associated diversity of biological activities, it is worth continuing research into this group of compounds. The aim of the current study was to evaluate the immunomodulatory properties of the water extract of young green barley (*Hordeum vulgare*) reached with polysaccharides by evaluating its influence on the viability and proliferation of NK-92 cells and above all their ability to kill human colon cancer LS180 cells representative for the common cancers worldwide. At the same time, the selectivity of the anticancer effect of polysaccharide-activated NK cells was also evaluated on the human normal colon epithelial CCD841 CoN cells. Furthermore, to investigate the major compounds related to the immunomodulatory effect, the structural characterization of extract polysaccharides was conducted.

#### **2. Results**

#### *2.1. Young Green Barley Extract Composition*

In the first stage, the chemical composition of young green barley extract was investigated. The total sugar and protein contents were examined using colorimetric methods (phenol-sulphuric acid assay, Pierce BCA protein assay). These studies revealed that the tested extract consisted mainly of sugars (64.2 ± 1.14%), while the protein amount (26.9 ± 4.09%) was also significant. Electrophoretic separation (SDS-PAGE) of young green barley extract (YGB) confirmed the presence of proteins with a molecular weight of around 55 kDa and 35 kDa, as well as proteins weighing less than or equal to 15 kDa. The obtained data could indicate that YGB is a carbohydrate-protein complex (Figure 1).

**Figure 1.** Electrophoretic separation (SDS-PAGE) of young green barley extract (YGB). The 12% polyacrylamide gel used after protein separation was stained with Coomassie Brilliant Blue. As a standard, a molecular protein mass marker (from 15 to 250 kDa) was used.

The FT-IR spectrum of YGB preparation ranged from 400 cm−1 to 4000 cm−1 , as shown in Figure 2. The results showed an intense and broad absorption peak at 3269 cm−1 for O-H and N-H stretching vibrations, a peak at 2933 cm−1 for C-H stretching vibrations, and a broad absorption band in the region of 950–1200 cm−1 , with a maximum at 1053 for coupled C-O and C-C stretching and C-OH bending vibrations. All these absorption bands are characteristic of polysaccharides [17]. The peaks at around 2933 cm−1 and 2880 cm−1 indicate the presence of C-H bonds (C-H stretching vibration of CH3 and CH2 groups from carbohydrate molecules, respectively) [18]. The broad symmetrical signal (ranging from 1740 to 1500 cm−1 ), with a maximum at 1593 cm−1 , might be indicative of proteins as well as adsorbed water (bending vibrations from amide I and amide II; absorbing groups: C=O, C-N, N-H, and H-O-H) [19,20]. The next intense broad signal at 1397 cm−1 corresponded with the absorbance of the CH2 and CH3 functional groups (deformational vibrations) from both sugars and proteins. The prominent and overlapping absorption bands between 1150 and 970 cm−1 were assigned to the C-O-C glycosidic bond vibrations and ring vibrations, as well as the C-O-H stretching vibrations of side-group bounds. These are commonly present in sugars [21]. The YGB preparation also showed absorption peaks at 940 and 860 cm−1 , which are especially characteristic of α-d-glucose [22]. A broad signal at 618 cm−1 could be indicative of pyranose rings [23]. A very intense band with a maximum at ~1050 cm−1 , and two sharp lines of similar intensity in the regions of 820 and 780 cm−1 indicate the presence of fructose from fructans [18].

**Figure 2.** FTIR spectrum of young green barley extract (YGB).

Standard sugar analysis of the YGB preparation revealed the presence of glucose (87%), and small amounts of mannose and galactose (7% and 6%, respectively). This method does not allow the detection of ketoses (which are, in this case, converted to two alditol acetates). Therefore, YEB preparation was hydrolyzed under mild conditions (1 M HClaq, 1 h, 50 ◦C), and the liberated monosugars were converted into methoxime peracetate (MOA) derivatives [24,25]. This procedure allowed us to identify fructose (42.7%) in an amount comparable to glucose (54.7%). A small amount of mannose (2.6%) and only traces of galactose (less than 0.5%) were also detected.

Table 1 contains the results of the methylation analysis and shows all derivatized compounds that were found. Fructose derivatives were found as terminal, →6)-linked and branched, →1,6)-linked forms, whereas glucose was terminal, →3)-linked, →4)-linked, and →6)-linked, as well as branched, →3,6)-linked. Mannose was exclusively terminal, and galactose—terminal, →3)-linked and →6)-linked. Among all the above-mentioned derivatives, terminal sugars prevailed, giving about 75% of all methylated end-products.

**Table 1.** Linkage analysis of young green barley extract (YGB). The sample was subjected to methylation, hydrolysis, reduction, and acetylation. The obtained permethylated alditol acetates were identified by GLC–MS, based on their mass spectra and retention times. The main permethylated alditol acetates are shown in bold type.

For the determination of fructan content, the PAHBAH reducing sugar method was used. The fructan content of young barley was calculated from the absorbance of the color complex and was then converted to a dry weight basis per 100 g of product. The average fructan content of the tested samples was 10.23 ± 0.22 g per 100 g dry weight basis (i.e., 10.2% of dry weight YEB extract).

The size of the oligomer/polymer of carbohydrates present in YEB was estimated using the mass spectrometry technique. The MALDI-TOF mass spectrum, in positive ion mode, is shown in Figure 3. A series of intensive signals appeared in the same intervals of 162.05 u, pointing to hexose as a repeating unit. In the MALDI-TOF mass spectrum of YEB (Figure 3A), only those polymers containing up to 10 hexose units (DP = 10) were shown, but, when tracing the spectrum at low intensities in the mass range from 1700 to 3000 *m/z*, it was possible to find oligomers up to DP = 19 (data not shown). The smallest oligomer visible in Figure 3A had *m/z* at 543.138. Its fragmentation pattern has been shown in Figure 3B. This MALDI MS-MS spectrum revealed the only fragment ion *m/z* at 381.062, which corresponded to a disaccharide potassium adduct, probably composed of glucose and fructose residues (sucrose, [G+F+K]+ ). This disaccharide may be here a kind of backbone to which the next fructose units are linked during the biosynthesis process, forming the fructan detected in the YGB preparation.

The complexity of the MALDI-TOF mass spectrum is displayed in Figure 3C, showing that the main signal (oligomer potassium adduct, [M+K]+ ) is accompanied by the sodium adduct ([M+Na]+ ), as well as these molecules that are deprived of water ([M-H2O+K]+ and [M-H2O+Na]+ , respectively). It should be noted that the protonated ([M+H]+ ) ions were not detected at the mass spectrum; thus, it can be concluded that the YGB preparation is a rich source of potassium and sodium salts.

#### *2.2. Young Green Barley Extract Impacts NK-92 Cell Viability and Proliferation*

In order to evaluate the immunomodulatory potential of the YGB, its influence on NK-92 cell membrane integrity, metabolic activity, and proliferation was assessed via the LDH (lactate dehydrogenase), MTT (thiazolyl blue tetrazolium bromide), and BrdU (bromodeoxyuridine) tests, respectively (Figure 4). The LDH test revealed that the tested extract was not cytotoxic against NK-92 cells. Furthermore, the extract, when in concentrations from 25 µg/mL to 500 µg/mL, significantly decreased the lactate dehydrogenase release from treated cells. The MTT test has shown that YGB, when in concentrations from 25 µg/mL to 250 µg/mL, did not impact NK-92 viability, while at concentrations of 500 and 1000 µg/mL, it decreased the lymphocytes' metabolic activity by 4.4% and 5.1%, respectively. The BrdU test revealed no changes in the proliferation of NK-92 cells treated with YGB when used in concentrations equal to or less than 250 µg/mL. On the contrary, the tested extract, when

used at concentrations of 500 and 1000 µg/mL, inhibited DNA synthesis in NK-92 cells, reducing cell proliferation by 9.8% and 11.4%, respectively.

**Figure 3.** MALDI-TOF mass spectrum of young green barley extract (YGB) obtained in positive ion mode (**A**); MS-MS of the ion at *m/z* 543.1 (**B**); details of the spectrum A in the range of 670–710 *m/z* (**C**).

**Figure 4.** The influence of young green barley extract (YGB) on NK-92 cell viability and proliferation. Cells were exposed to culture medium alone (control) or with YGB at the following concentrations of 25, 50, 100, 250, 500, and 1000 µg/mL for 48 h. Cell viability was determined by the examination of both cell membrane integrity, using an LDH assay (**A**), and metabolic activity using an MTT assay (**B**), while cell proliferation was measured via an immunoassay based on BrdU incorporation (**C**). The results are presented as a mean ± SEM of at least 4 measurements. \* *p* < 0.05 vs. control, one-way ANOVA test; post hoc test: Dunnett.

### *2.3. Young Green Barley Extract Enhances NK-92 Cells' Cytotoxicity in Human Colon Cancer LS180 Cells without any Side Effects on Human Colon Epithelial CCD841 CoN Cells*

Before studying the impact of YGB on NK cells' cytotoxic activity, the extract's influence on normal and cancer colon cells was examined via LDH, MTT, and BrdU assays. As presented in Figure 5 (left panel), YGB in the whole range of analyzed concentrations (from 50 to 1000 µg/mL) did not affect the viability of the human colon epithelial cells (CCD841 CoN), which was seen in all the performed assays. On the contrary, when tested at the highest concentration, the extract decreased the metabolic activity of human colon adenocarcinoma LS180 cells by 21.3% (Figure 5D), and simultaneously inhibited DNA synthesis in the investigated cell line by 10.7% (Figure 5F). At the same time, when tested at concentrations of 50, 250, and 1000 µg/mL YGB, the extract was cytotoxic against colon cancer cells, as was shown by the increase in LDH release to the culture medium by 26.5%, 31.8%, and 36.4%, respectively (Figure 5D).

In the next step, YGB's influence on the NK-92 cells' cytotoxicity was determined under the same conditions. First, NK-92 cells, when used alone, did not cause any side effects in the investigated human colon epithelial cells. Similarly, the NK-92 cells' impact on CCD841 CoN viability did not change in the presence of tested extract (Figure 5; left panel). On the contrary, NK-92 cells distinctly inhibited the metabolic activity and DNA synthesis of human colon cancer cells and disrupted the integrity of their cell membranes. Furthermore, the anticancer properties of NK-92 cells were enhanced by YGB in a dose-dependent manner. As presented in Figure 5B, the cytotoxic effect of NK-92 cells in response to treatment with YGB increased from 133.9% to 143.5% (50 µg/mL), 147.3% (250 µg/mL), and 162.5% (1000 µg/mL). The beneficial effect of tested compounds was also observed in another assay. The MTT test (Figure 5D) revealed that the metabolic activity of colon cancer cells treated with both NK-92 cells and the tested extract decreased from 74.7% to 54.9% (50 µg/mL), 51.0% (250 µg/mL), and 47.5% (1000 µg/mL), while the BrdU assay (Figure 5F) showed the significant antiproliferative properties of NK cells against LS180 cells, which intensified in the presence of YGB, as reflected in the decrease in DNA synthesis from 85.5% to 66.5% (50 µg/mL), 62.8% (250 µg/mL), and 59.5% (1000 µg/mL). It should be highlighted that the inhibition of the LS180 cells' metabolic activity, as well as the DNA synthesis caused by NK-92 in the presence of YGB in the whole range of tested concentrations, was significantly stronger than the decrease in cancer cell viability induced by the extract when used alone (comparison of corresponding YGB concentrations). A similar pattern of changes was observed in the results of the LDH test. Nevertheless, a comparison of results obtained from LS180 cells treated with only NK-92 cells, vs. LS180 exposed simultaneously to both NK cells and YGB, revealed statistically significant differences in cancer cell viability and proliferation at the whole range of tested extract concentrations, while a significant distortion of cell membrane integrity was only noted at 1000 µg/mL YGB.

#### *2.4. Young Green Barley Extract Enhances Colon Cancer Cell Death in Response to NK-92 Cells*

To confirm the observed cytotoxic effect in LS180, cells of NK-92, used separately and together, a YGB cell death detection ELISA assay (Figure 6A) and nuclear double staining were performed (Figure 6B). The results of the ELISA assay revealed that the investigated extract in the tested concentrations was not able to induce apoptosis in LS180 cells at the same time as a number of nucleosomes were released into the cell culture medium (a marker of necrosis) and increased by 69.9% (50 µg/mL), 86.5% (250 µg/mL), and 103.2% (1000 µg/mL). NK-92 cells, when used alone, killed LS180 cells and induced both programmed cell death and necrosis, which was demonstrated by the increase in the number of DNA fragments recorded in the cytoplasmic fraction of colon cancer cells (126.5% of control) and the medium collected from above the cell culture (264.2% of control). Similar to the MTT and LDH results, this research revealed that the anticancer properties of NK-92 cells were enhanced by YGB in a dose-dependent manner. The proapoptotic abilities of NK-92 cells in LS180 cells increased in response to YGB, from 126.5% (LS180 cells treated with only NK-92 cells) to 131.8% (50 µg/mL), 136.4% (250 µg/mL),

and 143.5% (1000 µg/mL); however, the statistically significant enhancement of NK-92 cells' ability to induce programmed cell death was observed only after NK cells were co-incubated with 1000 µg/mL YGB. Conversely, the number of necrotic colon cancer cells that were observed in response to NK-92 when co-incubated with the tested extract increased from 264.2% (LS180 cells treated with only NK-92 cells) to 302.9% (50 µg/mL), 337.6% (250 µg/mL), 388.8% (1000 µg/mL), and the reported YGB intensification of NK cells' cytotoxicity was statistically significant in the whole range of the investigated extract concentrations. Furthermore, LS180 cell-killing by NK-92 in the presence of YGB, in the whole range of tested concentrations, was significantly improved compared to the direct anticancer effect of the extract (comparison of corresponding YGB concentrations).

**Figure 5.** Influence of young green barley extract (YGB) on NK-92 cell cytotoxicity against normal and cancer colon cells. The human colon adenocarcinoma cell line LS180 and human colon epithelial cell line CCD841 CoN, and were exposed to culture medium alone (control) or the YGB (50, 250, and 1000 µg/mL) used alone, or to NK-92 cells in the absence or presence of YGB (50, 250 and 1000 µg/mL) for 48 h. NK-92 cells were used in the same concentration as treated cells. Changes in

investigated cells in response to YGB, used alone or together with NK cells, were determined by examining cell membrane integrity using an LDH assay (**A**,**B**), metabolic activity using an MTT assay (**C**,**D**), and DNA synthesis using the BrdU assay (**E**,**F**). Results are presented as mean ± SEM of at least 4 measurements. \* *p* < 0.05 vs. control; ˆ *p* < 0.05 colon cells treated with both NK-92 cells and YGB extract vs. colon cells treated with only NK-92 cells; # *p* < 0.05 colon cells treated with both NK-92 cells and YGB extract vs. colon cells exposed to YGB at the same concentration as those being tested in a one-way ANOVA test; Tukey post hoc test.

**Figure 6.** Cell death induction in human colon cancer cells, in response to NK cells co-incubated with young green barley extract (YGB). The impact of YGB (50, 250, and 1000 µg/mL) or NK-92 cells, used together or separately, on the viability of the human colon adenocarcinoma cell line LS180 was investigated after 48 h of incubation. (**A**) Enrichment of nucleosomes in the cytoplasmic fraction (an apoptosis marker) or cell culture medium (a necrosis marker) was determined by a cell-death detection ELISA assay. The results are presented as the mean ± SEM of at least 4 measurements. \* *p* < 0.05 vs. control; ˆ *p* < 0.05 cancer cells treated with both NK-92 cells and YGB extract vs. cancer cells treated with only NK-92 cells; # *p* < 0.05 cancer cells treated with both NK-92 cells and YGB extract vs. cancer cells exposed to YGB at the same concentration as tested. One-way ANOVA test; post hoc test: Tukey. (**B**) Untreated LS180 cells (control), as well as LS180 cells exposed to the extract with or without NK-92 cells, were double-stained (Hoechst and propidium iodide) and examined under fluorescence microscopy. Representative pictures are presented; the magnification is 400×.

The results described above were reflected in the microscopic analysis of LS180 cells that were double-stained with Hoechst and propidium iodide (Figure 6B). The conducted studies have shown single colon cancer cells undergo programmed cell death in both untreated and YGB-treated LS180 cells. Nevertheless, YGB, when used alone, significantly damaged colon cancer cell membranes, and the observed cytotoxicity was dose-dependent. On the contrary, NK-92 cells induced both necrosis and apoptosis in the investigated colon cancer cells; however, the cytotoxic effect was dominant. The anticancer effect of the NK cells distinctly increased in the presence of the tested extract, which was associated with elevated levels of both necrotic and apoptotic LS180 cells; however, the disintegration of cell membranes was the dominant mode of cancer cell elimination. The enhancement of colon cancer cell death by YGB-treated NK-92 cells was directly dependent on extract concentration.

#### **3. Discussion**

Young green barley water extract has been shown to contain more than 64% of saccharides, composed mainly of glucose and fructose. Methylation analysis shows that the majority of them are in the terminal position, which might suggest that the preparation contains short oligosaccharides. Interestingly, the presence of saccharose—the metabolic precursor of fructans—could not be observed in the MALDI-TOF mass spectrum. This means that saccharose can be quickly metabolized or exported outside of the leaves. Fructans are considered to be the main saccharide reserves in the vegetative tissues of grasses, including barley [26]. The oligosaccharides observed in the analyzed extract can be recognized as plant fructan. Mixed-type fructan (containing both (1→2) and (6→2)-linked fructosyl units were found in the YGB preparation, based on methylation analysis and the mass spectrometry technique. The fructan content in YGB was also determined and represented approximately 10.2% of the dry matter basis of the analyzed samples. This type of sugar polymer is typical for cereals, such as barley [27,28]. Plant fructans are usually shorter than bacterial ones and, in general, contain less than 50 fructosyl units. Nemeth and co-workers showed changes in the distribution of different chain lengths and the pattern of fructan structures in the barleys derived from different cultivars or breeding lines [29]. In the case of YGB extract, the fructose oligomers had fewer than 20 units, and the most frequent fraction that was found contained 4 sugars—this is probably nystose. Nystose was described earlier as a component of barley grains [30].

Despite the fact that the immunomodulatory properties of plant fructans are becoming better and better known, most of the studies focused only on their indirect influence on the immune system components, which is a consequence of their prebiotic properties [31,32]. Nevertheless, the results from recent studies dedicated to fructans isolated from a wide range of plants revealed that the mentioned carbohydrates may also directly impact immune cells and, consequently, modulate the immune system responses [31]. Unfortunately, there is a lack of evidence presenting the direct impact of barley fructans on the components of the immune system. Nevertheless, considering the fact that the main component of the investigated extract was also fructooligosaccharide, the discovery of a beneficial impact on immune cells was predicted.

It should be emphasized that the chemical examination of YGB revealed that, next to carbohydrates, the second main component of the investigated extract was proteins, the total amount of which was around 27%. The electrophoretic separation of young green barley extract revealed the presence of proteins with a molecular weight of around 55 kDa and 35 kDa, as well as proteins weighing less than or equal to 15 kDa. The obtained data indicated that YGB was a carbohydrate–protein complex. Considering the proven enhancement impact of protein ligands on both the anticancer and immunomodulatory properties of polysaccharides [33], discovered that the presence of proteins in YGB seems to be beneficial. It should be emphasized that, according to our best knowledge, the data presented herein is the first report published about the immunomodulatory properties of the barley carbohydrate-protein complex.

The immunomodulatory properties of young green barley extract were examined using NK-92 cells, the best-described human NK cell line, the therapeutic utility of which has been confirmed in several clinical trials [34,35]. NK-92 cells characterized the ability to produce cytokines, as well as high cytotoxic activity. Although they are highly cancerspecific, the cells can be further modified to express different cancer receptors to increase

the targeting and killing of cancer cells, as well as to express therapeutic antibody-binding receptors, which also enhances their anticancer activity. Furthermore, NK-92 cells are able to resist the suppression induced by many agents released by cancer cells, thanks to the absence of killer inhibitory receptors (KIR) [35,36]. The above-mentioned features, as well as their long-term growth potential, have made NK-92 cells an attractive model by which to study the function of human NK cells, and have become an excellent tool for evaluating the immunoenhancement properties of compounds of various origins; thus, this cell line was selected to examine the possibility of using polysaccharide-rich young green barley extract in oncological immunotherapy.

Evaluation of the direct impact of young green barley extract on NK cells revealed that the tested compounds are not cytotoxic in the whole range of investigated concentrations, and did not affect the viability and proliferation of NK-92 cells in a wide range of tested concentrations (from 25 to 250 µg/mL); however, at higher doses (500 and 1000 µg/mL) the extract decreased both lymphocyte metabolic activity and DNA synthesis. Several studies have reported the stimulation of leucocyte proliferation by barley polysaccharides; however, the beneficial effect on NK cell numbers was reported only by Han et al., who revealed the increase of NK cell proliferation in immunosuppressive BALB/c mice in response to water-soluble barley polysaccharide (BP-1; molecular weight 67 kDa) [11]. Nevertheless, it needs to be highlighted that NK-92 cells are human lymphoma-derived cells and, despite the fact that they are phenotypically similar to activated normal NK cells [36], the results obtained from studies conducted on this cell line should be interpreted in light of this data. Because of the reaction observed in response to higher doses of young green barley extract, the slight decrease of NK-92 cell viability and proliferation with a simultaneous lack of YGB cytotoxicity did not unequivocally determine the existence or absence of the immunomodulatory properties of the test substance.

In order to verify the possibility of using young green barley extract-derived carbohydrate-protein complex in oncological immunotherapy based on NK cells, YGB's influence on NK-92 anticancer activity was evaluated. Because immunotherapy should be both effective and safe, studies have been conducted on both normal and cancer cell lines (human colon epithelial CCD841 CoN cells and human colon cancer LS180 cells). An in vitro model of colon cancer was chosen because of the fact that this type of cancer is one of the most common cancers in terms of incidence and mortality rate in the world.

In the first step of these studies, the direct anticancer effect of YGB as well as NK cells used separately has been analyzed. The obtained results revealed that the examined compounds significantly decreased the viability and proliferation of colon cancer LS180 cells, as well as effectively damaging their cell membranes and causing necrosis induction. At the same time, YGB did not affect human colon epithelial CCD841 CoN cells. The observed high selectivity of the examined compounds can be understood as a lack of negative influence on normal colon cells, with an evident anti-cancer effect against colon cancer cells, which corresponds with the previous results of our research group [37,38].

Similar to YGB, NK-92 cells also revealed a high selectivity of action in relation to colon cancer cells. The recorded anticancer effect of NK cells was mostly associated with cytotoxicity; however, significant apoptosis induction was also noted. In terms of oncological immunotherapy, the observed ability of NK-92 cells to induce different cancer cell death would create the possibility of modulating the immunogenicity of the tumor microenvironment, which is important in the case of therapy efficiency and safety.

The discovered anticancer effect of NK-92 cells and YGB, when used alone, intensified when the lymphocytes and the extract were used together. The beneficial impact of NK cells co-incubated with the examined polysaccharide-rich young green barley extract was observed on metabolic activity, DNA synthesis, cell membrane integrity, and cell death induction in LS180 cells. The enhancement of NK cells' anticancer activity by YGB was dose-dependent; nevertheless, a significant improvement in the NK-92 cells' proapoptotic and cytotoxic abilities was noted only in the presence of tested compounds at the highest tested concentration (1000 µg/mL), while other investigated phenomena

were evidently improved by YGB in the whole range of tested concentrations. It should be highlighted that cancer cell elimination by NK-92 in the presence of YGB in the whole range of tested concentrations was significantly stronger than the decrease in cancer cell viability and proliferation, as induced by the extract when used alone at the corresponding concentrations. Nevertheless, the synergy of the anticancer actions of NK-92 cells and the tested compounds was observed in all performed studies except the LDH assay. The YGB in the whole range of investigated concentrations that were given to cancer cells together with NK cells caused a stronger inhibition of metabolic activity and DNA synthesis, as well as improved apoptosis induction, than would have been anticipated from the mere summation of the direct effects of the lymphocytes and tested compounds. In the case of necrosis, a synergy of action was observed in an NK cell co-incubated with YGB at a concentration of 1000 µg/mL. The observed enhancement of the NK cells' cytotoxic abilities in response to barley polysaccharides corresponds with previously published data [10]. Ex vivo studies conducted by Han et al. revealed that the oral administration of crude barley polysaccharides (BLE0; mainly composed of neutral sugars and uronic acid and containing minor levels of 2-keto-3-deoxy-mannooctanoic acid-like materials) significantly recovered the cyclophosphamide-induced reduction of NK cell activity against YAC-1 mouse T cell lymphoma cells [10].

In summary, the results of the present studies indicate that polysaccharide-rich young green barley extract may have immunomodulatory properties associated with the enhancement of NK cells' ability to recognize and eliminate human colon cancer cells without any side effects on normal colon epithelial cells. Furthermore, the obtained data also revealed the direct anticancer effect of the tested compounds, as well as high selectivity against neoplastic cells. Although the presented studies were performed only on cell lines, their importance is supported by the fact that they focused on NK92 cells with proven therapeutic utility in clinical studies. The discovered beneficial impact of the investigated extract on NK92 cells' anticancer properties brings hope for its therapeutic use, especially as an adjuvant in the currently applied immunotherapies for colon cancers. Despite the fact that over 90% of the extract components have been determined, further study is necessary to recognize other functional ingredients, next to the discovered carbohydrate-protein complexes, that may also influence the discovered immunoenhancement properties of YGB.

#### **4. Materials and Methods**

#### *4.1. Reagents*

Unless indicated otherwise, the chemicals used in this study were purchased from Sigma-Aldrich Co. LLC, Saint Louis, MO, USA.

#### *4.2. Preparation of Young Green Barley Extract*

The powdered young green barley juice (*Hordeum vulgare*) was purchased from Green Ways (Prague, Czech Republic). First, 5 g of the product was dissolved in 150 mL sterile water. Extraction was then carried out for 24 h on a rotator at room temperature. The resulting mixture was centrifuged (4075× *g*, 10 min, 20 ◦C) and the collected supernatant was filtered through a microbiological filter. The resulting filtrates were then subjected to a freeze-drying process. The lyophilizate thus obtained was stored at −20 ◦C. The stock solution of young green barley (25 mg/mL) was prepared by dissolving the lyophilizate in PBS (phosphate-buffered saline). The stock solution was stored at −20 ◦C. Working solutions of the extract were prepared by dissolving a stock solution in a culture medium. In this manuscript, young green barley extract has been expressed as YGB.

#### *4.3. Sugar Content Determination*

The total sugar content in YGB was determined using the phenol-sulphuric acid method, as previously described [39], using glucose as a standard. The tested extract (200 µL) was mixed with 5% aqueous phenol solution (200 µL) and concentrated H2SO4 (1 mL). After 10 min of incubation at room temperature, absorbance was recorded on

a microplate reader, the BioTek ELx800 (Highland Park, Winooski, VT, USA) at 490 nm wavelength. The results were expressed as the percentage of YGB.

#### *4.4. Protein Content Determination*

The protein concentration in YGB was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. Bovine serum albumin was used as a standard. Absorbance was recorded on a microplate reader, a BioTek ELx800, at 570 nm wavelength. The results were expressed as the percentage of YGB.

#### *4.5. Proteins Separation—SDS-PAGE*

YGB at a concentration of 25 mg/mL was solubilized in sample buffer (30% glycerol, 10% SDS, 0.5 M Tris-HCl, pH 6.8, 0.012% bromophenol blue, 5% β-mercaptoethanol) and boiled for 5 min. Then, 10 µL of the YGB sample, as well as 5 µL of Page Ruler Plus prestained protein ladder (Thermo Fisher Scientific, Waltham, MA, USA), were loaded onto the 12% polyacrylamide gel. Protein separation was carried out at 80 V for 30 min, followed by 200 V for 60 min for the resolving gel, using a Power Pac (Bio-Rad Laboratories, Hercules, CA, USA). The gel was stained for 30 min with Coomassie Brilliant Blue, dissolved in water/methanol/acetic acid (5/4/1, *v/v/v*), and the destaining of the gel was achieved using Coomassie solvent for 3 h. The estimation of the molecular mass of proteins was performed using the abovementioned molecular protein mass marker (from 15 to 250 kDa).

#### *4.6. Chemical Analyses of Sugars*

Monosugars were liberated from 1 mg of the YGB sample by hydrolysis, using 2 M trifluoroacetic acid (100 ◦C, 4 h), and were converted into alditol acetates via reduction with NaBD4 and acetylation, as described elsewhere [40]. The obtained alditol acetates were analyzed via gas-liquid chromatography coupled with mass spectrometry (GLC-MS) technique.

To obtain the acetylated methyl glycosides, the YGB sample was subjected to methanolysis (2 M HCl/methanol, 85 ◦C, 2 h) and acetylation (pyridine/acetic anhydride, 85 ◦C, 30 min).

To obtain the peracetylated derivatives of methyl oximes, the YGB preparation was mildly hydrolyzed (1 M HClaq 50 ◦C, 1 h) and dried under a vacuum. Methoxylamine hydrochloride (100 µL, 0.18 M in pyridine) was added and the sample was incubated at 70 ◦C for 1 h. Acetic anhydride was added (100 µL), then a sample was incubated at 45 ◦C for 1 h. The sample was dried, dissolved in ethyl acetate, and analyzed by GLC-MS. The standard aldoses (mannose, glucose, galactose) and ketoses (fructose, tagatose, sorbose) were prepared in the same way.

The linkage position between sugar components in the sample was established by methylation analysis [41] and the extraction of permethylated products into chloroform, followed by acid hydrolysis (2 M TFA, 100 ◦C, 4 h), reduction with NaBD4, and acetylation. The obtained partly methylated alditol acetates were analyzed by GLC–MS.

#### *4.7. Fructan Content Determination*

The fructan content was determined using the PAHBAH reducing sugar method, with the help of the Fructan Assay Procecedure Kit (Megazyme, Bray, Ireland), following the manufacturer's instructions. First, 100 mg increments of three independent lyophilized samples were used in triplicate. The PAHBAH color complex absorbance was recorded on a microplate reader, BioTek ELx800, at a 405 nm wavelength. Fructan content was calculated using the Megazyme Mega-Calc spreadsheet (Megazyme, Ireland).

#### *4.8. FTIR Spectroscopy*

An infrared absorption spectrum (FTIR-ATR) of the YGB was recorded using an FTIR spectrometer, the Nicolet 8700A (Thermo Scientific). The spectral range was from 400 to 4000 cm−1 .

#### *4.9. GLC-MS Analysis*

The sugar derivatives were analyzed by GLC-MS using a gas chromatograph 7890A (Agilent Technologies, Inc., Wilmington, DE, USA) connected to a mass selective detector (MSD 5975C, inert XL EI/CI (Agilent Technologies, Inc., Wilmington, DE, USA)). The chromatograph was equipped with an HP-5MS column (30 m × 0.25 mm) and used helium as a carrier gas. The temperature program was as follows: 150 ◦C for 5 min, increased to 310 ◦C (5 ◦C min−1 ); the final temperature was maintained for 10 min. The components were identified based on their mass spectra and retention time and then compared with the known standards.

#### *4.10. Mass Spectrometry*

The MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization—Time of Flight Mass Spectrometry) and MS-MS (tandem mass spectrometry) of YGB were performed with a SYNAPT G2-Si HDMS instrument (Waters Corporation, Milford, MA, USA) equipped with a 1 kHz Nd: YAG laser system (355 nm wavelength). Acquisition of the data was performed using the MassLynx software, version 4.1 SCN916 (Waters Corporation, Wilmslow, UK). To act as a matrix, 2,5-dihydroxybenzoic acid solution (20 µg/µL) in 50% acetonitrile was used. The sample was dissolved in 50% methanol to a concentration of 20 µg/µL and then mixed with matrix solution in the proportion of 1:1 (*v*/*v*) [42]. Spectra were recorded in a positive ion mode. For MS/MS experiments, isolated precursor ions were fragmented using a collision voltage of 15 V. MS data were collected for 120 s. Mass spectra were assigned with multi-point external calibration using red phosphorous (Sigma Aldrich, St. Louis, MO, USA).

#### *4.11. Cell Lines*

Human natural killer cells (NK-92) and human colon epithelial cells (CCD841 CoN) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human colon adenocarcinoma cells (LS180) were obtained from the European Collection of Cell Cultures (ECACC, Center for Applied Microbiology and Research, Salisbury, UK).

NK-92 cells were grown in Alpha Minimum Essential Medium without ribonucleosides and deoxyribonucleosides, and with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate, supplemented with 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 200 U/mL recombinant IL-2, 12.5% horse serum, and 12.5% fetal bovine serum (FBS). CCD841 CoN cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS. LS180 cells were grown in a 1:1 mixture of DMEM and nutrient mixture, Ham F-12, supplemented with 10% FBS. All media were supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL). Cells were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37 ◦C.

#### *4.12. Cell Treatment*

In the case of the MTT, LDH, and BrdU assays, cells were seeded on 96-well microplates at a density of 5 × 104 cells/mL. In the case of microscope evaluation, cells were seeded on Lab-Tek Chambers Slides (Nunc) at a density of 5 × 104 cells/mL.

Basic variant: Immediately after seeding on cell culture vessels, the NK-92 cells were exposed to a series of YGB dilutions. Conversely, adherent cells (CCD841 CoN, LS180) were treated with a series of YGB dilutions 24 h after cell seeding into cell culture vessels. YGB dilutions were prepared in a culture medium suitable for individual cell lines.

The variant in co-cultures: Adherent cells were seeded on cell culture vessels. The following day, the culture medium was removed, and the cells were exposed to NK92 cells in the absence or presence of a series of YGB dilutions. The proportion of adherent cells to NK cells was 1:1. The YGB dilutions were prepared in a culture medium suitable for NK-92 cells.

In both variants of the experiments, cells were treated with YGB, or NK-92 cells, or YGB + NK-92 cells for 48 h, and afterward, proper investigations were performed.

#### *4.13. Cytotoxicity Assessment—LDH Assay*

After treatment, the cells growing on microplates were centrifuged at 300× *g* for 10 min, at room temperature. Then, the culture supernatants were collected in new 96-well microplates, which were used to perform an LDH assay following the manufacturer's instructions (In Vitro Toxicology Assay Kit—Lactate Dehydrogenase-Based, Sigma Aldrich, St. Louis, MO, USA). Absorbance was recorded on a microplate reader (BioTek ELx800), at a wavelength of 450 nm.

#### *4.14. Assessment of Metabolic Activity—MTT Assay*

After cell treatment, the metabolic activity of cells was investigated using the MTT test. In brief, cells were incubated with MTT solution (5 mg/mL in PBS) for 6 h. Then, formazan crystals were solubilized overnight in SDS buffer (pH 7.4) (10% SDS in 0.01 N HCl), and the product was quantified spectrophotometrically by measuring the absorbance at 570 nm wavelength using the microplate reader (BioTek ELx800).

#### *4.15. Assessment of DNA Synthesis—BrdU Assay*

After cell treatment, DNA synthesis was measured with a colorimetric immunoassay, Cell Proliferation ELISA BrdU (Roche Diagnostics GmbH, Penzberg, Germany), according to the manufacturer's instructions. Absorbance was recorded on a microplate reader (BioTek ELx800) at a wavelength of 450 nm.

#### *4.16. Cell Death Detection—ELISA*

After cell treatment, cell death (both apoptosis and necrosis) was assessed using the Cell Death Detection ELISA PLUS kit (Roche Diagnostics, Mannheim, Germany), according to the manufacturer's instructions. Studies were conducted in cytoplasmic fractions as well as the cell medium collected from above the cell cultures, which allowed the determining of nucleosome quantity in apoptotic and necrotic cells, respectively. Absorbance was measured at a wavelength of 405 nm, using a microplate reader (BioTek ELx800).

#### *4.17. Cell Death Detection—Nuclear Double Staining*

After treatment, cell death was visualized using nuclear double-staining. Cells were incubated for 5 min with a fluorochrome mixture: Hoechst 33342 (0.24 mg/mL) and propidium iodide (PI) (0.15 mg/mL). The stained cells were observed under an Olympus BX51 System microscope and the micrographs were prepared using the CellFamily AnalySIS software.

#### *4.18. Statistical Analysis*

The obtained data were developed in the following programs: Microsoft Excel 2010 and GraphPad Prism 5.0. The results are presented as the mean value and standard error of the mean (SEM). The data were analyzed using a one-way ANOVA test with Dunnett's or Tukey's post hoc tests, and column statistics were used for comparisons. Significance was accepted at *p* < 0.05.

**Author Contributions:** Conceptualization, M.K.L., I.K., A.C. and W.R.; formal analysis, M.K.L., I.K. and A.C.; funding acquisition, M.K.L. and W.R.; investigation, M.K.L., I.K. and M.C.; methodology, M.K.L., I.K., M.C. and A.C.; visualization, M.K.L., I.K. and M.C.; writing—original draft, M.K.L., I.K. and A.C.; writing—review and editing, M.K.L., I.K., M.C., A.C. and W.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Green Ways International, Prague, Czech Republic (Project Agreement No. 713/11/2019, 2019).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Sample Availability:** Samples of the investigated extract are available from the authors.

#### **References**

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**1. Introduction**

People who agree to assume important management roles in organizations often bear many responsibilities to a varied set of stakeholders in their daily work. Given the impact managers have on their organizations, research has examined many facets of the challenging nature of this work. The better scholars can shed insight into managing and mitigating the stressors management positions hold, the better the manager will perform as both a professional and person. In turn, the better the manager performs, the better the organization will do as well (Bakker and Demerouti 2007). The job demands research tradition is one management area that holds particular significance for studying issues managers face in their daily work (Demerouti et al. 2001). Specifically, job demands have been defined as "the degree to which a given executive experiences his or her job as difficult or challenging" (Hambrick et al. 2005, p. 473). Job demands are not inherently a negative phenomenon in the workplace. If job demands are reasonably manageable, many managers may find the challenges interesting and satisfying, as the work offers them opportunity to apply and develop their expertise and experience (Gardner 1986; Garner and Cummings 1988; Janssen 2001; Scott 1996). After all, people in leadership positions usually reach their status from seeking and succeeding in situations others may avoid. However, researchers have also discovered that overly taxing job demands can entail great mental strain and stress (Karasek 1979; Van Yperen and Snijders 2000; Wall et al. 1996; Xie and Johns 1995) and incur physical health problems (Fox et al. 1993; Theorell and Karasek 1996; Warr 1990). The nature of whether a job demand is stimulating or taxing is dependent on three factors: task challenges,

performance challenges, and personal performance aspirations (Hambrick et al. 2005). The degree of stress a manager finds in addressing their tasks, organizational expectations, and personal aspirations can also impact the quality of their decision-making (Ganster 2005). Therefore, overly demanding jobs can lead to poor decisions by managers. This negative consequence is multiplied when made by leaders who supervise outcomes with great impact on their organization and/or society. Few professionals in society make more important decisions than nurse managers. They routinely are faced with "life or death" situations requiring decisions of how their frontline nurses are to proceed. Better practices for assisting nurse managers with the demands they face in their work will improve their decision making and, ultimately, will better serve their patients with quality care as well as address patient safety.

The job of the nurse is filled with much stress and chaos given the challenging demands within today's medical environment (Goldsby et al. 2020; Greggs-McQuilkin 2004). One of the major stressors confronting nurses is perceived time pressure (Teng et al. 2010). Time pressure impairs the decision making of nurses (Hahn et al. 1992), reduces their emotional well-being (Gärling et al. 2016), and leads to nurse exhaustion Gelsema et al. (2006). Furthermore, recent research in the psychological sciences suggests that increased time pressure can lead to more dishonesty (Protzko et al. 2019). Given such potential negative outcomes on nurses due to perceived time pressures, it seems logical that a nurse manager's ability to help nurses manage this time pressure and become better decision makers (that is, become better time managers) could enhance nurse well-being and performance. Research in the organizational time management literature shows a positive relationship between time management and job satisfaction, health, and performance outcomes (Claessens et al. 2007). Paralleling research in the nursing literature suggests that in order to improve the perception patients have of the quality of care they receive, nurse managers should embrace ways to lower perceived time pressure (Teng et al. 2010). However, while time pressure on nurse managers has been studied as a common problem in hospitals, a theory-based framework for better performance within those constraints has not been provided in the literature. We seek to address that literature gap by answering the following research question in the upcoming sections: How can nurse managers and nurses provide quality patient care by making better decisions under time pressure? This conceptual paper provides a step in that direction; that is, to help nurse managers reduce perceived time pressure by nurses and improve the decision making of nurse managers and nurses. In this article, we suggest how evidence-based time management practices (Garbugly 2013; Saunders 2014) and self-leadership theory (e.g., Neck et al. 2019) can help nurse managers to reduce perceived time pressure by the nurses that they manage. We capture these insights in a framework we call *The Time Pressure Mitigation Model for Nurse Managers* (see Figure 1). We propose that guidelines inherent in this model will also serve other managers who find themselves making decisions under time pressure constraints.

**Figure 1.** The time pressure mitigation model for nurse managers.

Our proposed model mitigates the time pressure nurse managers face based on three areas of research: psychosocial care, time management, and self-leadership. Based on the conceptual methodology of metatriangulation (Lewis and Grimes 1999; Saunders et al. 2003; Cristofaro, Matteo 2020. Unfolding Irrationality: How do Meaningful Coincidences Influence Management Decisions? forthcoming), we provide three metaconjectures for further consideration by organizational and psychological researchers. Metaconjectures are "propositions that can be interpreted from multiple paradigms" (Saunders et al. 2003, p. 251). Saunders et al. (2003), for example, applied the approach to examining power and information technology. The context of nurse managers is included in the title of the framework because the professional outcome pertinent to their roles is psychosocial care. Based on the research on nurse management studies, we conjecture that time pressure will impede good decision making and detract from providing quality psychosocial care. However, the research in time management and self-leadership warrant us to also conjecture that when practices from these two areas are successfully implemented, the negative effect of time pressure on decisions related to psychosocial care can be lessened. In other words, proper application of time management and self-leadership practices moderates the relationship between time pressure and psychosocial care by nurse managers.

In this conceptual paper, we first describe the context nurse managers face that affects their decision making. We then provide the model and its constituent parts. An example is then given that demonstrates how the model may work in a healthcare setting. We conclude with considerations for future development of the model.

### **2. The Context of Healthcare Settings**

Time is a major issue in healthcare today. The emphasis on quality of care, safety, standardization, and efficiency has to be managed within the constraints of an increase in the amount of patients being treated and a condensed length of stay (Bundgaard et al. 2016). Nurses are often in a continuous struggle to perform an increasing number of complex tasks under time-crunched conditions (Chan et al. 2013). Further complicating the nurse's job are the many decisions that must be made within that limited time (Saintsing et al. 2011). In a study of a medical admissions unit, it was found that a nurse confronts up to 50 important clinical decisions in a single 8-hour shift (Thompson et al. 2004). Other researchers have discovered similar patterns of clinical judgements and choices in controlled time frames. Thompson et al. (2008), for example, discovered that nurses in intensive therapy units encountered a clinical judgment or decision every 30 seconds. Along with that, in a study by Saintsing et al. (2011), nurses reported time constraints that limited their patient assessments with approximately 80% of the novice nurses acknowledging making mistakes due to time pressure. In this study, it was reported that each nurse made better decisions when there was no time pressure confronting their interactions. Additionally, Gonzalez (2004) demonstrated that people making decisions under limited time conditions performed worse than others faced with the same situations but with more time.

Accelerated information processing (Maule and Edland 1997) has been recognized as a natural response to time pressure when implementing a desired strategy (Payne et al. 1993). Furthermore, novice nurses described that peer-pressure is an indirect basis of time constraint (Ebright et al. 2004). Specifically, they feel pressure to leave no unfinished tasks for the incoming nurses that start the next shift, pressured to complete their allotted tasks so that incoming nurses start their shifts without leftover work. This self-inflicted time constraint is pervasive in healthcare, especially for new nurses who want to avoid being seen as incapable of thoroughly completing their responsibilities. The "must-do work" supersedes the "should-do work" (Bowers et al. 2001), thereby causing nurses to perform in a reactive fashion rather than being proactive with decision making (Hoffman et al. 2009). Nurses report that pressure to perform, workload, technology, and system issues produce barriers that prevent them from providing compassionate care. The often-chaotic environment weakens their capacity to care for others as well as themselves. This can result in dissatisfaction, burnout, and compassion fatigue (Roussel et al. 2020). Moreover, time pressure bears a high price of energy expenditure as nurses rely on coping mechanisms to bear the increased anxiety that comes with time pressure. In other words, nurses are not able to fully focus on their job, but must engage in self-coping to stand up to the pressure. This unfortunate predicament can drain energy over a work shift (Maule et al. 2000). Given this context of time pressure within a healthcare setting, we next explain the Time Pressure Mitigation Model for Nurse Managers and offer related metaconjectures for future study.

### **3. The Time Pressure Mitigation Model for Nurse Managers**

Time pressure is the perception that scarcity of time exists to finish obligatory tasks (Teng and Huang 2007). Nursing responsibilities have been rising along a range of complexity. This amplified workload can complicate nursing (Gurses et al. 2009). Too much to do without enough help was found to be the top source of stress in a study of Iranian nurses (Mosadeghrad 2013). In the study, occupational stress was found to lessen the quality of care due to having less time to demonstrate compassion for the patients in their care. Additionally, mistakes and practice errors occurred more often when occupational stress increased. Stress is related to time pressure in that it is recognized as inadequate time for accomplishing required tasks that compromises one's ability to cope. Under intense time pressure, it was found that individuals tend to escalate information processing, hinder decision-making quality, and experience information overload (Ben-Zur and Breznitz 1981; Hahn et al. 1992). Concurrently, the stress of time pressure has physical complications, such as increased blood pressure and a rise in human cortisone levels (Wellens and Smith 2006; Greiner et al. 2004). Thompson et al. (2008) found that time pressure reduced the nurses' capacity to assess patient needs in acute care and a ffected nurses' risk assessment decisions. Time pressure also creates negative emotions, increases anxiety, and leads to nurse emotional exhaustion (Gelsema et al. 2006) or burnout (Ilhan et al. 2008). When in a state of high anxiety, one's working memory resources are constrained, significantly diminishing resources for completing tasks and ultimately diminishing individual effectiveness. Patients are aware of this limited cognition, which can diminish client satisfaction and confidence in the hospital. Teng et al. (2010) discovered that nursing-perceived time pressure is negatively related to patient perceptions of dependability, accountability, responsiveness and assurance of the nurse. Thus, to enhance patient perception of care quality, nursing managers must develop means to lessen nurse-perceived time pressure.

### *3.1. Time Pressure and Psychosocial Care*

Psychosocial care, a holistic approach to nursing to meet the psychological and social needs of patients (Kenny and Allenby 2013), is an important healthcare outcome compromised by insu fficient time and heavy workloads (Legg 2011). Barriers, including lack of time, stand in the way of appropriate psychosocial care (Legg 2011). Recent studies reveal that o ffering good psychosocial care may improve patient overall health outcomes (Chen and Raingruber 2014). Additionally, appropriate psychosocial care reduces patient anxiety and stress and alleviates pain, thereby improving quality of life as well as a reduction in hospitalization cost due to a decreased need for medical resources (Kenny and Allenby 2013; Legg 2011). Studies by Legg (2011) and Rodriguez et al. (2010) found that good psychosocial care decreased the duration of hospitalization.

Unfortunately, psychosocial care does not often become an area of focus in hectic acute care settings (Legg 2011; O'Gara and Pattison 2015). In a study by Chen and Raingruber (2014), all the participants stated that, although often limited, positive interactions with patients and their family members were vital for providing psychosocial care. Communication within a time-pressured environment is hard to come by. As one nurse in the study said, "I think in order to know the needs of the patient, you need to communicate with them, and then you will know what they need." (p. 229).

The literature supports that e ffective interactions between nurses and patients result in increased rapport, trust and medical care, thus making therapeutic relationships possible (Belcher and Jones 2009; Josefsson 2012; Mcmillan et al. 2016). Furthermore, although nurses support spiritual care, they commonly said it was not possible when under time constraints (Balboni et al. 2014). Most participants in Legg (2011) study responded that time constraints due to excessive workloads was the top obstacle to o ffering psychosocial care. Time constraints shortened conversations with patients that would better uncover individualized needs. Other studies support this conclusion that most nurses contend with time-related pressures (Legg 2011; Chen and Raingruber 2014; DeCola and Riggins 2010; Lawless et al. 2010). A likely cause for this occurrence are the high patient workloads in hospitals and the distribution of tasks that require a specific schedule that needs completed before the end of one's shift (Lim et al. 2010). An enormous preoccupation with documentation also limits time available for psychosocial needs (Legg 2011). Most nurses in this study expressed that a preoccupation with timely documentation requirements limiting patient interaction was the cause of not attending to psychosocial needs.

Quality decisions within a healthcare context are also a ffected by time pressure. Good decisions are greatly dependent on the information considered by decision makers. However, new findings in the psychological sciences reveal that time pressure can cause nurses to misrepresent actual events and results, in order to appear more favorable to other people (Protzko et al. 2019). This was a ffected, though, by participants' beliefs on whether their true self was virtuous. Bear and Rand (2016) and Rand et al. (2014) proposed that one's automatic responses develop from internalizations of actions commonly agreed to as good to others in social exchanges. With this notion in mind, when time pressure is at hand, an individual might say what would appear to be the right response when indeed it might not be the truthful response. An example of this could be when a nurse reports to the nurse manager that she/he has followed protocol and checked all the necessary patient identifiers before giving medications during a busy shift, while in reality the nurse is telling the nurse manager what she/he thinks she/he wants to hear. This error in judgment could result in a medication error, which is one of the most common—and dangerous—mistakes in healthcare. When people are under time-crunched conditions, they often o ffer socially desirable answers or information as a default. A clash between a person's long-held self-conception and workplace role takes place. Thus, under time pressure, people often operate in opposition to their true self-concept, responding consistently with the internalized social norm of how the unit is supposed to operate from day-to-day (Everett et al. 2017). Positive self-presentation becomes a habitual tendency when time pressure is present (Protzko et al. 2019). When intentions and actual events are in misalignment, cognitive dissonance can place further stress on the nurse's psyche—sometimes lasting well beyond the date of the actual occurrence.

Given this discussion of the e ffect of time pressure on psychosocial care, we o ffer the following metaconjecture based on the literature:

*Metaconjecture 1: In situations where nurse managers face increased time pressure, providing quality psychosocial care will be compromised; i.e., the more time pressure a nurse manager experiences, the less psychosocial care their patients receive.*

### *3.2. Time Management for Nurse Managers*

Nurse managers must create a positive work environment even when they are confronted with ever-changing priorities. They must especially consider time pressure of their sta ff when designing their schedules. Workload should relate to a realistic assessment of individual nurses' capabilities and resources. Adequate sta ff can help in keeping nurses' assignments realistic to su fficiently manage workloads (Waterworth 2003). Nurse managers should initiate strategies to provide substantial support for the nurses to deal with the stresses that are at hand. Numerous studies have considered time pressure and work overload as major contributors to work stress among healthcare professionals. A burdensome workload intensifies job tension and reduces job satisfaction which, in turn, increases the probability of turnover. Although, Efron (2014) identifies poor leadership as the main reason for sta ff to leave and notes that sta ff quit the leader, not the organization. Roussel et al. (2020) report that the highest turnover takes place within one year of employment, with the cost of replacement at USD 75,000 due to recruitment, temporary sta ff, overtime, orientation and replacement. Moreover, the remaining sta ff are a ffected with heavier work assignments and overtime, which also leads to burnout. If the vacancy rate remains high, burnout may lead to more vacancies and, in turn, increase the potential for further burnout among the remaining sta ff leading to a downward spiral. It is as if a unit is unintentionally downsizing itself.

Inadequate sta ffing also impacts quality of care and patient outcomes (Aiken et al. 2002). Conscientious management of all these factors is key. Time management for nurse managers is an important issue (Mirzaei et al. 2012) because it directly a ffects people's health, availability of critical time, and can cause a decrease in e fficiency (Soleymani et al. 2011). It was found in a study by Ziapour et al. (2015) that training nurse managers according to time management practices delivers positive results in healthcare centers.

A main objective for the nurse is to make optimum usage of the time at hand. Nurses keep part of their focus on maintaining the expectations of the greater healthcare system while also providing individualized care in the most e fficient way they can. Thus, instead of developing a relationship with a patient, much of the allotted time is spent on the technical and instrumental responsibilities in nursing. As a result, compassionate nursing becomes harder to provide (Bundgaard et al. 2016). Therefore, productivity, and not compassion, becomes the key objective of the job. Numerous researchers cited Lakein (1973) when studying time management, emphasizing the practices of needs assessment, setting goals to meet these needs, prioritizing, and task planning necessary to meet the set goals. Practices proposed to extend intellectual e fficiency were suggested by Britton and Tesser (1991). Kaufman-Scarborough and Lindquist (1999) provided methods for strategizing activities by prioritizing them by their relative importance to the healthcare mission.

Collections of behaviors that are considered to aid e fficiency and lessen stress were suggested by Lay and Schouwenburg (1993). Based on a review of the time management literature, Claessens et al. (2007) suggest the following definition: "behaviors that aim at achieving an e ffective use of time while performing certain goal-directed activities" p. 262. Since the focus is on goal-directed activities that are accomplished in a manner that implies successful use of time, the following behaviors are included in their definition: (1) Time Assessment Behaviors—focusing on mindfulness of the past, present and future with self-awareness of time usage within the boundaries of one's abilities (Kaufman et al. 1991) and self-awareness of time handling by deciding which tasks suitably fit into one's abilities; (2) Planning Behaviors—with the goal of e ffective use of time, that includes goal setting, development, prioritizing, formulating a to-do list, and arranging tasks (Britton and Tesser 1991; Macan 1996); and (3) Monitoring Behaviors—with the objective of attending to how time is allotted, engagement of planned undertakings, and limiting the impact of disturbances by others in the completion of tasks and goals (Fox and Dwyer 1996; Zijlstra et al. 1999).

Nurses face time management problems due to the unpredictability and complexity of their assignments. Accomplishing tasks e ffectively and minimizing interruptions are essential to the nurse. The importance of routines and prioritization is key to time management in a healthcare setting (Waterworth 2003). Furthermore, in complex environments, routines allow for a way of maintaining order since actions have already been planned out and can decrease thinking time needed to make decisions. Routines bring about a sense of predictability, awareness of time control, and familiarity of experience that is pertinent to time management (Waterworth 2003). Furthermore, prioritization is a prerequisite for e ffective work performance for nurses. Sequencing work and its duration is necessary as well. It is imperative for timing and speed that there is synchronicity with others in the nursing environment. Determining what is urgent and important is a critical step to attaining high returns on time investments. Simply put, more hours worked does not mean more hours of productivity. Therefore, productivity experts o ffer many suggestions for being more deliberate and conscious of where time on activities is allocated. Some of the latest advice for nurses on time management includes (Garbugly 2013; Saunders 2014):

minute, but over the course of hours, days, and weeks, significant time can be lost in perceived "multitasking." Thus, it is more e fficient and productive to complete tasks with full attention and then move onto the next one needing accomplished.

Given this extensive review of research on time management theory and practices for better performance, we o ffer the following metaconjecture:

*Metaconjecture 2: In situations where nurse managers face increased time pressure, proper application of research-based time management practices can improve psychosocial care; i.e., time management practices positively moderate the negative relationship between time pressure and psychosocial care.*

### *3.3. Self-Leadership for Nurse Managers*

The research in self-leadership suggests that it can be an appropriate training tool for nurse managers in better performing their roles. Based in social cognitive theory, self-leadership can help nurse managers better manage their thoughts, behaviors, and environment to create a better workplace for improved results. Self-leadership (Manz 1986; Manz and Neck 2004) is a process in which people can regulate what they do, how they interact with others, and how they decide to lead themselves and others by using certain behavioral and cognitive strategies. Self-leadership strategies fall into three groups focused on behavior, natural rewards, and positive thought patterns (Manz and Neck 2004; Prussia et al. 1998; Neck and Houghton 2006). Strategies revolving around behavior improve the awareness a person has on what they are trying to accomplish, especially regarding tasks with which one might want to procrastinate (Manz and Neck 2004; Neck and Houghton 2006). Behavior-focused strategies are:

Natural reward strategies are designed to establish conditions that spur correct actions through focusing on the gratifying aspects of a task (Manz and Neck 2004; Neck and Houghton 2006). These strategies encourage a sense of competence and self-determination in the person practicing them, two key drivers of intrinsic motivation (Deci and Ryan 1985). The necessity for competence comprises the need to practice and increase a person's proficiencies, and self-determination implicates one's desire to be independent from pressures such as conditional rewards. When individuals feel negatively controlled by their environment and they associate their expected actions to external pressures, they are likely to be less motivated by the work itself. To avoid this negative perspective of work, two natural rewards strategies that can be practiced are:

Effective thought pattern strategies are devised to enable a positive stream of recurring thoughts and constructive thinking habits that can enhance a person's performance (Manz and Neck 2004; Neck and Manz 1992). Positive thought pattern strategies include:

conducted a meta-analysis of 35 empirical studies and discovered that mental imagery has a significant positive effect on individual performance (Manz and Neck 2004; Manz and Sims 1980, 2001). Mental imagery can be useful when a problem stems from time pressure. In that case, the nurse manager would picture herself in a calm manner listening to the nurses' concerns over the challenges at hand, offering timely encouragement, and providing useful, deliberate direction. Solutions can be created that can ultimately save time in the future.

When time pressure is at hand, having a deliberate strategy with self-leadership skills is key. While many factors in the surrounding environment can cause stress on a nurse manager, her/his state of mind is within her/his power. Dysfunctional thinking, however, often hinders the nurse manager in advancing a unit toward its preferred benchmarks (Goldsby et al. 2020). Fortunately, dysfunctional or self-limiting thinking can be changed to be more constructive with evidence-based self-leadership strategies.

A significant research finding in the past 30 years is that people can decide on the way they wish to think (Seligman 1991). In the book, Talking to Yourself, Dr. Pamela Butler proposes that people participate in "an ever-constant dialogue" with themselves so that they can pilot their behaviors, feelings, and even stress level (Butler 1983). Much of this self-dialogue is centered on where a person places their attention. Nurses often struggle over difficulties that are not within their power to change, such as situations resulting from time pressure. Many are burdened about consequences that they cannot anticipate. Then, when time pressure is at hand with the potential stress that comes with it, self-defeating thinking can be the cause of extra burden. Significant challenges stem from dysfunctional thinking patterns. The most common dysfunctional thinking patterns are (Manz 1992):

Psychologists point to cognitive distortions as sources of these mental states that can undercut personal effectiveness (Neck and Barnard 1996). Even forms of depression can be the result of these mindsets. When nurse managers can recognize their self-defeating self-talk when it is taking

place, they have the opportunity to alter and re-verbalize these personal dialogues. There is always potential for creating a more positive outlook that will enhance their performance and satisfaction (Goldsby et al. 2020).

Once the self-leadership practices of the nurse manager and nurses are improved, the interactions between the two parties can be honed as well. Social cognitive theory (SCT) (Bandura 1986)—the underlying theoretical foundation of self-leadership—explains that performance is the outcome of a three-way relationship between a person's thoughts, actions, and surroundings in which they find themselves. Self-efficacy, which is a self-assessment of a person's ability to achieve specific undertakings, is in particular an important concept of social cognitive theory. Thus, self-efficacy is also significant within the practice of self-leadership (Neck and Houghton 2006). Furthermore, a chief aspiration of self-leadership practices, including thought pattern strategies, is the development of high self-efficacy prior to performing an activity (e.g., Manz 1986; Manz and Neck 2004; Neck and Manz 1992, 1996). Thus, increased task-specific self-efficacy promotes superior performance expectations (Bandura 1991). Backed by empirical research, self-leadership has been found to be a very helpful process for achieving perceptions of high self-efficacy and task performance (Neck and Houghton 2006). According to self-leadership theory—to the degree that an activity or task is selected—a strong belief in self-determination coupled with the application of practiced skills in increasing a sense of proficiency can enhance a person's performance on a task (Neck and Houghton 2006). In other words, once a person truly believes something is within their hands to do and that they have the ability to do it, they have a much better chance of doing so. The aforementioned self-leadership strategies intentionally practiced over time increases that desired self-efficacy.

It should be noted though that self-leadership is not an isolated process. Improving not only the personal habits of thoughts and behavior but the interactions between all parties in the environment is crucial as well. After all, much of time pressure can be due to systematic factors within a unit. Systems improvements require the involvement of the whole team. Once the nurse manager has improved her/his practices of self-leadership, it is time to improve the environment he or she co-exists in with others in the unit. Turning a manager's employees into better self-leaders themselves is a process known as SuperLeadership. The best managers set the example of what a good self-leader does and empowers and coaches the rest of the team to reach that same level of self-performance. When achieved, a team can outperform others who must wait for a manager to inform them how to handle complex situations. In a sense, the SuperLeader has inculcated the values and goals into each team member to exceed what she/he can do alone (Manz and Sims 1989, 1991; Manz 1990, 1991, 1992; Neck and Houghton 2006). The best SuperLeaders of self-managing teams encourage and support their employees to learn and practice the self-leadership process (Neck and Houghton 2006). The nurse manager, after all, cannot find or fix all the factors and issues causing time pressure on the unit.

Given this extensive review of research on self-leadership theory and practices for better performance, we offer the following metaconjecture:

*Metaconjecture 3: In situations where nurse managers face increased time pressure, proper application of self-leadership practices can improve psychosocial care; i.e., self-leadership practices positively moderate the negative relationship between time pressure and psychosocial care.*


**5. Conclusions**

In conclusion, MeJA treatment decreased inflammation and oxidative stress in the brain of rats with adjuvant-induced arthritis. The improvement of oxidative stress occurred in consequence of reduced inflammation associated with increased antioxidant defenses. On the other hand, MeJA induced the hexokinase (HK) detachment from the brain mitochondria of both healthy and arthritic rats a phenomenon that can diminish glucose phosphorylation and metabolism in the brain. The effective doses of MeJA were relatively high, but the results of the present study make this compound a potential

precursor for developing anti-rheumatic and anti-inflammatory drugs. Finally, future investigations may take into account the role of Nfr2 in the actions of MeJA and the perspective of modifications in the metabolism of glucose in the brain tissue.

**Author Contributions:** Conceptualization, J.F.C. and A.B.S.-N.; investigation, H.V.P.-M., J.F.C., L.S.C., L.B., F.M.S.S. and G.A.G.; resources, J.F.C., A.B., R.M.P.; data curation, C.A.B.-A., A.B.S.-N. and J.F.C.; writing—original draft preparation, A.B.S.-N. and J.F.C.; writing—review and editing, A.B. and R.M.P..; visualization, A.B.; funding acquisition, J.F.C. and A.B.S.-N.

**Funding:** This work was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [Grant number:<PHONE_NUMBER>5].

**Acknowledgments:** Authors wish to thank the financial support of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


*3.1.1 Healthcare information organization*

Research on the subject of interoperability between blockchain applications in healthcare can be originated here [14]. The MedRec model [15] provides a proof-ofwork that allows blockchain designs and other decentralization standards to anchor and communicate with other medical record technologies. The architecture is organized using Ethereum smart contracts, which also provides a log that keeps track of medical data and allows users to see entire data, review care records, and exchange information. An innovative approach for structuring open APIs and system structure transparency, as well as conditioning with the supplier's existing framework, has also been described in this study [16]. An adaptive method for processing large-scale personal health data using a tree-like method is proposed [17]. A blockchain-based program has been adopted to secure documents stored in the cloud. A further platform built on the blockchain for healthcare data is BlockHIE [18], which includes off-chain archiving with on-chain validation to guarantee privacy as well as authenticity for records. With the use of blockchain technology, healthcare data may be transferred in a secure and auditable way [19]. Additionally, it employs blockchain to manage access-based health data [20, 21]. **Figure 3** represents the scope of CSP. Lastly, a blockchain-based technology for maintaining patient and physician databases in the field of healthcare analytics is shown. The benefits of a parallel healthcare system are brought in by a methodology that is given in Ref. [22] and is built on artificial systems, computational trials, and then a simultaneous implementation utilizing blockchain technology. To handle health data at the individual and institutional levels, several experts have suggested using private blockchain solutions [23].

**Figure 3.** *The scope of CPS.*


*3.2.1. EAP-CRA message and process details*

The proposed EAP-CRA packet is depicted in Table 2. The reasons for designing each of the fields are illustrated based on the associated requirements. The fields are transmitted from left to right. The first influencing factor of EAP-CRA is that it is based on the EAP protocol. Therefore, the fields, code, identifier and length are inherited from an EAP structure. The explanation of each field is listed below.

parameter which needs to be determined. The exact number for the time and trials will be decided in the actual implementation and depends on the protocol process time, line traffic and other unforeseen factors. One of the issues present in retry is the duplicate packets which must be handled by the receiving peer. Three retries will be performed, forming the base

EAP-CRA for WiMAX, WLAN and 4G LTE Interoperability

http://dx.doi.org/10.5772/54837

115

**Fragmentation**: EAP-CRA message may span multiple EAP-packets due to the multiple public and private key encryptions; hence there must be a method, to be engineered in the servers, for handling the fragmentation. As a base for work on the fragmentation, the length of the TLS record can be up to 16384 octets, while the TLS message may be 16 MB if it carries the PKI certificate of a server. However, to protect against denial of service attacks and reassembly lockup there must be maximum size set for the group of the fragmented messages. An example can be seen in what was implemented for EAP-TLS[19]. The exact numbers will be determined during implementation of the protocol, and will reveal the average length of long EAP-CRA messages. For the purposes of initial configuration, this number can be borrowed from EAP-

Since EAP is an uncomplicated ACK-NAK protocol, fragmentation support can be provided according to a relatively simple process. Damage or loss of fragments during transit is an inevitable risk for any communication. In EAP, these fragments will be retransmitted, and because sequencing information is included in EAP's identifier field, a fragment offset field

EAP-CRA fragmentation support will be provided by adding flag fields to the EAP-CRA packets inside the EAP-Response and EAP-Request. Flags include the Length (L), More fragments (M), and Start (S) bits. The L flag indicates the presence of the four octet Message Length field. It *must* be set in the first piece of a fragmented EAP-CRA message or set of messages. The M flag will be set in all except the last fragment showing that there are more frames to follow. The S flag will only be for the EAP-CRA start message sent from the EAP server to the peer. The T flag refers to the source type of the EAP-CRA message; whether it is coming from an 802.1x authenticator or from an authentication server. If there is a fragmented message, both server and the other peer must acknowledge the receipt of a packet with the flag set to M. The response can be an empty message to the other peer showing that the message

For our experiments we setup three different scenarios to compare the time taken to authen‐ ticate a user. Edu-roaming, EAP-CRA and direct authentication with a single RADIUS authentication server were considered. RADIUS servers were installed on Windows 2003

Microsoft Internet Authentication Service (IAS) with Microsoft EAP-PEAP was used in these experiments. IAS is the Microsoft implementation of a Remote Authentication Dial-In User Service (RADIUS) server and proxy in Windows Server 2003. As a RADIUS server, IAS performs centralized connection authentication, authorization and accounting for many types

Server standard edition and all platforms had 2 GB RAM and 2GHz dual core CPU.

configuration for the EAP-CRA.

TLS which is 64 KB.

has not been received.

**3.3. Experiments**

like that of IPv4 is not necessary.

**Table 2.** CRA Packet

The Code field is one octet and identifies the type of EAP packet. EAP Codes are assigned as 1 for Request, 2 for Response, 3 for Success and 4 for Failure. The Identifier field is one octet and aids in matching responses with requests. The Length field is two octets and indicates the length of the EAP packet including the Code, Identifier, Length and Data fields. Octets outside the range of the Length field should be treated as Data Link Layer padding and should be ignored on reception. The Flags field includes the following fields:

L = Length included, M = More fragments, S = EAP-CRA start, R = Reserved, T = Source Type

**Table 3.** Add Caption


**4.4 The impact of different concentrations of some heavy metals on some growth indicators of** *Lantana camera* **under** *in vitro* **culture conditions**

### *4.4.1 Cadmium (Cd)*

The data in **Table 7** indicate that there is no significant effect of the heavy metal cadmium concentrations in plantlet height, compared with the control treatment. It is also noted from the same table that there is no significant effect in each of the characteristics of the leaf numbers and the shoot dry weights among all treatments. While the addition of the cadmium heavy metal to the MS medium had a significant effect, as the plantlets treated with a concentration of 0.8 mg L−1 were significantly superior in the total shoot fresh weights, reaching 0.461 g, compared with the other treatments [27].

The data of the phytoremediation in **Table 7** for the *Lantana camera* plant show that there is no significant effect of different cadmium concentrations among all treatments in each of the shoot numbers plantlet−1, leaf area (cm2 ), and total chlorophyll content of the leaves (mg.100 g−1 fresh weight) (**Figure 3**).

The reason may be attributed to the use of plants to absorb these heavy metals from the culture media and translocate them to the vegetative organs or convert them into volatile compounds using the phytovolatilization technique. This technique exploits the ability of some plants to convert some heavy elements into volatile compounds for disposal [38].

The reason for this may be because the plant is a natural phytoremediator, as it can accumulate the contaminant, break it down, or assemble it in its biomass, and it is characterized by being a fast-growing plant and having a large biomass and having a widespread root system [39].

The data in **Table 8** show the effect of cadmium on the root growth indicators of the *L. camara* plantlets. It is noted that there are no significant differences in the number of main roots per plantlet in all treatments. It is also observed from the same table that there were no significant differences among all cadmium treatments in each of the root length and dry weight characteristics, while the data are shown in the same table

*The Efficiency of Phytoremediation of the Big-Sage Plant in Accumulating Some Heavy Metals… DOI: http://dx.doi.org/10.5772/intechopen.109640*

### **Figure 3.**

*Effect of different concentration of cadmium on plantlet growth of* Lantana camara *shrub [37].*

### **Table 8.**

*Effect of different concentrations of cadmium on some root growth of* Lantana camara *shrub [37].*

that there was a significant effect in the fresh weight of the roots in the MS medium to which the cadmium heavy metal was added. The MS medium supplemented with 0.8 mg L−1 cadmium was significantly superior in the root fresh weight, reaching 0.114 g. This is explained as the ideal concentration of *L. camara*. Despite the toxicity of the lead element, the big-sage shrub showed a phytoremediator for this heavy metal.

This can be explained by our findings is the ability of the big-sage plant to accumulate and be tolerant to cadmium heavy metal. Al-Wahaibi [40] indicated the characteristics of the accumulating plants when they absorb heavy elements, they stimulate the form of chelating compounds that surround the atoms of the contaminating elements and keep them within the vacuoles found in the cells of plant tissues.

### *4.4.2 Cobalt (Co)*

**Table 9** shows the effect of different concentrations of the heavy element cobalt on the vegetative growth indicators (**Figure 4**). The data showed that there was no significant effect on the characteristics of each of the plant's height (cm), leaf numbers, and the fresh and dry weights of the shoots (g) among all treatments [27].

**Table 9.** *Effect of different concentrations of cobalt on some vegetative growth of* Lantana camara *shrub [37].* *The Efficiency of Phytoremediation of the Big-Sage Plant in Accumulating Some Heavy Metals… DOI: http://dx.doi.org/10.5772/intechopen.109640*

**Figure 4.** *Effect of different concentration of cobalt on plantlet growth of* Lantana camara *shrub [37].*

**Table 9** includes the effect of different cobalt heavy metal concentrations on the shoot numbers per plantlet. There was no significant effect of the element cobalt in this characteristic among all treatments. The addition of cobalt to the MS medium had no significant effect on the total chlorophyll content of leaves in all treatments.

The different concentrations of the heavy element cobalt had a significant effect on the leaf area. The treatment with 0.8 mg L−1 cobalt showed a significant effect on the leaf area, reaching 3.00 cm2 compared with other treatments, except for the treatment with 0.6 mg L−1 cobalt, which did not differ significantly from it, reaching 2.70 cm2 .

The reason for this is that the heavy metal ions that enter the cell are associated with the chelators and companions. These chelating compounds remove the toxicity of metals by transporting minerals to the cytosol, while the companion transfer minerals to the organelles to reach the proteins that require metal. There are many chelating metal compounds and well-known chelators in plants, including phytochelatins, metallothioneins, organic acids, and amino acids [41].

The data of **Table 10** showed the effect of adding different cobalt concentrations of the MS medium on root growth indicators of *L. camara*. It was observed that there

### **Table 10.**

*Effect of different concentrations of cobalt on some root growth of* Lantana camara *shrub [37].*

*The Efficiency of Phytoremediation of the Big-Sage Plant in Accumulating Some Heavy Metals… DOI: http://dx.doi.org/10.5772/intechopen.109640*

was no significant effect on some root growth characteristics, including the main root numbers per the plantlet, root length, and root dry weights.

The contamination of the MS medium with cobalt had a significant effect on the total root fresh weights. The treatment at 0.8 mg L−1 cobalt was significantly superior in this characteristic, reaching 0.055 g compared with the treatments at 0.6 mg L−1 cobalt and the control, which reached 0.026 g.

The reason for this may be that plants exposed to high levels of cobalt, more than the permissible levels of heavy metals, show symptoms of toxicity due to excessive treatment of cobalt, which is more than what most species need. Moreover, cobalt toxicity rarely occurs when plants are exposed to low levels [42]. Therefore, the *L. camara* shrub showed tolerance for this heavy metal.

*4.4.3 Lead (Pb)*

The data in **Table 11** show that there are no significant differences in the characteristics of vegetative organs, plantlet height (cm), leaf numbers, and fresh and dry weights of the shoot (g) of *L. camara* shrub grown in MS media that supplemented with different concentrations of lead heavy metal (**Figure 5**) [27].

The data in **Table 11** show that there were no significant differences when the MS medium was contaminated with lead after 1 month of the experiment in each of the characteristics of the number of leaves per shoot, leaf area (cm2 ), and total chlorophyll content of leaves (mg 100 g−1 fresh weight).

The data of **Table 12** indicate that there are no significant differences when adding lead at the different concentrations in the MS medium in each of the characteristics of the main root numbers per the plantlet, root length (cm), and the fresh and dry weights of the root parts (g).

This can be explained by the limited transport of lead through the root, as a result of the precluding caused by the Casparian strip in the root endodermis, which prevents the translocation of lead through the endodermis to the central vascular cylinder tissues.

### **Table 12.**

*Effect of different concentrations of lead on some root growth of* Lantana camara *shrub [37].*

Whereas the accumulation of lead depends on the species, variety, and plant organ, and then increases in the accumulation within the root organs compared with the vegetative organs, and then a decrease occurs in some characteristics of the vegetative organs such as total fresh weight of the shoots when the concentration of lead is increased, which causes a difference in the characteristics of the roots at the expense of the characteristics of the vegetative parts [43].