Systematic Comparison of Structural Characterization of Polysaccharides from Ziziphus Jujuba cv. Muzao

To investigate the structural information differences of Ziziphus Jujuba cv. Muzao polysaccharides, ten samples were successfully extracted from aqueous and alkaline solutions, prepared via DEAE-Sepharose Fast Flow through different eluents and Sephacryl S-300 columns, and systematically analyzed. Their characteristics were studied and then compared using chemical testing, high-performance gel permeation chromatography (HPGPC), gas chromatography (GC), methylation analysis, and NMR spectroscopy. The data achieved demonstrated that different jujube polysaccharide fractions possessed different structural characteristics, and most of them belonged to pectic polysaccharides. Overall, the structural information difference of jujube polysaccharides was preliminarily illuminated, which could not only promote the potential application of Z. Jujuba cv. Muzao polysaccharides but also provide an effective way to analyze the structures of polysaccharides from other genera jujube fruit.


Introduction
Chinese jujube (Zizyphus Jujuba Miller), named "Dazao" or "Hongzao" in Chinese, is mainly found in Asia's subtropical and tropical regions, particularly in the eastern regions, the Yellow River Valley regions, and the northwest [1]. Traditional Chinese medicine (TCM) often employs jujube as a crude medicament since it is a fruit that is advantageous and lucrative. According to Bencao Gangmu, written between 1552 AD and 1578 AD, jujube fruit was believed to improve people's "Qi" (the vital energy) and "Blood" (body circulation) [2]. Nutritional findings have reported that various functional compositions within jujube are being proposed, including total phenolics, individual phenolic compounds, triterpenic acids, polysaccharides, α-tocopherol, carotene, vitamin C (Vc), nucleosides, nucleobases, etc. [3]. According to pharmacological investigations, jujube has various medical qualities, such as anti-obesity, anticancer, anti-inflammatory and antioxidant, as well as immunostimulating, gastrointestinal protective, and hepatoprotective properties. It also inhibits the production of foam cells in macrophages [3][4][5].

PZMPs' and SAZMPs' Separation and Purification
Deproteinization, extraction with hot water/alkaline extraction, precipitation of ethanol, and freeze-drying were used to produce ZMP and AZMP from Z. Jujuba cv. Muzao. Regarding dried jujube fruit power, the overall yield of ZMP and AZMP was 3.82% and 3.30%, respectively. The ZMP and AZMP fractions were progressively separated using Fast Flow (DEAE-Sepharose) and gel filtration columns (Sephacryl S-300) to purify them further. Figure 1 displays a flow diagram of the separation process for ZMP and AZMP fractions. According to the elution curve, PZMP1 and SAZMP1 were determined as neutral polysaccharides, and PZMP2-4 and SAZMP2-4 were acid polysaccharides [14]. The yield was a crucial parameter for extraction efficiency for futural structure research. In a previous study, PZMP yield ranged from 0.76% to 14.61%, with a total yield of 37.64%, based on ZMP level, while SAZMP yield ranged from 2.26% to 10.22%, with a total yield of 24.23%, based on AZMP level [14]. All the total extraction yield of purified ZMP and ZAMP fractions was calculated at 2.24% according to dried jujube fruit power. It is possible that the different eluents and extraction techniques used in this research contributed to the heterogeneity in the yield of jujube polysaccharides [15].

Physical and Chemical Characteristics of PZMPs and SAZMPs
The total carbohydrate, protein content, and phenol of PZMPs and SAZMPs are given in Table 1. Phenol-sulfuric acid assay results revealed that PZMPs and SAZMPs contained higher carbohydrates (>95%), excluding only 92.64% total sugar of PZMP4. The higher the carbohydrate, the more accurately the jujube polysaccharides' structure could be analyzed [16]. Additionally, traces of total phenol and protein were discovered in all PZMP and SAZMP samples, and only PZMP4 included a higher protein content (3.09%). The result was similar for ZSP4b extracted through Z. Jujuba cv. Jinsixiaozao, which might be caused by a higher concentration of NaCl-PBS eluent [15]. The total phenolic contents of SAZMPs were not discovered using the Folin-Ciocalteu reagent technique, and the contents evaluated in PZMPs increased gradually from 0.139% to 0.950%. The ten polysaccharide fractions of Z. Jujuba cv. Muzao may have distinct chemical compositions according to these facts.

Physical and Chemical Characteristics of PZMPs and SAZMPs
The total carbohydrate, protein content, and phenol of PZMPs and SAZMPs are given in Table 1. Phenol-sulfuric acid assay results revealed that PZMPs and SAZMPs contained higher carbohydrates (>95%), excluding only 92.64% total sugar of PZMP4. The higher the carbohydrate, the more accurately the jujube polysaccharides' structure could be analyzed [16]. Additionally, traces of total phenol and protein were discovered in all PZMP and SAZMP samples, and only PZMP4 included a higher protein content (3.09%). The result was similar for ZSP4b extracted through Z. Jujuba cv. Jinsixiaozao, which might be caused by a higher concentration of NaCl-PBS eluent [15]. The total phenolic contents of SAZMPs were not discovered using the Folin-Ciocalteu reagent technique, and the contents evaluated in PZMPs increased gradually from 0.139% to 0.950%. The ten polysaccharide fractions of Z. Jujuba cv. Muzao may have distinct chemical compositions according to these facts.

Weight of Molecular and Composition of Monosaccharide Analysis
Jujube polysaccharide molecular weight distribution is essential for biological activity. As shown in Table 1, a wide range of Mw (8.23-141 kDa) was found in PZMPs and SAZMPs. Compared with water-extracted jujube polysaccharides, the Mw of alkaline (0.1 M NaOH solution) extracted polysaccharides was generally lower, which may be explained by the alkaline solution's influence on the glycosidic bond's breakdown, accelerating the destruction of the jujube saccharide molecule [26]. Low Mw jujube polysaccharides may have significant potential benefits for intestinal absorption, as well as for food digestion and the development of intestinally beneficial bacteria [16]. Different molecular weights of ZMP were attributed to different extraction conditions [10].
Information on the monosaccharide composition is far more crucial for understanding jujube polysaccharides' physiochemical and structural characteristics. As can be seen in Table 1, almost all of the PZMPs and SAZMPs were made up of seven distinct types of monosaccharides with varying amounts. Rhamnose, galacturonic acid, galactose, and arabinose were among the principal constituents in ZMP and SAZMP samples. Additionally, the structural types of jujube polysaccharides were determined by calculating the relative content ratio of (Ara + Gal)/Rha (0.69-38.98). While PZMP1 and AZMP1 contained no uronic acid, the amount of galacturonic acid assessed in other jujube polysaccharide fractions rose steadily, which was connected with the rising ion exchanging capacity of the NaCl eluent [14]. These data reconfirmed that different purified jujube polysaccharides possessed other structural characteristics, and SAZMP4 contained the most acidic group.

Analysis of Methylation
Determining glycosyl linkages in jujube polysaccharides may be achieved using GC-MS to examine methylation, which is a reliable and effective method. The findings are shown in Table S1 based on the CCRC Spectral Database's default data. The following conclusions can be drawn: (1) Different PZMPs and SAZMPs had several primary forms of sugar bonds, and the most prevalent derivatives matched the monosaccharide composition discovered by GC. (2) 1,3,5-, 1,5-and 1,3-linked Araf were the central glycosidic bonds in all ZMP and AZMP samples. (3) In the PZMPs, the galacturonic acid proportion increased with the concentration of eluent sodium chloride [14]. No uronic acid was found in the AZMPs (SAZMP3 and SAZMP4), which might be because the alkaline solution reacted with the acidic group of uronic acid and could not be detected [27]. (4) The fact that the total amount of terminal Araf /Galp/Glcp/Rhap residues was about equivalent to the total amount of branching residues indicates that all PZMPs and SAZMPs underwent successful methylation.

Analysis of NMR
NMR analysis was performed on the PZMPs and SAMPs to shed light on their structural details further. Table S2 summarizes all the relevant NMR data for various glycosidic connections. The following conclusions could be made: (1) Pure jujube polysaccharides had various main anomeric proton signals; 3-8 main anomeric proton signals were found in the PZMPs. (2) The protons in the C-residues with chemical shifts between 5.30 and 3.60 ppm in the 1 H NMR spectra were identified. (3) The same glycosidic bond types might have different C/H proton signals under the same/different NMR conditions. (4) The NMR glycosidic bonds of C/H signal assignment were similar to methylation results. (5) Through NMR and methylation analysis, the structural characteristics and structural types of jujube polysaccharides could be determined, and most of the jujube polysaccharides belonged to pectin polysaccharides [28,29].

Analysis of Chemicals
The phenol-sulfuric acid technique was used to determine the total sugar content of PZMPs and SAZMPs, using glucose as the benchmark [30]. Using the Bradford technique and the reference concentration of bovine serum albumin, the amount of protein was measured [31]. With minor modifications, gallic acid was utilized as the reference material to determine the total phenolic content using the Folin-Ciocalteu reagent [32].

Gel-Permeation Chromatography and Monosaccharide Analysis
PZMPs and SAZMPs were identified as monosaccharides and quantified using GC analysis, as previously mentioned [33]. The monosaccharide derivatized product was determined using GC (Shimadzu 2014 C, Kyoto, Japan) and a high-performance capillary column (DB-17, 30 mL × 0.25 mm ID, film thickness of 0.25 µm, Agilent, Santa Clara, CA, USA).
PZMPs and SAZMPs were analyzed using HPLC utilizing Agilent-LC 1200 equipment and a TSK-gel G3000PWxl/G5000PWxl column (7.8 mm × 300 mm) to establish average molecule weights and uniformity [34]. A calibration curve was used to estimate the molecular weights of PZMPs and SAZMPs compared to dextran.

NMR Analysis
An NMR spectrometer (AVIII-600, Bruker Corporation; Billerica, MA, USA) was used to acquire the NMR spectra in one and two dimensions of PZMPs. SAZMPs swapped for deuterium were diluted in D 2 O (0.5 mL), and NMR spectra ( 1 H and 13 C) were obtained using a Brucker AVANCE III 500 MHz NMR [37].

Conclusions
The present study prepared a series of Z. Jujuba cv. Muzao polysaccharides through various extraction and purification methods and found some interesting interrelations concerning the physicochemical properties of varying Z. Jujuba cv. Muzao polysaccharides. The molecular weight ranged from 8.23 to 141 kDa, and the monosaccharide composition was composed of various molar ratios of rhamnose, arabinose, galactose, galacturonic acid, etc. Arabinofuranosyl had methylated sugar derivatives of Z. Jujuba cv. Muzao polysaccharides, which belong to pectic polysaccharides. The present work might methodically aid in a more significant comprehension of the many Z. Jujuba cv. Muzao polysaccharide structures and offer a thoroughly relevant bibliography for more structure-function correlations and precise structural details for other species of jujube polysaccharides.