Purification, Characterization and Bioactivities of Polysaccharides from the Stalk of Abelmoschus manihot (L.) Medic

Abstract

Two new types of hydro soluble polysaccharides, denoted AMP-1 and AMP-2 were first extracted from the stalk of Abelmoschus manihot (L.) Medic by DEAE Sephadex A-52 chromatography and Sephadex G-100 chromatography in sequence. The structure characterization and monosaccharide composition were analyzed by SEM, DSC, FT-IR and GC. The polysaccharide fractions had good thermal stability, while possessing significant variance in surface features. Meanwhile, it was found that the two polysaccharides contained different monosaccharide compositions with diverse molar ratios. The antioxidant assays manifested that AMP-1 and AMP-2 showed 1, 1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radicals and superoxide anion radicals scavenging activities. In addition, the polysaccharides possessed the ability to bind to sodium cholate, sodium deoxycholate and sodium taurocholate. These results suggested that AMP-1 and AMP-2 have broad prospects in the development of value enhancement products with hypolipidemic and free radical inhibitor activity.

Introduction

To strengthen the exploitation and utilization of plant resources in environmental protection would create huge market potential and environmental and economic benefit (Barros et al., 2019). Significant interest has been paid by consumers in recent times towards polysaccharides as functional ingredients in diets due to the various beneficial health effects. Polysaccharides from plants, fungi, yeasts and algae possess the extensive beneficial properties for promoting health, such as being antioxidants, hypolipidemic, anti-cancerous and provide immunomodulatory activities (Li et al., 2016; Yu et al., 2019). A great deal of literature has demonstrated that the bioactivity of polysaccharides was intimately associated with the chemical component and structure, for instance, the monosaccharide component, chain conformation, glycosidic bonds, etc. (Nie et al., 2019)

Abelmoschus manihot (L.) Medic (A. manihot), belonging to the family of Malvaceae, which was one of the most normally used Chinese medicines, has made a great difference in treating chronic disease (Pan et al., 2018). A. manihot has a large content of nutritional components, like flavonoids (Yan et al., 2015), amino acids (Du et al., 2015), trace elements (Rubiang-Yalambing et al., 2016), dietary fiber and polysaccharides (Zheng et al., 2016). Previous studies of A. manihot have merely focused on the flowers or fruits. As far as the author knows, there has been no detailed studies to date regarding the purification, characterization and bioactivities of polysaccharides from the stalk of A. manihot.

Thus, for the purpose of exploiting and utilizing A. Manihot polysaccharides in the field of functional foods and pharmaceutical further, crude polysaccharides were distilled from the stalk of A. manihot (AMP) and fractioned purified by a DEAE Sephadex A-52 cellulose anion exchange column chromatography and Sephadex G-100 size-exclusion column chromatography in sequence (designated AMP-1 and AMP-2) subsequently. The chemical component, preliminary structure and bioactivities of the two polysaccharides were comparatively analyzed. The information derived from the literature could underlie a scientific ground to develop the functional foods basing on the A. manihot polysaccharides.

Materials and Methods

Plant materials and reagents    Seeds of A. manihot were obtained from Tianjin Kehai Zhongda Technology Co. Ltd and were cultivated in Tianjin University of Science and Technology. The stalk of A. manihot was harvested and cut to 5–10 cm after washing with deionized water. The material was dried at 35 °C, smashed and screened through mesh of 60 meshes, and then stored at 4 °C until further analysis.

Standard monosaccharides (xylose, rhamnose, galactose, arabinose, mannose, fructose, glucose), trifluoroacetic acid (TFA), sulfuric acid (H₂SO₄), ascorbic acid (Vc) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). DEAE Sephadex A-52 chromatography and Sephadex G-100 size-exclusion chromatography were purchased from Whatman (Maidstone, UK). Sodium cholate, sodium deoxycholate and sodium taurocholate were obtained from Solarbio (Beijing, China). All other chemicals were analytical grade.

Extraction and separation of polysaccharide fractions    AMP was extracted from the stalk powder of A. manihot. In order to dislodge pigments and lipids, stalk powder was pretreated with petroleum ether for 48 h at room temperature while removing the supernatant. At 80 °C, the residue was refluxed by distilled water in proportion of 1:60 (w/v) for 1.5 h and filtered. The water extract was concentrated under a vacuum after centrifuging at 1 400 g for 10 min. Then, the supernatant was precipitated with 80% (v/v) final concentration of ethanol for 12 h at 4 °C. The precipitation was gathered by centrifuging at 3 150 g for 15 min, and then dispersed in distilled water. The protein was removed through enzymatic methods. The papain in the concentrated solution was 2.0% and the mixture was reacted at 45 °C for 2.5 h. Enzymes were inactivated by boiling for 10 min. Afterwards, the mixture was centrifuged at 900 g for 15 min. The extract was discolored with hydrogen peroxide and the polysaccharides (AMP) were obtained through ethanol precipitation.

The crude AMP was edulcorated by sequential DEAE Sephadex A-52 column chromatography and Sephadex G-100 column chromatography. In brief, AMP (5 mg/mL) was applied to a DEAE Sephadex A-52 column (2.5 × 40 cm) column, and eluted first by 250 mL distilled water with a flow rate of 1.0 mL/min (10 mL/tube). Afterwards, the liquid was eluted with 2 mL/min aqueous NaCl solution which the ionic strength of increased gradually from 0.1, 0.2, 0.3, 0.5 to 0.8 mol/L. Eluents were mixed in accordance with the content of total carbohydrate determined by the phenol-sulfuric acid method. After dialyzing and concentrating, water and 0.2 mol/L NaCl eluates were regarded as two major components, which were nominated Fraction A and Fraction B, severally. Sephadex G-100 (2.5 × 40 cm) columns were used to further purify two related components. Ultimately, two purified fractions (AMP-1 and AMP-2) with high content of polysaccharides were combined, desalted, dialyzed and lyophilized for analysis.

Characterization of polysaccharides

Scanning electron microscopy (SEM) observation    The surface topography of AMP, AMP-1 and AMP-2 were determined by a scanning electron microscope (SEM, SU1510, Hitachi High-Tech Corp., Japan) to obtain the microstructure and surface characteristics of each sample. The samples were taken at a magnification of 400 at an acceleration potential of 5.0 kV.

Differential scanning calorimetry (DSC)    Differential scanning calorimetry (DSC) of the polysaccharide samples was conducted on a DSC-60A instrument (Shimadzu Co., Ltd., Kyoto, Japan). Two milligram samples were placed in the plate and heated from 25 °C to 200 °C at the rate of 5 °C/min under 10 mL/min of liquid nitrogen.

Fourier-transform infrared spectra (FT-IR)    IR spectroscopic changes of the polysaccharides were measured by a Nicolet iS50 spectrometer (Thermo Nicolet, USA). One milligram lyophilized sample were blended with 100 mg of KBr, ground and pressed into flaky for FT-IR analysis with a wavelength range of 4 000 to 400 cm⁻¹.

Analysis of monosaccharide composition    The composition of monosaccharides in AMP, AMP-1 and AMP-2 was detected by gas chromatography (Agilent Technologies, Palo Alto, CA, USA) on the basis of their acetylated derivatives depending on the well-established procedures (Chen et al., 2008). The samples (0.1 µL) were injected into an Agilent 7890A GC system equipped with a DB-17 capillary column (30 m × 0.32 mm × 0.50 mm) and a flame ionization detector (FID). The temperature of the column was set to rise from 100 °C to 280 °C, with a heating rate of 10 °C/min, and was maintained for 12 min. The injection temperature was 280 °C, the detector temperature was 280 °C, and the flow rate of N₂ carrier gas was 1.0 mL/min. As referenced, seven monosaccharides, with different concentrations, (arabinose, glucose, xylose, mannose, ribose, rhamnose, and galactose) were elected as standards.

Bioactivities of polysaccharides

Determination of DPPH Free Radical Scavenging    Six milligram DPPH were dispersed in 100 mL of anhydrous ethanol. The certain concentrations (0.5–10 mg/mL) of AMP, AMP-1 and AMP-2 were prepared by distilled water. After mixing, the samples were stored in the dark for 30 min. Where after, the absorbance at 517 nm was determined using spectrophotometer. The activity to scavenge was calculated according to the equation 1:   

where A₀ was the absorbance of DPPH without sample; A₁ is the absorbance of sample and DPPH; and A₂ was the absorbance of sample without DPPH.

Hydroxyl free radical scavenging activity    The measurement of hydroxyl free radical scavenging activity was on the basis of literature with the slight modifications (Smirnoff and Cumbes, 1989). Hydroxyl free radicals were generated from FeSO₄ and H₂O₂, and their effect on hydroxylating salicylate was detected. The reaction system (2.5 mL) was consisting of 0.15 mL of sodium salicylate (20 mM), 0.35 mL of H₂O₂ (6 mM), 0.5 mL FeSO₄ (1.5 mM), and 1.0 mL polysaccharides with different potency. Ascorbic acid was considered as a positive control. The absorbance of hydroxylated salicylate complexes was determined at 510 nm after incubating at 37 °C for 1 h. The percentage of scavenging effect was calculated as per equation 2:   

where A₀ was the absorbance of solvent control, A₁ was the absorbance of the sample or ascorbic acid, and A₂ was the absorbance of the reagent blank without sodium salicylate.

Superoxide anion radical scavenging activity    Sample solution (1.0 mL) was mixed with 2.25 mL of 50 mM Tris–HCl buffer (pH 8.2) and incubated at 25 °C for 20 min. Afterwards, 2 mL of pyrogallol (25 mM) which was pre-heated at 25 °C, was blended with the reaction system thoroughly. The absorbance of the mixture was performed at 325 nm. The auto-oxidation ability of pyrogallol was reckoned based on the equation 3:   

where A₀ was the absorbance of control (without sample) and A₁ was the absorbance of the sample.

Bile Acid Binding Capacity

•Bile acid binding process

The ability of polysaccharides to bind bile acids was slightly modified by reference to published methods (Gao et al., 2015). One milliliter sample solution was added to 7.5 mL sulfuric acid solution (60% volume fraction) and bathed at 70 °C for 20 min. The samples then were immediately placed into the ice-bath for 5 min. Absorbance was determined at 387 nm using an ultraviolet spectrophotometer.

•Preparation of cholate standard curve

A sodium cholate hydrate having a concentration of 1 mmol/mL was prepared as a standard solution in a 100 mL volumetric flask; 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution was taken in tubes and filled the volume to 1 mL with physiological saline, followed by additional 7.5 mL of sulfuric acid solution (volume fraction 60%). The mixtures were reacted in the water-bath at 70 °C for 20 min. Afterwards, the mixtures were bathed in the ice for 5 min immediately. The absorbance was determined at 387 nm using the UV spectrophotometer. The standard curve for sodium deoxycholate and sodium taurocholate were plotted by the same method.

•Bile acid binding test of polysaccharides in vitro

AMP, AMP-1 and AMP-2 solutions were prepared at a concentration of 3 mg/mL using NaCl. One milliliter of artificial gastric was added to the sample for 1 h at 37 °C. After simulating the process of gastric digestion, the pH was regulated to 6.8 with 0.1 N NaOH. At the same time, 4 mL of 10 mg/mL pancreatic enzymes solution were introduced into the solution. The mixtures were shaken and incubated for 1 h at 37 °C in the temperature controlled shaker bath.

After the end of simulated gastrointestinal, 1 mL mixture and 4 mL of 1 mM cholate solution were shaken at 37 °C for 2 h, and then was centrifuged at 900 g for 20 min. The content of the cholate in the supernatant obtained in the previous step was determined and computed basing on the standard curve. All samples were analyzed in triple times.

Statistical analysis    All results were presented as the mean ± standard deviation (SD) obtained from the triple times experiments. The data was analyzed by the way analysis of variance (ANOVA) with SPSS (Version 20), and means of differences among treatment were examined using Tukey Kramer test at p < 0.05.

Results and Discussion

Extraction and purification of polysaccharides    Crude polysaccharide (AMP) which the final yield was approximately 24.3%, was extracted from the stalk of A. manihot in the order of hot water extraction, ethanol precipitation, deproteination, decolorization and freeze-drying. As is shown by Fig. 1, two major fractions (A and B) were exhibited by DEAE Sephadex A-52, which was eluted with 0.2 M NaCl solutions and H2O, separately. The neutral fraction (Fraction A) and salt-eluted fraction (Fraction B) were further isolated by a Sephadex G-100 column eluted with deionized water to obtain AMP-1 and AMP-2, and basing on the amount of crude AMP, it was calculated that their final yield was 6.13% and 9.58% separately. Due to the low content of the product, it was not separated and purified the fractions eluting by 0.1 M, 0.3 M, 0.5 M and 0.8 M NaCl solutions.

Fig. 1.

Stepwise elution curve of crude AMP.

Stepwise elution curve of crude AMP on anion-exchange chromatography column DEAE Sephadex A-52 (A) and elution curve of polysaccharides fractions (AMP-1 and AMP-2) from DEAE-SephadexA-52 on size-exclusion chromatography column of Sephadex G-100 (B and C).

SEM analysis    SEM is considered as a powerful tool to characterize the conformation of individual macro-molecules, which can intuitively aid in observing their physical properties. The surface topography of AMP, AMP-1 and AMP-2 at magnifications of ×400 were shown in Fig. 2 (A–C). The topography of crude polysaccharides and purified polysaccharides varies greatly in size and shape. The morphology of AMP was massive and had obvious bulges at the boundaries (Fig. 2A). The surface of AMP-1 was characterized by fragmentation and coarse structure (Fig. 2B). AMP-2 was composed of big and dense flakiness which was significantly different to other samples (Fig.2C). The results were similar to the study by Yu et al., (2017) to a certain extent. Therefore, the distinct surface topography of the polysaccharides could be attributed to the constitution of complexes and specific extraction methods.

Fig. 2.

Images of AMP (A), AMP-1 (B) and AMP-2 (C) observed at magnifications of ×400.

DSC analysis    Differential scanning calorimetry (DSC) was utilized to investigate the exothermic or endothermic changes with the increase of temperature. The potential for the use of polysaccharides in applications is mostly dependent on their thermal properties such as heat flow (Pelgrom et al., 2012). The curves of polysaccharides were given in Fig. 3. The first endothermal valleys were approximately 74 °C, 110 °C and 80 °C, respectively, corresponding to different polysaccharides (AMP, AMP-1 and AMP-2), this was basically relevant to the movement of bound water (Zhang et al., 2016). In addition, no obvious endothermal and exothermal changes were observed at the range from 110 °C to 200 °C. The results imply that AMP, AMP-1 and AMP-2 were structurally stable and have good thermal stability; this can be considered advantageous in retaining the stability of functional properties during production and storage.

Fig. 3.

DSC curves of AMP, AMP-1 and AMP-2

FT-IR analysis    FT-IR spectroscopy is considered to be an effective tool to evaluate the preliminary qualitative of polysaccharides. AMP-1 and AMP-2 showed distinguishable infrared spectra with characteristic signals. As given in Fig. 4, the range of FT-IR spectra was 4 000–400 cm⁻¹. It was found that the absorption bands of AMP-1 and AMP-2 showed a trend from medium to strong, which had a strong O-H stretch at 3 000–3 500 cm⁻¹, a C-H stretch at 2 800–3 000 cm⁻¹ and 1 200–1 400 cm⁻¹. All of these peaks are the typical absorption of polysaccharides. The bands at 1 431 cm⁻¹ and 1 416 cm⁻¹ for AMP-1 and AMP-2 indicated the presence of a bending vibration peak of C-H. For AMP-1, an absorption at 1 148 cm⁻¹, generally indicative of the furan type sugar ring in the neutral sugar, was in complete agreement with the monosaccharide component. The bands at 1 637 cm⁻¹ were due to COO-bending. For AMP-2, there was an absorption at 1 722 cm⁻¹ due to COO-, indicating the existence of uronic acid in the AMP-2 structure. The characteristic absorptions at 1 258 cm⁻¹ and 1 148 cm⁻¹ could be aligned to the presence of the S=O stretch, which indicated that the sulfate ion was contained in salt-eluted fractions. Additionally, bands at 917 cm⁻¹ and 822 cm⁻¹ were regarded as characteristic absorption for β-configuration and α-configuration, respectively (Luo et al., 2010). It has been recognized that β-glycosidic linkage is an essential structure in immune stimulation and anti-tumor activity (Kozarski et al., 2012).

Fig. 4.

FT-IR spectra of AMP-1(A) and AMP-2(B) in the range of 4000–400 cm⁻¹.

Monosaccharide component analysis    To investigate the types of monosaccharide ulterior, AMP, AMP-1 and AMP-2 were hydrolyzed and analyzed via GC. Fig. 5 revealed the monosaccharide composition of the three polysaccharides. The molar ratios of the three polysaccharides were tabulated in Table 1. It manifested that they were hetero polysaccharides which were consisting of arabinose, xylose, mannose, rhamnose, glucose and galactose in different ratios. A significant difference was that the AMP contained more types of monosaccharide than AMP-1 and AMP-2. The results indicated that the AMP mainly included rhamnose, arabinose, xylose, mannose, glucose and galactose, with a molar percentage ratio of 0.28:0.63:4.87:0.92:5.21:1.87. AMP-1 contained arabinose, mannose, glucose and galactose with a molar percentage ratio of 1.28:0.48:7.20:1.42. AMP-2 was composed of rhamnose, arabinose, mannose, glucose and galactose in the molar ratio of 1.57:0.33:0.24:0.43:1.56. The monosaccharide compositions of the polysaccharides in the work were unlike to those reported by Zheng et al., (2016) who found that A. manihot polysaccharides was composed of fucose, mannose, glucose and galactose. Such differences might be due to differences in source materials, isolation protocols and purification procedures.

Fig. 5.

Monosaccharide composition of AMP (A), AMP-1(B) and AMP-2(C).

Table 1. Monosaccharide composition of AMP-1, AMP-2, and AMP (mol %).

Polysaccharides	Rhamnose	Arabinose	Xylose	MannoseG	lucose	Galactose
AMP	0.28	0.63	4.87	0.92	5.21	1.87
AMP-1	-	1.28	-	0.48	7.2	1.42
AMP-2	1.57	0.33	-	0.24	0.43	1.56

Bioactivities of polysaccharides

DPPH radical scavenging activity    The DPPH radical, which was extensively applied to assess the free radical scavenging activities of natural antioxidants (Kou et al., 2013), is a steady free radical with a typical absorption at 517 nm and purple color. In the current study, the effects of AMP, AMP-1 and AMP-2 fractions on scavenging DPPH free radicals were investigated and the results were displayed in Fig. 6A. With regard to all the samples, the ability to scavenge DPPH performed a concentration-dependent manner at the range of 0.5 to 10.0 mg/mL. Notably, the highest percent inhibition of AMP-2 observed in the experiment was 84.3% at 10.0 mg/mL. Compared with crude AMP-1, the purified fractions of AMP-2 exhibited a similar anti-oxidative capacity. Moreover, it was apparent that AMP showed the lower ability to scavenge DPPH radicals than that of AMP-1 and AMP-2 after analysis in groups from the concentration of 2.0 mg/mL (p < 0.05, Tukey-Kramer test). The high DPPH radical scavenging activity of purified polysaccharides might provide deeper insights into the clinical efficacy of A. manihot in inflammatory disturbance and tissue repair. The results were analogy to those of the polysaccharides which were separated from a Chinese medical herb (Ramulus mori) (Zhang et al., 2008).

Fig. 6.

In vitro antioxidant activities of the polysaccharides (the data was conducted within group). Scavenging effects of AMP-1, AMP-2, and AMP at different concentrations towards DPPH radicals (A); scavenging activity of AMP-1, AMP-2, and AMP towards hydroxyl radicals (B); scavenging activity of AMP-1, AMP-2, and AMP towards superoxide anion radicals (C).

Hydroxyl radical scavenging activity    It was well-known that hydroxyl radicals are the most injurious free radicals among the reactive oxygen species (ROS), which can destroy various substance in the body, for instance, nucleic acid, lipids carbohydrates, and amino acids (Giese, et al., 2015; Shen, et al., 2016). Therefore, it is important for human to enhance the antioxidant defenses in cells and food systems to avoid the effects of hydroxyl radicals. With concentration increasing from 0.5 to 10 mg/mL, the scavenging activity of AMP increased slightly, whereas the activity of AMP-1 and AMP-2 exhibited a markable concentration-dependent manner (Fig. 6B). The high capacity to scavenge hydroxyl radical could be owing to the monosaccharide composition and chemical bonds of polysaccharides.

Scavenging activity on superoxide anion radical    The superoxide anion is a kind of weak free radical, which can be produced by mitochondrial electron transport systems. However, it can indirectly initiate lipid peroxidation, resulting in a stronger ROS, such as hydroxyl radicals (Chen and Huang, 2019). Consequently, the activity of CVPS to scavenge the in-vitro superoxide anion free radical was also studied. As vividly shown in Fig. 6C, whether the DPPH radical or hydroxyl radical scavenging activity, the scavenging activity of superoxide anion radicals was enhanced with the concentration of polysaccharide increasing. For AMP1 and AMP2, the trend was more pronounced when the solution was at a higher concentration (2.0–10.0 mg/mL). Compared with AMP, the activity of AMP-1 and AMP-2 was much higher from the concentration of 1.0 mg/mL (p < 0.05, Tukey-Kramer test). In the words, these results demonstrated that both AMP-1 and AMP-2 had antioxidant activities in a concentration-dependent manner to a certain extent, which had potential value in developing new antioxidants. It was recognized that antioxidant activity of the polysaccharides might be relevant to physiochemical characteristics including monosaccharide composition, molecular size, conformation, and the content of protein and sulfate. According to the previous literature, the high content of Ara, Gal and sulfate were beneficial for the antioxidant activity of polysaccharides (Mukherjee et al., 2019; Wang et al., 2017). Interestingly, there were no report about the correlation of antioxidant activity and monosaccharides in polysaccharides in these studies, although this phenomenon has been reported by the other researchers. These results suggested that the antioxidant activity of AMP-1 and AMP-2 was an effect of a combination of several factors, and the differences in antioxidant activities between AMP-1 and AMP-2 could not be attributed to a single specific factor.

Bile acid binding capacity For many years, Chinese people have been taking A. manihot as a tea as a means of anti-hyperlipidemia, with the perspective that polysaccharides may play a major role in reducing blood fat. In this study, sodium cholate, sodium deoxycholate and sodium taurocholate were used to assess the binding capacity of bile acids (Shi et al., 2016).

The ability of polymers to combine with bile acids may improve the elimination of bile acids and assist the conversion of cholesterol to bile acids in the liver. Furthermore, it could decrease the levels of total plasma and LDL cholesterol, thereby declining the risk of cardiovascular diseases (Niu et al., 2013; Wu et al., 2019). A possible mechanism of action of polysaccharides is as follows: polysaccharides can combine with bile acids to enhance their intestinal excretion function, preventing the reabsorption of bile acids in the small intestine back into the liver, and stimulate the conversion of cholesterol to bile acids in the liver to maintain the balance of the bile pool. Cholesterol in the blood enters the liver through related receptors, which reduces the levels of total cholesterol and low-density lipoprotein cholesterol in the blood, thereby reducing the risk of cardiovascular disease (Gunness and Gidley, 2010). The capacities to bind to bile acid of polysaccharides were shown in Table 2. The result revealed that AMP, AMP-1 and AMP-2 could bind to sodium cholate, sodium deoxycholate and sodium taurocholate but differences existed in terms of quantity. The binding capacities of AMP with sodium cholate and sodium deoxycholate were higher than that of AMP-1 and AMP-2. This may be due to the presence of protein or glycoprotein polymer in AMP which has an adsorption to bile acid. The purification process destroys the proteins and glycoprotein structure, resulting in the lower binding capacities. In addition, due to the complex composition of AMP, there may be a synergistic effect between the various monosaccharides. After purification by the chromatography, the types of polysaccharides were reduced. To note, AMP had a better binding capacity on sodium taurocholate than two other bile acids. The side chains of sodium taurocholate were sulfated groups which were more readily available to be ionized and more likely to be exposed in the active position. The results of the study confirmed that crude polysaccharides in the stalk of A. manihot could be explored as a potential novel antilipemic material, which might have great application value in pharmaceutical and food industries.

Table 2. The capacity of AMP-1, AMP-2, and AMP to bind to bile acid (the data was conducted within group).

Bile acid binding capacity	AMP	AMP-1	AMP-2	Cholestyramine
Sodium cholate (µmol/L)	0.49 ± 0.002a	0.30 ± 0.001b	0.24 ± 0.003b	1.91 ± 0.008c
Bile acid binding relative to cholestyramine %	25.72 ± 0.006a	15.46 ± 0.034ab	12.41 ± 0.047b	100 ± 0.020c
Sodium deoxycholate (µmol/L)	0.49 ± 0.001a	0.35 ± 0.003b	0.30 ± 0.001b	2.06 ± 0.014c
Bile acid binding relative to cholestyramine %	23.87 ± 0.019a	17.32 ± 0.038ab	14.33 ± 0.066b	100 ± 0.037c
Sodium taurocholate (µmol/L)	0.87 ± 0.004a	0.57 ± 0.003a	0.89 ± 0.002a	2.44 ± 0.022b
Bile acid binding relative to cholestyramine %	35.62 ± 0.029a	23.34 ± 0.035b	36.51 ± 0.057a	100 ± 0.046c

Note: Binding capacity of polysaccharides for bile acid was denoted with per mL solution. Binding capacity of cholestyramine for bile acid was denoted with 100 mg dry weight of cholestyramine.

Conclusion

In conclusion, two novel kinds of hydro soluble polysaccharides (AMP-1 and AMP-2) were isolated from the stalk of A. manihot for the first time. The preliminary structural characteristic was characterized by physicochemical properties, SEM, DSC, FT-IR and monosaccharide composition. The two polysaccharide fractions had good thermal stability, but possessed significant variance in their surface features. Furthermore, it was found that three polysaccharides contain different monosaccharide compositions with different mole ratios. All polysaccharides displayed good ability to scavenge DPPH, hydroxyl radicals and superoxide anion radicals. The antioxidant activities of AMP were consistently lower than AMP-1 and AMP-2. In addition, the two polysaccharides possessed the binding capacity for sodium deoxycholate, sodium cholate and sodium taurocholate. The presence and great activity of purified polysaccharides from the stalk of A. manihot may potentially provide an innovative approach for applications in health-related fields like functional foods and medicine.

Acknowledgments    This work was supported by the National Natural Science Foundation of China (31701526), the Student Lab Innovations Fund project from Tianjin University of Science and Technology (1714A302), Novel Coronavirus Prevention and Control Project from Tianjin University of science and technology (2020STCV0008) and Undergraduate Innovation Project from Tianjin University of science and technology (201910057195).

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