Purication, Characterization, and Self-Assembly of the Polysaccharides from Allium Schoenoprasum

Background: Allium schoenoprasum is a world-wide common vegetable while only its leave is used in the food factory. Its stalk is largely discarded, for potential heavy metal accumulations, which eventually lead to an environmental contamination. To fully utilize this vegetable and minimize its metal content, the major polysaccharide content is puried and characterized with chemical and computational approaches. Results: The major polysaccharide component from the stalk of Allium schoenoprasum (AssP) was extracted and puried. The gel ltration chromatography puried AssP exhibited a molecular weight of around 1.6 kDa, which was veried by MALDI-ToF-MS. Monosaccharide analysis revealed its composition as rhamnose: arabinose: galactose: glucose: mannose: fructose with a molar ratio of 0.0264:2.46:3.71:3.35:1.00:9.93, respectively. Multiple NMR analysis revealed its backbone as α-Ara/Glu/Gal-(1 → 2)-linked and β-D-Fru-(4 → 5)-linked sugar residues. There was no tertiary structure of this polysaccharide, however, it self-assembled into a homogenous nanoparticle with a diameter of ~600 nm. The solution behavior of this AssP polysaccharide was simulated, and there was no specic binding site on one molecule for another. Association of this polysaccharide was concentration dependent. As the AssP concentration increased, the spherical particles increased their sizes and eventually merged into cylindrical micelles. The diversity of AssP hydrodynamic behavior endowed potential versatility in its applications. Conclusions: AssP was characterized as a polysaccharide with identied monosaccharide compositions and linkage between them. Although there is no tertiary structure in one AssP molecule, self-assembly of AssP molecules could form nanoparticles or micelles depending on its solution concentrations. The unique AssP solution behavior itself a potential biomaterial for nanoparticles preparations. (30 mg/mL). The diversity of AssP solution particle forms suggested a strong versatility in application.


Background
Allium schoenoprasum, also known as Chinese chive, is a widely seen vegetable and seasoning on the dining table worldwide since ancient times. It was shown to exhibit antioxidant activity, with highest activity from its leaf of the cultivated plants and highest activity in its roots from the tissue culture plants [1][2][3]. The diallyl sul de content in the chive oil inhibits a wide spectrum of foodborne pathogens, while the phenolic compounds, steroidal saponins, exhibited antiproliferative effects [4,5]. Extracts from the chive leaves could inhibit Ehrlich carcinoma, and attenuates erythrocyte deformability in sickle cell anemia patients [6,7]. However, it was also found that cadmium and selenium could accumulate in this plant to a toxic level [8][9][10].Thus, study on the extracts from Allium schoenoprasum, which could e ciently eliminate the heavy metal while maintains its biological function, became signi cant.
The stalk of the Chinese chive was shown to contain about 16% polysaccharides [11]. The structure of this polysaccharides puri ed from the Allium schoenoprasum stalk (AssP) was preliminarily studied, and it showed an effect of reducing blood lipids of wistar rats [11]. More and more natural polysaccharides were investigated [12][13][14][15][16] and most of the studies were focusing on their biological functions while few reported their solution behaviors as encapsulating and delivering reagents. Here, we puri ed the polysaccharides from A. schoenoprasum, further studied its structure from primary to tertiary with different conclusions from previously reported, and its novel solution behavior both in the test tube and in silico. Results indicated this polysaccharide, with identi ed structures, could potentially server as a nanoparticle material in multiple circumstances.

Results
Extraction, puri cation, and molecular weight characterization of AssP Hot water was used to extract the polysaccharide from the stalk of the Chinese chive, and the crude polysaccharide was ethanol precipitated for its safety and simplicity for medicine and food products [17]. After pectin removal by CaCl 2 titration, the polysaccharide was dialyzed to remove small molecules. The polysaccharide was further puri ed with gel ltration chromatograph, and each fraction of elution was detected. In our experimental setup, AssP eluted at around 270-330 mL with little impurities (Fig. 1, A).
Multiple methods were employed to measure the molecular weight of AssP, including GPC and MALDI-TOF/TOF mass spectrometry. Firstly, polysaccharide molecular standard of pullulan with a variety of molecular weight (6600, 21700, 113000, 348000, 805000 Da) was loaded to a GPC column and each elution time was obtained.
The log value of their molecular weight was plotted against each corresponding elution time and a standard curve with R 2 of 0.99 was obtained. The puri ed AssP was analyzed with identical procedures, and its elution peak corresponding to a time range of 9.1-9.8 min centered at 9.35 min ( Fig. 1, B). This corresponded to a molecular weight range of 3121-434 Da centered around 1543 Da. To further identify the molecular weight of AssP, MALDI-TOF/TOF was employed to measure its accurate molecular weight. A cluster of peaks with 162 Da differences appeared on the mass spectrum in a range of 527.14-2147.38 Da, which is consistent with the GPC analysis ( Fig. 1, C).

Structural characterization of AssP
To determine the monosaccharide composition of AssP, the polysaccharide was acid hydrolyzed before ion chromatography analysis. Monosaccharide standards of galactosamine, rhamnose, arabinose, galactose, glucose, mannose, fructose were analyzed in parallel to establish the curve for calculation of each mole fraction based on previous research [11]. The chromatogram of each above monosaccharide exhibited peak area that yielded a linear relationship with their concentrations that enabled us to calculate the corresponding mole fraction (Fig. 2, A, Additional File 1: Fig. S1, Table S1). Molar ratios of monosaccharides were calculated as 0.0264:2.46:3.71:3.35:1.00:9.93 of rhamnose: arabinose: galactose: glucose: mannose: fructose in the puri ed AssP (Fig. 2, B). It is worthy to mention that in the previous study [11], the ratio of 5.1:22.6:31.5:1 as arabinose: galactose: glucose: mannose was obtained for AssP. This is probably due to the incompetency of the 1-phenyl-3methyl-5-pyrazolone (PMP)-derivation method they used for monosaccharide analysis, which could not detect the presence of fructose.
As shown in Fig. 3, A, the UV-Vis spectrum of gel ltration puri ed AssP yielded a signi cant absorption at 192 nm while no absorbance at 260 or 280 nm, indicating non-detectable nucleotide or protein in the puri ed AssP [18]. As shown in Fig. 3, B, functional groups of the AssP were characterized with FT-IR. The strong and broad bands at ~3392 cm -1 indicates the hydroxyl (O−H) stretching vibration of the polysaccharide chain. Bands at 2932 cm -1 were ascribed to the asymmetric C−H stretching vibration. Absorption at 1644 cm -1 was assigned to the carbonyl stretching of the polysaccharide. The peak at 1416 cm -1 corresponded to the C=O stretching and C−O bond from the carboxyl group and C−H bending vibrations. The band at 1024 cm -1 corresponded to the C−OH bonds, and the band at 928 cm -1 corresponded to the asymmetric stretching of pyranose ring in the polysaccharide chain.
Integrative analysis of a variety of 1D and 2D NMR spectrums lead to the identi cation of chemical shifts of sugar residues and their possible glycosidic linkage as listed in Table 1. HSQC spectrum was applied to identify the anomeric carbons, COSY and H2BC spectroscopy were applied to identify the 1 H and 13 C chemical shift of adjacent sugar residues, while HMBC and HSQC-TOCSY spectroscopy were applied to further identify the 1 H and 13 C correlation in between sugar residues or within one sugar residue. A total of 4 anomeric carbons could be identi ed, indicating the presence of sugar residues in 4 different chemical environments. Residue a exhibited one less carbon than residues b, c, and d. Thus, residue a was designated as the arabinose residue, and identi cation of other sugar residue b, c, and d were listed in Table 1. Since the chemical shifts between b and c are not distinguishable, they were designated as the same sugar residue. As illustrated in the monosaccharide component analysis and 1 H spectroscopy (Fig. 4, A), sugar residues b and c should be galactose, and d should be glucose, respectively. In the one dimensional 13 C, (Fig. 4, B), there was 3 clusters of quaternary carbon signals at around 103 ppm, and these signals were correlated with residues a, b, c, and d. Combined with all the information, it was designated as the characteristic signals of fructose. With information from all the spectroscopy, fructose e, f, and g were identi ed and listed in Table 1.
The hydrocarbon coupling constants of anomeric carbons in residue a, b, c, and d were all around 170 Hz, indicating the presence of α-glycosidic bonds [19]. In the HMBC spectroscopy (Table 1), correlation signals were found between a1 and f2, b1 and f2, d1 and e2, c1 and e2, suggesting linkage between a(1→2)f, b(1→2)f, d(1→2)e, and c(1→2)e. In the NOESY spectroscopy (data not shown), correlation between b5, c5, d5 and f4, g4 were observed. With all the information, the main backbone of the puri ed AssP could tentatively be α-L-Ara- among which G is Glc or Gal and the ratio between these two is approximately 1:1.

Tertiary structure and solution behavior of AssP
Polysaccharides containing triple-helical structures form a Cong-red-polysaccharide complex, and the maximum absorption wavelength would have red-shifted compared with Congo-red solution. Addition of strong alkali disrupts the hydrogen bonding thus the red-shift of the Congo red-polysaccharide complex is weakened [20]. The tertiary structure of puri ed AssP was examined with Cong red assay as shown in Fig. 5, A. The maximum absorption wavelength of the AssP-Conge red mixture did not yield any dramatic change (491 to 489 nm) as the concentration of NaOH increased, indicating the absence of triple-helical structure of AssP in solution.
The polysaccharide solution of each puri cation step was examined with DLS to reveal the solution behavior of AssP at each stage (Fig. 5, B). Water extraction of the Chinese chive yielded a mixture of multiple-size polysaccharide solution, with particle size ranging from 100 nm to 5000 nm. After precipitation with ethanol, the large proportion of polysaccharide with size ranging from 100 to 500 nm were homogenized to ~300 nm, while the largest particle at ~5000 nm did not change. Removal of pectin further homogenized the polysaccharide solution to a ~150 nm size particle solution as indicated by the narrower peak. Notably, gel ltration which further puri ed AssP rendered this polysaccharide to form a more homogenous, but larger size (with average size of ~600 nm) particle in solution. This was not mentioned in any studies previously, and it indicated a great potential of AssP being used as a natural encapsulating/delivering reagent in the future.
The AssP molecule in solution exhibited horseshoe-like con guration, and two AssP clamped each other at their grooves (Fig. 6, A). The third polysaccharide molecule ended clamping to the rst one, with no speci c binding site on neither of the AssP molecules. Up to ten AssP molecule were simulated to interact in solution. The fth molecule joined the crowding by interacting with the third and the forth AssP, with no direct interaction with the very rst one. Aggregation of further more AssP molecules exhibited a one-dimensional growth phenomenon where the binding of a later polysaccharide molecule always took place on the same site of the very rst one.
While stacking of these molecules yielded a certain curvature, nucleation at one side of the rst AssP molecule would certainly form a spherical structure.
To visualize the resulting scenario of AssP aggregation, its solution state was simulated with sugar residues represented by coarse-grained bead to calculate their interactions (Additional File 1: Fig. S2, Table S2). This method emphasized on the solution distribution and particle formation (Fig. 6, B). At the very beginning of time (t=1step), the AssP molecules distributed homogenously in the aqueous solution. As time passed (t=500 steps), the polysaccharide molecules aggregated into irregularly sized particles. Large spherical particles began to show up at a time point (t=3000 steps), and the system tended to favor this type of solution behavior where eventually all the AssP molecules are distributed in the uniformly sized spherical particles (t=30000 steps). This is so far consistent with our DLS study (Fig. 5, B). However, it is likely a metastable state. At an approximate of in nite time point (t=50000 steps), the AssP spherical particles began to disassembly into differently sized particles, suggesting an eventually unstable assembly of this polysaccharide nano-particle.
The solution particle was also concentration dependent as the equilibrium state (t=50000 steps) of different concentrations of AssP were simulated (Fig. 6, C). At low concentrations, such as 1 mg/mL, the polysaccharide molecules form small spherical particles. As the concentration increased (10 mg/mL), the small particles approached each other and merged into larger particles. The spherical particles would merge into cylindrical micelle at higher AssP concentrations (20 mg/mL), and the diameter of the cylinder would increase as the concentration further increased (30 mg/mL). The diversity of AssP solution particle forms suggested a strong versatility in application.

Discussion
Hot water extraction of polysaccharides from terrestrial plant has been the most e cient, low cost and environmental-friendly method in a variety of applications [21,22]. One of the main purpose of this study is to remove potential heavy metal accumulation in this vegetable, thus careful use of edible chemical reagents were paid special attention through the puri cation steps. Size exclusion chromatography identi ed a single peak after extraction steps as of water extraction, evaporation/concentration, ethanol precipitation, pH adjustment, calcium precipitation and dialysis. This comparatively simple set of puri cation steps suggests potential feasibility in industrial puri cation of AssP.
The molecular weight, monosaccharide composition, and glycosidic linkages between sugar residues were identi ed. Unlike these more abundant polysaccharides from terrestrial biomass (e.g. xylans or cellulose) which has much larger molecular weights, AssP is composed of only ~10 sugar residues [23,24]. This of course contributes to the high solubility of AssP in water. What is more, the monosaccharide composition in AssP is more even while the glycosidic bonds are more diverse. Although there is no tertiary structure in AssP, DLS experiments indicate that AssP eventually self-assembled into homogenous nanoparticles with a diameter of 600 nm in solution. The particle size is not static but constantly changing during the puri cation steps, suggesting a concentration and time dependent solution behavior of AssP. Simulation results indicate that association of AssP molecules in solution exhibit a non-speci c manner where binding of one molecule to the other one is not at a certain sugar residue. Meanwhile, there is a certain pattern of AssP molecule association that the later associating molecules always binds to the complex from the same side and form a curvature. As more and more molecules associated, this curvature eventually end up as a spherical nanoparticle. More interestingly, AssP form spherical nanoparticles at concentration of 10 mg/mL and lower or micelles at concentration of 20 mg/mL and higher. This phenomenon of concentration dependent nanoparticle formation was reported with polyelectrolyte complex dispersions (PECs) formed nanoparticles [25]. However, it is rarely seen in biomass-based nanoparticles.
Biopolymeric nanoparticles are often categorized into polysaccharide nanoparticles and protein nanoparticles, which all nd applications in drug delivery in controlled or sustained or targeted manner [26]. Nanoparticles made from carbohydrates or polysaccharide played a wide role in pharmaceutical industry due to their excellent biocompatibility, which are often used to deliver proteins, peptides and nucleic acids [27][28][29]. Fabrication of biomass-based nanoparticles often involves a variety of technique including desolvation, coacervation, emulsi cation-diffusion and electrohydrodynamic atomization. AssP could self-assembly into either spherical nanoparticles or micelles at different diameters with ne tune of solution concentrations without the above mentioned fabrication process. This is, to the best of our knowledge, not reported previously and suggesting a promising application of AssP as a self-assembly biomaterial of nanoparticles for different scenario.

Conclusions
The major polysaccharide component of a tradition vegetable Allium schoenoprasum was puri ed and characterized. Different from the previous report, our study revealed a monosaccharide composition of this AssP was arabinose: galactose: glucose: fructose of approximately 1:2:2:5. The main backbone of this polysaccharide is α-Ara/Glu/Gal-(1→2)-linked and ß-D-Fru-(4→5)-linked sugar residues, and it forms ~600 nm nanoparticles in water solution. AssP exhibited concentration dependence in forming different solution assemblies, and this behavior diversity could serve in different applications.

Materials and chemicals
Air dried A. schoenoprasum stalk was kindly offered by Jiangsu Natural Food Co., Ltd. (Xinghua, Jiangsu Province, China). Calcium chloride dehydrate, absolute ethanol, sulfuric acid and aqueous ammonia of analytic grade were purchased from Beijing Chemical Works (Beijing, China).

Extraction and puri cation of AssP
Puri cation of chive polysaccharides was following the methods developed by Zhang Y [11] with modi cation.
The grinded, dry chive stalk was ltered through a 20 mesh screen, rinsed with 30-folds (by weight) of water for 2 hours at 85 ℃. The residue was removed with gauze, and ltrated to get clear solution before further concentrated with a rotary evaporator (RE-200, Yarong Biochemical Instrument, Shanghai, China). Addition of 4folds (by volume) of anhydrous ethanol was performed to precipitate the polysaccharides overnight. After removal of the supernatant, the polysaccharide was resuspended in water and ammonia solution was added to adjust the pH to 8.5. Then 10% calcium chloride solution was titrated until white precipitation appears in the solution. The solution was centrifuged to remove the pectin precipitation, and the titration was repeated until no more precipitation was seen. The solution was then loaded into a dialysis bag with molecular cutoff of 500Da, dialyzed against water 6 times at 4 ℃ for a total of 48 hours. The AssP solution was then concentrated, ltered through a 0.22 μM lter and puri ed on a HiPrep TM Sephacryl TM S-400 HR gel ltration chromatography column (GE Healthcare, USA). The ow rate of water was set at 1 mL/min and every 5 mL of elution was collected. The polysaccharide content in each elution was monitored by measuring its absorbance at 200 nm on a DS-11 FX+ spectrophotometer (DeNovix, Wilmington, DE, USA). The polysaccharide concentration was measured with the phenol-sulfuric acid method as previously [20].

Gel permeation chromatography
The puri ed AssP of 1 mg/mL was analyzed with a PL aquagel-OH Mixed-H GPC/SEC column (300×7.5mm, Agilent) on a 1260 In nity II GPC/SEC system coupled with a G7800A refractive index detector. The mobile phase contains pure water which was set at a ow rate of 1.0 mL/min and the column was incubated at 30℃.

Mass spectroscopy
The AssP sample was mixed in equal volume with 2,3-dihydroxybenzoic acid (DHB) and spotted onto a MALDI target pre-spotted with 5-chloro-2-mercaptobenzothiazole (CMBT). After air drying, crystallized spots were detected with a MALDI-TOF/TOF mass spectrometry (AB SCIEX 5800, AB Sciex, CA, USA). Polysaccharide mass maps were acquired in the positive re ectron mode with an accelerating voltage set at 20 kV. Spectra were obtained by accumulating 3500 laser shots for quanti cation with the laser intensity set at 6659. Calibration mixtures were used to calibrate the spectrum.

Monosaccharide composition analysis
AssP was totally hydrolyzed with equal volume of 0.4 mol/L tri uoroactic acid (TFA) at 110 ℃ for 2 hours in a sealed glass tube. The hydrolysate was dried under reduced pressure at a temperature lower than 40 ℃. The same volume of ethanol was added to the dry hydrolysate to dissolve residual TFA and evaporated under the above condition. This was repeated 3 times to scavenge TFA. The dried hydrolysate was dissolved in 1 mL of ultrapure water and stored at 4 ℃ for later use. The monosaccharide standard was made to 1μg/mL solutions.
Both the AssP hydrolysate and the monosaccharide standard were ltered with a 0.22 μm membrane lter before analyzed on a Dionex CarboPac PA10 analytical column (250mm×4mm, Thermo Scienti c) installed on a Dionex ICS-5000 + ion chromatography System coupled with a DC detector (Thermo Scienti c). The mobile phase contains a mixture of 80:20 (v:v) of water and 250 mM/L NaOH with a owrate of 1.0 mL/min. The molar ratio of the monosaccharide component was calculated by area via the standard curve established with pure monosaccharide of known concentrations.

UV-Vis spectroscopy
The UV-Vis spectra of AssP (0.25 mg/mL) was recorded on a Hitachi U-3900H spectrophotometer (Tokyo, Japan) in a quartz cuvette with a light path of 1 cm. The absorbance was recorded between 190 and 800 nm with a step size of 1 nm.

FT-IR spectroscopy
FT-IR spectra of AssP was recorded on a PerkinElmer FT-IR spectrometer (Spectrum 100, Fremont, USA). An aliquot of 5.0 mg freeze-dried AssP was blended with 200 mg potassium bromide into powder and pressed into pellets. The sample was scanned at room temperature under dry air, and 16 scans of the transmittance were recorded between 4000 and 452 cm -1 with a step size of 4 cm -1 . The collected spectra was processed with Spectrum 10 software(PerkinElmer).

Nuclear magnetic resonance
The AssP was dissolved in 3% (w/w) D 2 O and incubated for 3 hours before the solution was freeze-dried. This step was repeated three times. The deuterated AssP was dissolved again in D 2 O of about 100 mg/ml for NMR measurement. All NMR spectra were acquired at 850 ( 1 H) or 212 MHz ( 13 C) on a Bruker NMR instrument 850 MHZ spectrometer using 5mm CPTCI probe at 27 °C. One-dimensional proton, 13 C and two-dimensional COSY, edit-HSQC, HSQC-TOCSY, NOESY, HMBC and H2BC spectra were acquired. 1-D proton spectrum was averaged from 16 scans, and 1-D 13 C spectrum was acquired in 2048 scans. The 2-D COSY spectra were acquired in 4 scans per increment and 256 increments, the edit-HSQC spectra in 8 scans and 256 increments, the HSQC-TOCSY spectra in 16 scans per increment and 256 increments, the NOESY spectra in 8 scans per increment and 256 increments, and the HMBC spectra in 32 scans and 128 increments. The relaxation delays for the NMR experiments were 2s. Mixing time for the TOCSY and NOESY spectra were 80 and 300ms, respectively.

Dynamic light scattering
Particle size of the AssP was determined with a dynamic light scattering (DLS) instrument (Zetasizer Nano-ZS90, Malvern Instruments Ltd., Worcestershire, UK), and the measurement was carried out at 25℃. The particle size was calculated by the Stokes-Einstein equation and reported as a cumulative mean diameter (size, nm) for size distribution [30].

Congo red test
The tertiary conformation of AssP was analyzed by the interaction with Cong red. Brie y, 2 mg polysaccharide sample was mixed with 2 mL distilled water and 2 mL Congo red solution (80 μmol/L). Different volumes of NaOH solution (1 mol/L) were added to the mixture gradually to obtain the nal NaOH concentration of 0-0.5 mol/L. The mixed solution without polysaccharides was scanned as a control. The maximum absorption wavelength at different concentrations of NaOH solution were determined in the range of 200-600 nm after equilibration at room temperature for 10 min [31].

Molecular interaction simulation
Discovery Studio v16 (BIOVIA, USA) was employed to perform the molecular simulation of interactions between AssP molecules with the CDOCKER molecular docking model. The 3D molecular structure of Assp was created in a new molecule window, and the CHARMm force eld was added before the Standard Dynamics Cascade was conducted [32]. The oligosaccharide conformation with the lowest total energy was selected and water molecules were removed to prepare the acceptor molecule le. The whole AssP molecule was selected as the binding site while another Assp molecule added as the ligand to conduct the CDOCKER simulation. After the rst round of simulation, the optimized AssP dimer was conceived as the receptor and a third Assp molecule was introduced as the ligand to perform the next round of CDOCKER simulation. The above steps were repeated until the 10 th Assp molecule was introduced into the complex.
Hydrodynamic behavior simulation The dissipative particle dynamics (DPD), a mesoscopic scale simulation technique, was employed to simulate the hydrodynamic behavior of AssP molecules in aqueous solution. Coarse-Graining models was used to represent the AssP and water molecules. For Assp, the coarse-grained structure is AF(GF) 4 , where bead A, F and G represents arabinoside, fructoside and glucoside/galactoside, respectively. Bead W represent water molecule. The Flory-Huggins parameters (x ij ) between component i and j were determined by approximation with solubility parameter δ i with the following formula [33]: where V is the arithmetic average of the molar volumes of components i an j. δ i and δ j are the solubility parameters of components i and j, respectively. R is the ideal gas constant and T (K) is the temperature of the system.
The solubility parameters of each component was calculated by the Amorphous Cell modules. The 3D structure of each component was constructed and energy minimized with the Minimizer in the Discover module. The AssP system was constructed with the Construction function in the Amorphous Cell module, and energy minimized with the Discover module. The Dynamics function in the Discover module was employed to calculate the system density which was then used to construct the AssP system again. Once the latest polysaccharide system was energy minimized, the nal density was used to calculate the solubility parameter with the Analysis in the Amorphous Cell module. Calculation of the repulsion parameters (a ij ) between component i and j were determined by the Flory-Huggins parameter x ij with the following formula [34]: To perform the DPD Simulation, a cubic simulation box with 30×30×30 R 3 c side lengths was constructed. The Default spring constant between the beads was set to 4.0, and the time step was set 0.05. The PDP simulation was carried out with the DPD module implemented in MS. Puri cation and molecular weight characterization of AssP. A, the extracted AssP was concentrated and puri ed on a S-400 HR gel ltration chromatography column. Each 5 mL of elution was collected, and the content of total carbohydrate in each fraction was monitored by its absorbance at 200 nm. B, gel permeation chromatogram of the polysaccharide standards (not shown) and puri ed AssP (right y axis). The retention time and log value of molecular standards (empty circles) regressed into a linear relationship with a R2 value of 0.99338 (left axis). The retention time of AssP (dotted line vertical to the x-axis) corresponds to a molecule weight of 1543Da (empty square, intercept with the standard curve). C, MALDI-TOF/TOF mass spectrum of puri ed AssP.

Figure 1
Puri cation and molecular weight characterization of AssP. A, the extracted AssP was concentrated and puri ed on a S-400 HR gel ltration chromatography column. Each 5 mL of elution was collected, and the content of total carbohydrate in each fraction was monitored by its absorbance at 200 nm. B, gel permeation chromatogram of the polysaccharide standards (not shown) and puri ed AssP (right y axis). The retention time and log value of molecular standards (empty circles) regressed into a linear relationship with a R2 value of 0.99338 (left axis).
The retention time of AssP (dotted line vertical to the x-axis) corresponds to a molecule weight of 1543Da (empty square, intercept with the standard curve). C, MALDI-TOF/TOF mass spectrum of puri ed AssP.

Figure 5
Tertiary structure characterization of AssP. A, the maximum absorbance wavelength of Congo-red (square) and Congo-red AssP mixture (circle) solution as the NaOH concentration changes. B, the dynamic light scattering intensity of AssP solution as a function of cumulative mean diameter at each puri cation step:1, water extraction, 2, after ethanol precipitation, 3, after pectin removal, 4, after gel ltration puri cation.

Figure 6
Simulation of AssP interactions and its solution behavior. A, the conformation of AssP and simulation of interactions between two, three, four and ten AssP molecules. The rst AssP molecule in a horseshoe shape (red) was bound with a second one (orange). Sequential binding of the third one (yellow) and the forth one (green) are all involved in the direct interaction with the rst AssP molecule. The following fth (cyan), sixth (blue), seventh Simulation of AssP interactions and its solution behavior. A, the conformation of AssP and simulation of interactions between two, three, four and ten AssP molecules. The rst AssP molecule in a horseshoe shape (red) was bound with a second one (orange). Sequential binding of the third one (yellow) and the forth one (green) are all involved in the direct interaction with the rst AssP molecule. The following fth (cyan), sixth (blue), seventh