Structural characterization and hypolipidemic activities of purified stigma maydis polysaccharides

Abstract This study aimed to investigate structural features and antihyperlipidemic effects of the stigma maydis polysaccharide, termed SMP‐1. This polysaccharide was composed of D‐mannose, L‐rhamnose, D‐glucose, D‐galactose, L‐arabinose, D‐xylose, and D‐galacturonic acid, with a molar ratio of 1.00:0.21:1.41:1.44:0.70:0.44:0.56. The SMP‐1 was mainly bonded by (1 → 6) and (1 → 3) linkages, with various monosaccharides being evenly distributed in the main and side chains. Moreover, SMP‐1 had neither triple‐helical structure nor molecular aggregation. Importantly, the SMP‐1 could effectively bind the bile acids in vitro and significantly lower the total cholesterol, triglyceride, low‐density lipoprotein cholesterol levels, and moderately increase the high‐density lipoprotein cholesterol level in poloxamer 407‐induced hyperlipidemic mice. Moreover, pretreatment with SMP‐1 (≥300 mg/kg) could remarkably reduce fat accumulation and restore hepatocyte morphology in the liver of hyperlipidemic mice. Altogether, these findings indicated that SMP‐1 could be developed as a safe and effective food supplement for preventing and treating hyperlipidemic disorders.

In this study, a water-soluble stigma maydis polysaccharide (SMP-1) was isolated by the hot water extracting-alcohol precipitating method, followed by purification with D315 anion exchange resin and Sephadex G-100 resin successively. The purified SMP-1 was then characterized by a series of analyses including high-performance gel permeation chromatography (HPGPC), Fourier transform infrared (FT-IR) spectrophotometry, precolumn derivatization highperformance liquid chromatography, periodate oxidation analysis, partial acid hydrolysis, atomic force microscope (AFM), and Congo red staining. Afterward, the antihyperlipidemia activity of SMP-1 was evaluated via testing bile acid-binding capacity in vitro and serum lipid-lowering effects in vivo. Collectively, this study would provide a solid evidence for the antihyperlipidemic effects of the stigma maydis polysaccharide, thus offering a safe and effective functional food supplement for the treatment of hyperlipidemia and other associated chronic diseases.

| Materials and chemicals
Stigma maydis was purchased from the local market (Zhenjiang, China). D315 macroporous resin and Sephadex G-100 were purchased from Jiangsu Synthgene Biotechnology Co., Ltd.
(Nanjing, China). Taurocholic acid sodium salt (purity >95%), glycodeoxycholic acid sodium salt (purity >95%), and other standard substances (purity >99%) were purchased from Aladdin Industrial Corporation. Test kits used for the determination of TC, HDL-c, LDL-c, and TG were purchased from Nanjing JianCheng Bioengineering Institute. All other chemicals and reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. and they were of analytical grade.

| Experimental animals
Male ICR mice (18-22 g) were obtained from the Laboratory Animal Center of Jiangsu University, with animal quality certificate number: SCXK (Su) 2013-0011. The animals were housed in cages for one week to adapt to the environmental conditions with free access to laboratory food and water before experiment. The experimental protocol was approved by the Institution of Animal Ethical Committee, and the study was carried out in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals.

| Extraction and purification of stigma maydis polysaccharide
The stigma maydis was extracted with hot water according to previous studies (Deng et al., 2012;Wang, Tian, et al., 2017). Then, the trichloroacetic acid solution (20%, 1:1 v/v) was added to the extracted solution to remove free proteins. The deproteinized solution was dialyzed in a dialysis bag (cut-off molecular weight, 3,500 Da) against running water for 72 hr. After lyophilization, the crude stigma maydis polysaccharide was obtained.
The crude stigma maydis polysaccharide (5 g) was dissolved in distilled water and further purified using D315 macroporous resin (D 40 cm × 100 cm), with gradient eluting by 0, 0.1, 0.3, and 0.5 M of NaCl solution (500 ml each), respectively. The fraction eluted with distilled water was collected for another round of purification with the Sephadex G-100 resin (D 24 cm × 80 cm). The distilled water was used as the eluent to produce a homogeneous fraction of the stigma maydis polysaccharide, SMP-1. After that, the SMP-1 fraction was collected, concentrated, dialyzed, and lyophilized. The resulting SMP-1 was kept in a sealed container with a drying agent for the subsequent experiments.

| Chemical analysis
The carbohydrate content of SMP-1 was determined with phenol-sulfuric acid method using D-glucose as the standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1951). The carbazole-sulfuric acid spectrophotometric method was used to determine the uronic acid contents, with D-Galacturonic acid as the standard. The Bradford method was also used to investigate the protein content of SMP-1 using bovine serum albumin as the standard (Shi et al., 2015).

| Molecular weight determination
The molecular weight (Mw) of SMP-1 was measured using the HPGPC method (Wang et al., 2011) equipped with a high-performance liquid chromatography instrument (Agilent 1260), a TSK-guard column PWH (7.5 × 75 mm), a TSK-gel G4000PW The molecular weight of SMP-1 was obtained through the regression equation of the molecular weights on a ln scale versus the retention time.

| UV and FT-IR analyses
Powdered SMP-1 (1 mg) was dissolved in distilled water. The UV spectrum was recorded within the wavelength ranging from 200 to 800 nm on a UV-1700.
For FT-IR analysis, completely dried SMP-1 (1 mg) was mixed with suitable amount of KBr, ground and pressed into a transparent pellet. The FT-IR spectrum of the sample was then recorded on a Bruker TENSOR 27 spectrometer (Bruker Optik).

| Periodate oxidation
The periodate oxidation that is usually performed to determine the glycosidic linkage position of polysaccharides was conducted according to a previous study . In brief, the SMP-1 (10 mg) was dissolved in distilled water (<30 ml), to which the potassium periodate (KIO 4 ) solution (15 mM) was added to a total volume of 30 ml. The mixture reacted at the room temperature (~26°C) under continuous stirring in a dark room for 8 days. An aliquot (0.1 ml) of the reaction solution was collected every 12 hr for the UV measurement at a wavelength of 223 nm, until the optical density value became stable. An equal volume (0.1 ml) of distilled water was added to the reaction system after each sample collection. Glycol (2 ml) was added to the mixture to terminate the reaction. The periodate consumption was determined using the calibration curve (y = 0.0404x + 0.0191, R 2 = 0.9981, where x represents the concentration of potassium periodate solution and y is the absorption value at 223 nm) of the different standard KIO 4 solutions (0, 8, 16, 24, 32, 48, 64 mM). Then, 10 ml of the resultant solution was mixed with phenolphthalein indicator to titrate the amount of formic acid against NaOH solution (0.01 M).

| Partial acid hydrolysis
The partial acid hydrolysis was carried out using the method described in an earlier report (Song et al., 2017). The TFA has been previously reported to partially hydrolyze the polysaccharides and remove branch chains (Wang et al., 2015). Briefly, the SMP-1 (25 mg) was hydrolyzed with 5 ml of TFA (0.05 M) at 100°C for 10 hr. Excess acid was removed using a vacuum evaporator. Then, the residues were dialyzed (cut-off molecular weight, 3,500 Da) against distilled water for 72 hr. The nondialyzable fraction (inside of the bag, SMP-1 a ) and dialysate (outside of the bag, SMP-1 b ) were collected, concentrated, and freeze-dried, respectively. The resulting fractions were then processed for monosaccharide composition analysis according to the procedures described in the Section 2.4.4.

| Congo red assay
Congo red method is usually performed to determine the triple-helix structure of the polysaccharides (Chen, Zhang, Gao, Huang, & Wu, 2017). In brief, 2.0 ml of the SMP-1 solution (1 mg/ml) was mixed thoroughly with 2.0 ml of Congo red reagent (100 μM). Then, the NaOH solution (2 M) was gradually added to the mixture to obtain a series of NaOH concentrations (0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 M). At each NaOH concentration, the maximum absorption of the mixture was measured with ultraviolet-visible scanning (300-700 nm), using the distilled water as control.

| Atomic force microscopy
Atomic force microscopy is employed to visualize the topographies of polysaccharides . Briefly, SMP-1 was dissolved in ultrapure water and filtered through 0.45 μm membrane filter. The filtrate (5 μg/ml) was placed on a freshly cleaved mica disk and dried in ambient air for 4-8 hr at room temperature. The AFM images were obtained using a MFP-3D (Asylum Research) microscope in tapping mode with commercial RTESP antimony doped Si tips (Bruker).
The images were analyzed using NanoScope analysis software.

| Evaluation of bile acid-binding capacity in vitro
The potential bile acid-binding capacity of SMP-1 was measured according to previously described procedure with slight modification (Camire & Dougherty, 2003). Two main in vitro existing forms of bile acid (taurocholic acid sodium salt and glycodeoxycholic acid sodium salt) were used to detect the bile acid-binding property of SMP-1. Three groups were set: cholestyramine positive control (A), polysaccharide control group (B), and polysaccharide experimental group (C). The reaction time used for the quantity-effect relation test with different concentrations of SMP-1 (5, 10, 15, and 20 mg/ ml) was 30 min. The concentration of SMP-1 used for time-response relation test with different time intervals (15, 30, 60, and 120 min) was 5 mg/ml. Afterward, the conjugation rate was determined as follows: Conjucation rate(%) = (C − B)∕A × 100%.

| Treatment of poloxamer 407 induced hyperlipidemia
Poloxamer 407 (P407)-induced acute hyperlipidemic mice have been proven to be an effective model for evaluating potential antihyperlipidemic compounds (Korolenko et al., 2016). A single intraperitoneal (i.p.) injection of P407 at a dose of 300 mg/kg can readily produce significant hyperlipidemia within approximately 4 days (Omari-Siaw, . were treated with the drug or sample described in each group by oral gavage. One hour later, animals in all groups (except NC) received a single i.p. injection of P-407 dissolved in normal saline at a dose of 300 mg/kg. Twenty-four hours after the P-407 treatment, the mice were fasted for 12 hr with free access to water. Afterward, the blood samples were collected from the orbital vein into heparin-coated tubes and centrifuged at 2,600 g for 10 min at 4°C to obtain serum samples.

| Determination of serum lipid profiles
The serum lipid profiles including TC, TG, LDL-c, and HDL-c in serum were determined with commercial test kits using an Epoch Microplate Spectrophotometer (BioTek Instruments).

| Histopathological analysis
After the blood samples were collected, the mice were sacrificed via cervical dislocation. The liver was removed from each mouse, washed twice with phosphate-buffered saline (PBS, pH = 7.4), and fixed in 5% formaldehyde overnight at 4°C. The liver samples were then washed thrice with double distilled water and embedded in paraffin wax. Afterward, the samples were sectioned into slices (5-μm thickness) using a rotary microtome (leica-RM2235). The transverse sections were collected on glass slides, de-paraffinized, and stained with hematoxylin and eosin (H&E; Beyotime Institute of Biology) and observed with an electron microscope (Nikon Eclipse 90i microscope) at 200× magnification.

| Statistical analysis
All values were expressed as means ± standard deviation (SD) of three replicates. Statistical analyses were performed using SPSS 19.0 software (SPSS Inc.). The differences between the various groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p-value of less than 0.05 or 0.01 was, respectively, considered as either statistically significant or very significant.

| Chemical components and molecular weight analysis
The carbohydrate content of SMP-1 was 79.71 ± 2.07% according to the results of phenol-sulfuric acid method. The contents of protein and uronic acid in SMP-1 were 0.92 ± 0.09% and 14.56 ± 0.67%, respectively.
The chromatogram recorded by HPGPC showed that SMP-1 gave rise to a single symmetrical peak (Figure 1a), indicating that SMP-1 was homogeneous. According to the calibration curve of lnMw = −0.8035t + 25.441 (R 2 = 0.995, where Mw was the molecular weight, and t was the retention time), the average Mw of SMP-1 was about 12.93 kDa.

| UV and FT-IR analysis
The UV analysis demonstrated that there was almost no absorption at 260 or 280 nm (Figure 1b

| Monosaccharide composition analysis
The results of precolumn derivatization HPLC showed that the monosaccharide composition of SMP-1 hydrolysate contained Man, Rha, Glc, Gal, Ara, and Xyl, as well as GalA, with a relative molar ratio of 1.00:0.21:1.41:1.44:0.70:0.44:0.56 (Figure 1d). The monosaccharide composition of CPS was also consistent with the previous study (Zou et al., 2017) but with different molar ratios, which could be due to the differences during extraction and analysis.

| Periodate oxidation
The position of glycosidic linkage of SMP-1 was evaluated using the consumption of KIO 4 and NaOH. The generated formic acid was titrated with NaOH (0.01 M). As a result, the sugar residue (1 M) of SMP-1 consumed 0.81 M of KIO 4 , and 0.44 M of formic acid was produced per molar sugar residue. According to the principle of periodate oxidation (Q. Peng, Li, Xue, & Liu, 2014), these results indicate that the SMP-1 residues are mainly bonded by (1 → 6) and (1 → 3) linkages with molar ratio of 1:1.47, and no (1 → 2) or (1 → 4) linkages are found in SMP-1.

| Partial acid hydrolysis
The monosaccharide compositions of the nondialyzable (SMP-1 a ) and the dialyzable (SMP-1 b ) fractions of SMP-1 were separately analyzed. The results showed that both fractions of SMP-1 were of the same set of monosaccharides (Man, Rha, Glc, Gal, Ara, Xyl, and GalA) with similar molar ratios (Table 1), suggesting that seven different types of monosaccharides are evenly distributed in the main and side chains of the polysaccharide (Song et al., 2017).

| Congo red test
As previously reported, the polysaccharides with a helical conformation could give rise to a bathochromic shift of the maximum absorption λ of the Congo red-polysaccharide complex, in comparison with pure Congo red (Xu et al., 2018). The absorptions of Congo red-SMP-1 complex and pure Congo red were determined at various NaOH concentrations. As shown in Figure 2a, the maximum absorption λ of the Congo red-SMP-1 complex was similar to that of the pure Congo red, which decreased in tandem with the increase of NaOH concentrations. This finding suggests that the conformation of SMP-1 in solution is not triple-helical.

| Atomic force microscopy
Atomic force microscopy is a powerful tool used to detect the surface topology and local mechanical properties of biological molecules (Li et al., 1999). Figure 2b shows the 2D and 3D images of SMP-1. The images revealed evenly distributed sharp peak-shaped structures with the height ranging from 0.6-5.3 nm. This result indicates that SMP-1 has almost no molecular aggregation.

| In vitro bile acid binding
Some components of food and herbs conjugate with bile acid and prevent its absorption, thereby producing certain hypolipidemic effects (Kahlon, Chapman, & Smith, 2007a, 2007b. Bile acid exists in the form of cholates such as taurocholate and glycocholate, and therefore, the capacity to bind cholates can be used to evaluate the hypolipidemic activities of active components in vitro. The bile acid conjugation rate of SMP-1 was evaluated using the SMP-1 concentration and the reaction time as the variable, respectively. As shown in Figure 3a, when different concentrations of SMP-1 (5, 10, 15, 20 mg/ml) reacted with the cholates for 30 min, the SMP-1 showed a strong binding capacity to both taurocholic acid sodium (65.96%-83.64%) and glycodeoxycholic acid sodium (71.76%-103.50%). Interestingly, as the SMP-1 concentration increased, the cholate-binding capacity decreased in general.
TA B L E 1 The molar ratios in partial acid hydrolysis analysis of SMP-1

| In vivo hypolipidemic activities
The serum lipid profiles (TC, TG, LDL-c, and HDL-c in serum) are shown in Table 2. There was a remarkable (p < 0.01 or p < 0.05) elevation in serum TC, TG, and LDL-c, but a significant (

| Histopathological analysis
To investigate the beneficial effects of SMP-1 on liver, the main organ involved in lipid metabolism, the histopathological analysis was performed. As shown in Figure

| Structure-function relationship
As aforementioned, SMP-1 was mainly composed of Gal (25%), Glc (24.5%), and Man (17.4%), followed by Ara (12.2%), GalA (9.7%), Xyl (7.6%), and Rha (3.6%). The monosaccharide composition of SMP-1 was similar to that of the previously reported Ginkgo biloba leaf polysaccharide which ameliorated changes in the lipid profile in serum and liver tissue of rats with nonalcoholic fatty liver disease (Yan et al., 2015). In addition, SMP-1 contained a relatively high content of GalA, a major component of pectin that is well known for its hypolipidemic function (Ridley, O'Neill, & Mohnen, 2001;Vergarajimenez, Conde, Erickson, & Fernandez, 1998). In a word, the hypolipidemic activity of SMP-1 is probably due to its monosaccharide components that are known for blood lipid regulation. Further studies will TA B L E 2 Effect of SMP-1 on serum lipid profile levels in P407-induced hyperlipidemic mice be carried out to clarify other possible relationships between the structure of SMP-1 and its hypolipidemic activity.

| CON CLUS ION
In this study, a water-soluble polysaccharide from the stigma maydis (SMP-1) was isolated, characterized, and investigated on its hypolipidemic activity in P407-induced hyperlipidemic mice model. The SMP-1 (average Mw = 12.93 kDa) was a homogeneous polysaccharide with no triple-helical structure and no molecular aggregations in solution. Importantly, the SMP-1 could effectively bind the bile acids in vitro and significantly lower the TC, TG, and LDL-c levels and moderately increase the LDL-c level in serum of the P407-induced hyperlipidemic mice. Moreover, pretreatment with SMP-1 (≥300 mg/kg) could remarkably reduce the fat accumulation in the liver of the hyperlipidemic mice. Collectively, these findings demonstrate that the stigma maydis polysaccharides can be developed as a safe and effective food supplement for preventing and treating the hyperlipidemia disorders. Committee for the kind guidance in the animal experiments.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L R E V I E W
The experimental protocol was approved by the Institution of Animal Ethical Committee, and the study was carried out in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals.