An Acidic Polysaccharide with Anti-Inflammatory Effects from Blackened Jujube: Conformation and Rheological Properties

An acidic polysaccharide fraction (BJP-4) was isolated from blackened jujube, and its advanced structures and anti-inflammatory activity were investigated. X-ray diffraction showed that BJP-4 exhibits both crystalline and amorphous portions. Atomic force microscopy data suggested that it contains a large number of spherical lumps. Circular dichroism and Congo red experiments revealed that it has no triple-helix conformation. In steady shear flow results, the BJP-4 solution was a pseudoplastic non-Newtonian fluid with acid-base stability. BJP-4 (20 mg/mL) showed liquid-like properties (G″ > G′), while it performed weak gel-like behavior at a high concentration (40 mg/mL) (G′ > G″). The anti-inflammatory effects of BJP-4 were further evaluated through in vitro experiments. BJP-4 could down-regulate the over-secretion of inflammatory factors (NO, IL-6, IL-1β, TNF-α, iNOS and COX-2) in RAW264.7 cells due to LPS stimulation. Moreover, it demonstrated that BJP-4 restrained the NF-κB signal pathway by regulating TLR4 expression, reducing IκBα phosphorylation level and NF-κB p65 nuclear translocation. In summary, this present study contributes to the application of blackened jujube polysaccharides in the foods and medicine field.


Introduction
Inflammation is the body's initial response to the stimulation of invasive harmful substances such as bacteria, viruses, and other pathogens [1]. It is generally believed that inflammation is the basic pathological process of disease and is associated with the pathogenesis and progress of various diseases [2,3]. Inflammation-related factors, including interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) play a critical role as promoters and indicators [4]. In these mediators, TNF-α, IL-6, and IL-1β are crucial cytokines for pro-inflammation, which can directly express the level of inflammation. The iNOS can promote nitric oxide (NO) secretion in cells, thereby stimulating the expression of various proteins and enzymes that play a key role in inflammation [5]. In addition, COX-2 is an inducible synthase, which is the key enzyme in initiating inflammatory responses [6]. Therefore, modulating inflammatory factors is one of the effective pathways to suppress the inflammatory response.
RAW264.7 macrophages, as the major members of the body's immune function, perform vital physiological actions during inflammation and are greatly used for cell biology research [7,8]. Studies have confirmed that when Toll-like receptor 4 (TLR4) received exogenous stimulation, the inhibitor kappa B alpha (IκBα) was phosphorylated and degraded, and then dissociated from nuclear factor kappa B (NF-κB) [9]. Then, the NF-κB p65 subunit enters the nucleus after being activated, further initiating the expression of related enzymes (COX-2 and iNOS) and the production of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α [10]. Therefore, the TLR4/NF-κB signaling pathway is consistently recognized by most researchers as a critical pathway for treating inflammation.
Jujube, broadly cultivated in China, has many bioactive compounds such as polysaccharides, polyphenols, vitamins, and adenosine cyclophosphate, of which polysaccharides account for more than 60% of dry weight [11]. Jujube polysaccharides were reported to exhibit various pharmacological effects, including antioxidant, anti-inflammatory, antitumor, and hepatoprotective activities [12]. Notably, a polysaccharide from Z. jujuba cv. Junzao, rich in galacturonic acid and with a molecular weight (MW) of 153.3 kDa, exhibited significant anti-inflammatory effects by inhibiting the secretion of TNF-α, NO, and COX-2 in RAW264.7 macrophages [13]. In addition, it was reported that a polysaccharide from the fruit of Z. jujuba cv. Muzao, which mainly contained arabinose (4.52%) and galactose (2.64%) with an MW of 89.90 kDa, inhibited the levels of IL-6 and TNF-α in RAW264.7 cells [14]. Blackened jujube is an elaborate food made of dried jujube fermented at 70-75 • C under a certain humidity for 5-7 days [15,16]. The in vitro antioxidant capacity of blackened jujube polysaccharides (BJPs) was reported, confirming the free radical scavenging activity and total reduction power, and exhibiting therapeutic effects on H 2 O 2 -stimulated HUVECs [17]. Considering the close relationship between anti-inflammatory capacity and oxidation imbalance, we conjecture that BJPs have excellent anti-inflammatory activity.
The biological functions of polysaccharides not only change with the primary structure (MW, monosaccharide composition, and sugar chain sequence) but are also closely related to the conformation [18][19][20]. For example, it is generally believed that polysaccharides with triple helix structures exhibit more excellent biological activities [21]. The structure results of jujube polysaccharides are quite different, mainly affected by jujube varieties, the extraction technology, purification process, and detection method. The advanced structure and anti-inflammatory effects of BJPs have been poorly reported; thus, exploring their structure-effect relationships was of great significance.
Following the previous method, BJP-4 was extracted using ultrasonic-assisted hot water and purified by a DEAE-cellulose 52 anion-exchange column and Sephadex G-100 gel column. In addition, BJP-4 had a high yield (12.36%) and excellent antioxidant activity with an IC 50 of 0.54 mg/mL and 0.58 mg/mL for DPPH and ABTS+ radical scavenging [17], respectively. As so, this fractionation was selected for further study. In this study, the conformational analysis of BJP-4 is systematically estimated by X-ray diffraction (XRD), atomic force microscopy (AFM), circular dichroism (CD), and Congo red experiments. Then, the anti-inflammatory effect of BJP-4 is evaluated with Lipopolysaccharide (LPS)-treated RAW264.7 cells as the model. These results could provide the theoretical link between the advanced structure and anti-inflammatory activity of blackened jujube polysaccharide and demonstrate its potential value for development as a novel anti-inflammatory drug. The effects of mass concentration and pH conditions on the rheological properties of blackened jujube polysaccharide are also investigated to provide guidance for its application in the food field.  Crystallization of BJP-4 was measured using XRD (D8 Advance, Bruker, Germany). Briefly, 2 mg of BJP-4 was weighed and placed on the sample plate, and carefully flattened. The operating conditions were applied as follows: Cu Kα radiation, 40 kV, 40 mA, 2θ = 5 • − 80 • , and a speed of 3 • /min [22].

AFM Measurement
BJP-4 samples were prepared at a concentration of 1 µg/mL then transferred to the mica carrier and dried at 25 • C. The AFM (BioScope Catalyst NanoScope V, Bruker, Billerica, MA, USA) was used to tap the dry mica flake and present images [23].

Congo Red Experiment
The Congo red experiment was performed as previously reported [25]. Briefly, 1.

Rheological Measurements
The rheological characterization was performed on a rheometer (MCR 102, Anton Paar, Germany) at 25 • C with a parallel board (diameter 50 mm, clearance 1 mm).

Steady Shear Analysis Effect of Concentration on Viscosity Characteristics of BJP-4
A series of BJP-4 sample solutions with concentrations of 2.5, 5.0, 10, 20, 40 mg/mL in deionized water was prepared. Sample flow curves were measured in viscosity and flow curve templates, where the shear rate range was set to 0.1-100 s −1 .

Viscosity Properties of BJP-4 at Different pH
The pH values of 20 mg/mL BJP-4 solution were adjusted to around 3.0, 7.0, and 11 using 1.0 M NaOH and HCl. The equipped solutions were measured as mentioned in the Effect of Concentration on Viscosity Characteristics of BJP-4.

Dynamic Oscillatory Analysis
Two types of BJP-4 solution (20 mg/mL and 40 mg/mL) were obtained as detailed in the Effect of Concentration on Viscosity Characteristics of BJP-4. Before dynamical oscillatory frequency measurement, the sample strain value was obtained using the linear viscoelastic region mode with a scanning range of 0.1-100%. According to the strain value, oscillatory measurements were implemented in the frequency range of 10 −2 -10 1 Hz at 25 • C and then the elastic modulus (G ) and viscous modulus (G ) of the polysaccharide solutions were recorded. The loss factor (tanδ) was derived from the following Equation (1). (1)

Cell Culture and Viability
A DMEM medium containing penicillin (100 U/mL) and 10% FBS was used to incubate RAW264.7 macrophages at 37 • C with 5% CO 2 .
Assessment of the cytotoxicity of BJP-4 in a certain concentration range (12.5-200 µg/mL) was performed using the CCK-8 method. Briefly, cells were cultured to the logarithmic growth phase and transferred to 96-well plates at a concentration of 3 × 10 3 cells/well, and then treated with 1 µg/mL LPS and the corresponding concentration of BJP-4. After 24 h of treatment in an incubator at 37 • C with 5% CO 2 , CCK-8 was added and incubated for 2 h. The microplate reader (ELX-800, Biotek, Winooski, VT, USA) was applied to determine the optical density (OD) of the solution at 450 nm. Cell viability was computed as follows:

Measurement of NO and Cytokines
Cells culture was as described in Section 2.4.1. Cell culture supernatant was aspirated and cytokine expressions were detected using ELISA kits based on the instructions.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
The gene expression levels in the RAW264.7 macrophages were determined by qRT-PCR, as previously reported [26]. TRIpure reagent was utilized to extract total cellular RNA, and then a spectrophotometer (Nanodrop 2000c, Thermo Fisher Scientific, Waltham, MA, USA) was applied to detect RNA concentration. The cDNA was synthesized from total RNA through Transcriptor Reverse Transcriptase. The amplification of the qRT-PCR used SYBR Green Master Mix on a system Exicycler 96 (Bioneer, Daejeon, South Korea). The amplification conditions were 94 • C for 5 min, 60 • C for 20 s (40 cycles), 72 • C for 150 s, and 40 • C for 90 s. The specific primers' sequences are listed in Table 1. The experimental analysis was performed using the 2 −∆∆Ct method. Table 1. Primer sequences of RT-PCR analysis.

Gene
Product Size (bp)

Western Blot
The cell treatments were the same as in Section 2.4.1. The Western blot analysis was based on the method in the literature [27]. Briefly, the proteins were isolated and quantified using protein extraction and BCA protein assay kits, respectively. Various concentrations of polyacrylamide gels (5-14%) were prepared and subjected to protein electrophoresis, after which the proteins were transferred to polyvinylidene difluoride (PVDF) film. PVDF films were removed and immersed in Tris-buffered saline with Tween 20 (TBST) and shaken for 5 min, then the membranes were immersed in 5% (m/v) skim milk powder solution and shaken for 1 h. Conditions of the primary antibody incubation were TLR4 (1:500), IκBα (1:1000), NF-κB p65 (1:1000), p-IκBα (1:1000), p-NF-κB p65 (1:1000), iNOS (1:500), COX-2 (1:1000), β-actin (1:1000), and Histone H3 (1:1000), overnight at 4 • C. After that, the films were removed from the hybridization bag and soaked in TBST for four washes, and then the goat anti-rabbit IgG HRP secondary antibodies were incubated at a dilution ratio of 1:5000 for 45 min at 37 • C. After washing six times, the ECL reagent was added to the membranes, which were photographed by the WD-9413B imaging system (Beijing LIUYI Biotechnology Co., Ltd., Beijing, China).

Statistical Analysis
Results are denoted as mean ± standard deviation (SD). The significance of differences was evaluated using a one-way analysis of variance (ANOVA) followed by Tukey's test using SPSS software with p < 0.05.

Morphological Properties of BJP-4
XRD can provide key information about the structure of biomaterials, such as the crystalline or amorphous properties of polysaccharides. As shown in Figure 1A, BJP-4 had a "bun-shaped" peak in the range of 10 to 15 • . In addition, there were two sharp and narrow characteristic peaks that appeared at 21.1 • and 22.4 • , corresponding to the crystalline nature of BJP-4 [28]. Meanwhile, there were small diffraction absorption peaks in the 40-50 • range related to the uronic acid found in acidic polysaccharides [29]. In summary, it indicated that BJP-4 exists in both a partially crystalline structure and amorphous fraction. This result was the same as previously reported for Althaea officinalis L. root [30]. AFM is commonly applied to observe the morphological characteristics of biological substances. As shown in Figure 2B,C, there were numerous spherical lumps of BJP-4, suggesting molecular aggregation. These can be explained by the interaction of carboxyl and hydroxyl groups of polysaccharides and the formation of molecular interactions with water molecules, which cannot be ignored [31]. The morphology of BJP-4 was different from that of Muzao [32], Ruoqiangzao [29], and Linzexiaozao [11], which might be related to galacturonic acid and hydrogen [28].

Advanced Structure of the Polysaccharide BJP-4
CD is considered to be a classical method for analyzing the conformational variation in polysaccharide secondary structures. The CD spectra of BJP-4 was shown in Figure 1D. No negative absorption peak was found over the 185-400 nm wavelength range, indicating that BJP-4 contained no helical structure. However, a large positive absorption spike was recorded at 190 nm, which proved to be an asymmetric structure in BJP-4. This was primarily caused by the folding, flipping, winding, and irregular morphology of polysaccharide molecules in an aqueous solution [33]. Congo red analysis was conducted to identify whether a triple helix structure was involved in BJP-4. The maximum absorption (λ max ) changes of BJP-4 and Congo red complex were shown in Figure 1E. No red shift in λ max of the complex was observed in Congo red solution as blank control, indicating that there was no triple helix structure in BJP-4, which was consistent with the results of CD. This phenomenon was in accordance with the previous research [30]. Numerous studies have shown that the triple helix structure and MW of polysaccharides significantly affect their pharmacological activity. For example, under the condition of similar molecular weight, the triple helix structure had better antitumor effects than the single helix structure of lentinan [34]. However, the structure-function relationships of polysaccharides are difficult to reveal systematically and more studies are needed to provide theoretical references.

Steady Shear Flow Properties
The apparent viscosities of the BJP-4 solutions at different concentrations (2.5-40 mg/mL) are displayed in Figure 2A. From the 0.1-100 s −1 shear rate range, the apparent viscosity of BJP-4 decreased with increasing shear rate and exhibited shear thinning behavior, which was a typical pseudoplastic non-Newtonian fluid. This behavior was mainly due to the change of the link point between molecules as the shear rate increased, which made the resistance between molecules smaller and led to a decrease in viscosity. The apparent viscosity of BJP-4 showed a significant concentration-dependent effect, where the viscosity of 40 mg/mL BJP-4 could reach 2000 mPa·s at a shear rate of 0.1 s −1 . This phenomenon was explained by the increasing number of polysaccharide molecules per unit volume and the enhancement of molecular association, resulting in an increase in the degree of polymerization and an increase in the flow resistance of the solution. Similar shearthinning characteristics have been reported for polysaccharides extracted from Basil seed gum, Dendrobium officinale, and Extreme salt-tolerant Bacillus subtilis LR-1 [35][36][37]. Figure 2B shows the viscosity characteristics of BJP-4 (20 mg/mL) samples with different pH values ranging from 0.1-100 s −1 . The viscosity of BJP-4 at pH = 3.0 and pH = 11 was less than that at pH = 7.0, which was attributed to the hydrogen bond cleavage of BJP-4 polysaccharide in an acidic or alkaline environment, thus promoting the decomposition of polysaccharide and reducing its apparent viscosity. However, the comprehensive results showed that the BJP-4 polysaccharide solution has good acid-base stability, so it could be used in acidic and alkaline foods.

Dynamic Oscillation Shear Test
The linear viscoelastic region was measured for different concentrations (40 mg/mL and 20 mg/mL) of BJP-4 solution showing results of 1-10% and 30-100%, respectively. In this experiment, frequency oscillation tests were carried out under 5% and 50% strain values, respectively. As can be seen in Figure 2C-F, the modulus G (loss modulus) was less than the modulus G (storage modulus) at a concentration of 40 mg/mL and exhibited a strong elastic gel-like behavior with tanδ < 1 at all frequencies. On the contrary, 20 mg/mL of BJP-4 showed viscous behavior. This phenomenon was similar to Yam polysaccharide, which exhibited "liquid-like" behavior below 14% (w/v) and "gel-like" behavior for 14-20% (w/v) [38].

Evaluation of BJP-4 on Cell Viability and NO Production
The assessment of the cytotoxicity of BJP-4 on RAW264.7 cells used a CCK-8 assay. As presented in Figure 3A, no significant cytotoxicity of BJP-4 was observed below 50 µg/mL. However, 100 µg/mL of BJP-4 treatment caused a 22.7 ± 9.2% reduction in cell viability compared to the blank control (p < 0.05). In RAW264.7 cells, excessive NO production can lead to a systemic inflammatory response [39,40]. As can be seen in Figure 3B, the NO secretion of RAW264.7 cells after induction by LPS increased by 13.19 ± 1.0 µM, which was extremely significantly higher than that of the blank control group (p < 0.01), showing that the inflammation model was successfully established. It was noteworthy that BJP-4-treated groups showed better inhibition of NO secretion only at doses higher than 25 µg/mL (p < 0.05), and the anti-inflammatory effect of BJP-4 was concentration-dependent over a range of dose concentrations (25−200 µg/mL). In particular, under the stimulation of 25 and 50 µg/mL BJP-4 secreted 13.9 ± 1.5 and 11.3 ± 1.5 µM of NO, respectively, which was 18.2 ± 8% and 33.5 ± 8% less than those of the positive control (LPS). Combining the above results, three optimal concentrations of BJP-4 (12.5, 25, 50 µg/mL) were chosen for follow-up tests.

Regulation of BJP-4 on mRNA Expression of Multiple Cytokines
The effects of stimulation of RAW264.7 cells with different products (LPS and BJP-4) on their mRNA expression levels of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β, COX-2, and iNOS) were further examined by qRT-PCR. As seen in Figure 4A-C, LPS treatment alone significantly increased TNF-α production by 5.09-fold, IL-1β production by 5.19fold, and IL-6 production by 4.08-fold, respectively, in comparison with the blank control (p < 0.01). BJP-4 was found to interfere with the mRNA expression of pro-inflammatory cytokines in a similar fashion to its impacts on secretion levels, exhibiting a dose-dependent down-regulation of gene expression. Notably, the gene expressions of TNF-α, IL-1β, and IL-6 by BJP-4 (25 µg/mL) treatment were reduced to 38.70%, 44.30%, and 31.37% of the LPS group, respectively (p < 0.01), and suppressed 68.96%, 63.58%, and 57.35% by BJP-4 (50 µg/mL), respectively (p < 0.01). The iNOS, a synthase that promotes NO production, is secreted by macrophages after inflammation. COX-2, an inducer enzyme, is expressed by macrophages subjected to multiple stimuli (e.g., pro-inflammatory factors). As can be seen from Figure 4D,E, the addition of LPS resulted in overexpression of iNOS and COX-2 gene levels in RAW264.7 macrophages, which increased by 4.05-and 4.31-fold, respectively, using the blank control group as a reference (p < 0.01). The inhibitory effect of three concentrations of BJP-4 on cytokine gene expression was enhanced as the dose increased (p < 0.01), especially when it was 50 µg/mL; the iNOS and COX-2 mRNA expressions accounted for 37.78% and 38.05% of the LPS group, respectively (p < 0.01). Additionally, similar trends were observed for the protein expression of iNOS and COX-2. Similar results were also observed in RAW264.7 macrophages with LPS-triggered inflammation in response to the supplementation of Moringa oleifera roots, Umbilicaria yunnana, and Rubus chingii Hu polysaccharides [45][46][47].

Conclusions
In the present study, a blackened jujube polysaccharide BJP-4 was found to have both semi-crystalline and amorphous portions and was identified to have no triple-helix conformation. In addition, BJP-4 exhibited non-Newtonian shear-thinning flow behavior and had good acid-base stability at pH 3.0-11. The dynamic shear rheological properties showed that BJP-4 exhibited weak gel behavior (G < G ) at a high concentration (40 mg/mL). BJP-4 is a non-gel polysaccharide and could be used as a food thickener. Further analysis found that BJP-4 had an excellent anti-inflammatory effect against RAW264.7 cells, which could obviously restrain the secretion of NO, IL-6, TNF-α, IL-1β, COX-2, and iNOS. Moreover, BJP-4 remarkably down-regulated the protein expression of TLR4, p-IκBα/IκBα, and p-NF-κB p65/NF-κB p65, suggesting that the TLR4/NF-κB signaling pathway performs a crucial action in the anti-inflammatory effect of BJP-4. This research could contribute to theoretical support for the conformation-activity relationship of blackened jujube polysaccharides, which is of vital relevance to the exploration of novel foods and anti-inflammatory drugs.
Because of the complex structure of polysaccharides, the anti-inflammatory activity for a single fraction of blackened jujube polysaccharides and whether its structure-activity relationship is relatively limited remains to be revealed and requires further systematic study. Moreover, the anti-inflammatory activity of polysaccharides and their mechanisms of action are more investigated at the cellular and animal levels, and there is a need for further clinical trial studies.