Effects of steam on polysaccharides from Polygonatum cyrtonema based on saccharide mapping analysis and pharmacological activity assays

Effects of steam on polysaccharides from Polygonatum cyrtonema based on saccharide mapping analysis and pharmacological activity assays | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of steam on polysaccharides from Polygonatum cyrtonema based on saccharide mapping analysis and pharmacological activity assays Zherui Chen, Baojie Zhu, Jing Zhao, Zhixin Chen, Wen Cao, Junqiao Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1645421/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background: Polygonatum cyrtonema, one of origins of Polygonata Rhizoma ( HuangJing in Chinese), is traditionally steamed repeatedly before being used as herbal medicine in China. However, there had no standardization of steaming of HuangJing . Therefore, comprehensive study for effects of steam on polysaccharide from Polygonatum cyrtonema based on saccharide mapping, a powerful method developed for polysaccharides analysis, and pharmacological activity are still necessary in order to explore the effect of steam on the physiochemical and biological activities of its polysaccharides and determine the standard for Polygonatum cyrtonema . Methods: To explore the effect of steam on the physiochemical and biological activities of P. cyrtonema polysaccharides (PCP), six polysaccharides named PCP0, PCP1, PCP2, PCP3, PCP4 and PCP5 were extracted from the herb consecutively steamed for 0 to 5 times, respectively. Their molecular weight distribution, monosaccharide composition and PACE fingerprints were investigated through HPSEC-MALLS-RID, HPAEC-PAD and saccharide mapping based on polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and HPTLC, respectively. In addition, their antioxidant ability and immunostimulatory activities on RAW 264.7 cells in term of NO production and phagocytosis were compared. Results: Results suggested that molecular weights could be changed during steam, which increased by first steaming and then decreased with further steaming though all polysaccharides molecular weight were 10 5 -10 7 Da. They all showed irregularly spherical conformation in aqueous solution based on AFM imaging. Monosaccharide composition and PACE fingerprints was significantly different after steaming, i.e., galactose increased while glucose and mannose decreased, and β-1,4-Gal p appeared while β-1,4-man p increased after steaming. Steamed PCP significantly increased scavenging activity against ABTS radicals, while PCP0 had better immunostimulatory effect on RAW 264.7 in terms of NO production and phagocytosis. Conclusions: In summary, steam significantly affected the chemical composition and bioactivities of polysaccharides from P. cyrtonema . Considering the conflict results of steaming on antioxidant and immunopotentiation activities of PCP, 2 times of continuously steam is the optimal choice under the modern process condition. Polygonatum cyrtonema Polysaccharides Steam Chemical composition Bioactivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Polygonatum cyrtonema Hua is one of origins of Polygonata Rhizoma ( HuangJing in Chinese), which has a long history of use in China as a tonic. It has high medicinal and edible value in medicine and food, and is beneficial to lowering blood sugar [ 1 ], blood lipids [ 2 ] and immune potentiation [ 3 ]. Polysaccharides are considered as its major active components [ 4 ], which showed the effects of anti-oxidation [ 2 , 5 – 7 ], anti-aging [ 8 ] and immunomodulation [ 9 – 14 ]. According to the theory of traditional Chinese medicine, the processing of Chinese raw materials is usually essential which can improve the efficacy or reduce the toxicity of crude drugs [ 15 ]. According to Ben Cao Gang Mu , HuangJing should be processed with “nine steaming and nine drying” ( Jiu-Zheng-Jiu-shai in Chinese), which means repeatedly steaming and drying until the herb turns black inside before being used as medicine. Yi Lin Zhuan Yao , a medical book published in Qing dynasty, recorded that HuangJing had the adverse effect of "stinging the throat". The process of “nine steaming and nine drying” was always used to eliminate or alleviate the adverse reactions, reduce irritation to throat, and improve its efficacy. However, there had no standardization of steaming of HuangJing . In ancient China, people believed the number of steaming times is judged based on its color and shape. Recently, small molecular components in P. sibiricum (another plant origin of HuangJing ), including aldehydes and alkanes decreased and ketones, nitrogen heterocycles increased during steaming process [ 16 ]. Though effect of steam on compositional monosaccharides, molecular weights, antioxidant and immunomodulatory activities of polysaccharides from HuangJing has been investigated [ 17 – 20 ], comprehensive study for effects of steam on polysaccharide from Polygonatum cyrtonema based on saccharide mapping, a powerful method developed for polysaccharides analysis, and pharmacological activity are still necessary. Consider to the complexity of polysaccharides, the establishment of simple and fast qualitative and quantitative methods with good accuracy and high specificity has been the key and bottleneck for the quality control of polysaccharides from traditional Chinese medicines. The analytical methods of polysaccharides, including conventional quantitative methods (i.e., colorimetric assays) [ 21 ] or recent qualitative methods (i.e., HPLC, GC, FT-IR) [ 22 , 23 ], show low specificity and poor accuracy for quantification or barely revealed limited structural information. In view of this, our research group proposed the strategy of saccharide mapping for qualitative analysis and quantitative detection of polysaccharides [ 24 , 25 ]. For this method, high performance size exclusion chromatography coupled with multi angle laser light scattering and refractive index detection (HPSEC-MALLS-RID) as one of the most powerful techniques can quickly and accurately determine the content and relative molecular weight of natural polysaccharides and their different components based on their universal refractive index increment (dn/dc) [ 25 ]. In addition, saccharides mapping based on polysaccharide analysis using carbohydrate gel-electrophoresis (PACE) is simple, reproducible, high resolution and high throughput. It has been proved to be one of the most effective methods for the quality control of natural resources polysaccharides [ 24 , 26 , 27 ]. In this study, six polysaccharides were extracted and obtained from P. cyrtonema consecutively steamed for 0 to 5 times, respectively, and their molecular weights and distributions, UV absorption, saccharide mapping based on polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and HPTLC, as well as antioxidant and immunostimulatory activities were investigated and compared. Materials And Methods Materials and chemicals Fresh cultivated rhizomes of P. cyrtonema were collected from Jinzhai Senfeng Agricultural Technology Development Co., Ltd., Anhui, China. After removing fibrous root, the rhizomes of P. cyrtonema were cleaned, dried and then cut into thin slices (3 mm ± 1 mm). Species identification was performed by Professor SP Li, one of corresponding authors. Voucher specimens of these samples were deposited at the Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China. ABTS, potassium persulfate and ascorbic acid (≥ 99%) were purchased from International Laboratory (San Bruno, CA), Fluka (Selzer, Germany) and Aladdin (Shanghai, China), respectively. Polygalacturonic acid (PGA), galacturonic acid (GA), konjac glucomannan (KG), dextran (DEX), pectinase (EC 3.2.1.15), endo-1,4-β-D-mannanase (EC 3.2.1.78) and endo-1,4-β-D-galactanase (EC 3.2.1.89) were purchased from Megazyme (Wicklow, Ireland). 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Griess reagent, lipopolysaccharides (LPS) and fluorescein isothiocyanate-dextran (FITC-Dextran) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Endotoxin detection specific limulus test kit was purchased from Bioendo TEShnology (Xiamen, China). Cell counting kit 8 (CCK8) was purchased from MedChemExpress. Phosphate-buffered saline (PBS), Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin (P/S) were purchased from Gibco-Invitrogen (Paisley, Scotland, UK). Nylon membrane filters (0.22/0.45 µm) were purchased from Millipore (Billerica, MA). Deionized water was prepared using a Millipore MilliQ-Plus system (Millipore, Billerica, MA). All the other reagents were of analytical grade. Preparation of P. cyrtonema and polysaccharides Steam of P. cyrtonema P. cyrtonema slices were steamed in an autoclave at 115 ℃ for 2 h (0.07 MPa) each time, followed by vacuum drying to be collected as the sample named as PC1. Afterwards, PC1 was further steamed and dried under the same condition to obtain the second sample named as PC2. Similarly, PC3, PC4 and PC5 were prepared, respectively. All those samples included the raw material without steaming (PC0) were grounded and sieved. Extraction of the polysaccharides Each sample (PC0-PC5) of 50.0 g was soaked at room temperature for 2 h, followed by hot water extraction (95 ºC) for further 2 h, material-to-liquid ratio was 1:15. The water extracts were precipitated with ethanol at final concentration of 75% to harvest polysaccharides. The supernatant (PCS) and precipitate were collected. Then small molecular weight substances (less than 3 KDa) were removed after ultrafiltration. The polysaccharides were dried with freeze-drier to obtain PCP0, PCP1, PCP2, PCP3, PCP4 and PCP5, respectively. Sample pretreatment of PCP Partial and complete acid hydrolysis of PCP According to a previously reported method with minor modification [ 28 ], PCP solution (2 mg/mL) was treated with trifluoroacetic acid (TFA) at a final concentration of 1.0 mol/L and incubated at 80°C for 2 h to gather partial acid hydrolysates (PAH). At the same time, the PCP solution (4 mg/mL) of each sample was mixed with equal volume of 4 mol/L TFA for complete acid hydrolysis at 105 ºC for 4 h to gather complete acid hydrolysates (CAH). Enzymatic hydrolysis of PCP Three enzymes of pectinase, β-1,4-galactanase and β-1,4-mannanase were selected to depolymerize PCP (2 mg/mL) at 40°C for 12 h. After incubation, enzymes were inactivated at 80°C for 20 min. As for PACE analysis, the enzymatic PCP hydrolysates should be freeze-dried and derivatized with ANTS. While no additional treatment is required for HPTLC and monosaccharides analysis. Polysaccharide standards including PGA, GA and KG were treated with those enzymes, respectively, under the same conditions. PCP solution without TFA or enzymes treatment was used as blank control. Physicochemical characterization of polysaccharides Molecular weights and chain conformation analysis The molecular weights and their distribution of PCP were determined by HPSEC-MALLS-RID according to our previous report [ 29 ]. HPSEC-MALLS-RID detection method is composed of multi-angle light scattering (MALS) detectors (Wyatt Technology Co., Santa Barbara, CA, USA), Agilent 1260 series LC/DAD system (Agilent Technologies, Palo Alto, CA, USA) and a refractometer (RID, Optilab rEX, Wyatt Technology Co.) in series at 35 ºC. The chromatographic columns are TOSOH gel columns TSK-GEL G5000PWXL (300 mm × 7.8 mm) and TSK-GEL G3000PWXL (300 mm × 7.8 mm), and the mobile phase is 0.9% NaCl solution. The flow rate is 0.5 mL/min with 100 µL injection volume. All the PCP were dissolved in mobile phase at 2 mg/mL and filtered through 0.45 µm filter membrane before injection. ASTRA 7.3.2 software was used to process the data. Compositional monosaccharides analysis The complete acid and pectinase hydrolyzed samples as well as PCS were analyzed by HPAEC-PAD system (Thermo Scientific™ Dionex™ ICS-5000 + , Dionex, USA). Ten monosaccharide standards including Fuc, Ara, Rha, Gal, Glc, Xyl, Man, Fru, GalA and GlcA were used to calculate the content of each monosaccharide in the samples. All the samples were filtered through 0.45 µm membrane before analysis. The mobile phase consisted of 88% deionized water and 12% 10mM NaOH, running for 22 minutes at a flow rate of 0.4mL/min under a CarboPac PA200 (3 mm × 250 mm) analytical column with a system temperature of 25°C (Supplementary Table 1). PACE analysis of partial acid and enzymatic hydrolysates PACE was performed according to previous report [ 30 ]. Briefly, all lyophilized derivatized hydrolysates of PCP were redissolved in isovolumetric urea (6 mol/L), and separated by Mini-Protean Tetra System, a vertical slab gel electrophoresis apparatus from Bio-Rad. Gels were imaged using an In-Genius LHR CCD camera system (Syngene, Cambridge, UK) under UV 365 nm. Quantity-One software (Ver4.6.2, BioRad) and Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Matlab version, Ver1.315, developed by the Research Center of Modernization of Chinese Herbal Medicine, Central South University and the Hong Kong Polytechnic University) were used for similarity analysis. HPTLC analysis of complete acid and pectinase hydrolysates Merck silica gel 60 plates pre-washed with methanol were used for HPTLC analysis. The method was modified according to previous report [ 30 ]. In brief, the PCP0-PCP5 samples after complete acid and pectinase hydrolysis were prepared into 8 mg/mL, respectively. SP-III electric thin-layer strip sampler (KEZHE SHANGHAI, China) was used for semi-automatic sampling. The bands were 7 mm wide, 5 mm distance, and 10 mm from the bottom edge. Then the plate was firstly developed to 90 mm with 1-butanol/isopropanol/acedic acid/water, 7:5:1:2 (v/v/v/v) as developing reagent at room temperature. Then the plate was dried and placed in the same chamber to develop 95 mm with the same developing reagent as described above. Finally, the developed plates were dried and colorized with aniline-diphenylamine-phosphoric acid solution, 10% sulfuric acid ethanol solution and 0.2% ninhydrin solution, respectively, then heated at 105 ºC and photographed under white light. AFM analysis PCP0-PCP5 (1 mg/mL) were fully dissolved in ultrapure water and diluted to a concentration of 1×10 − 2 µg/mL. Using droplet deposition method, pipette 5µL of solution onto the surface of newly cut mica sheet and dry it at room temperature. After the sample was dry, used BioScope Resolve AFM (Bruker Co., Santa Barbara, USA) for AFM measurement. NanoscopeAnalysis 1.8 software was used for image analysis. Antioxidation of PCP against ABTS radicals In brief, 7 mmol/L ABTS aqueous solution and 2.5 mmol/L potassium persulfate aqueous solution were mixed in a ratio of 1:1, and then stand in the dark for 12 hours. This solution was diluted with deionized water to reach a 0.7 ± 0.05 absorbance value at 734 nm and obtained the ABTS working solution. PCP0-PCP5 solution (0.5, 1, 2, 4, 8 mg/mL) and ascorbic acid (0.015, 0.03, 0.06, 0.12, 0.24, 0.5, 1, 2, 4, 8 mg/mL) were prepared. In a 96-well plate, 200 µL of ABTS working solution and 10 µL of the sample solution were added to each well, and the reaction was kept in the dark for 6 min. After the reaction, the absorbance was measured, and the ABTS clearance rate formula was as followed: C (%) = [1-(A 1 -A 2 )/A 0 ] ×100 C was the clearance rate, A 0 is the control absorbance, A 1 is the sample absorbance, and A 2 was the background absorbance, which was to eliminate the interference of tested solution. Effects of PCP on macrophage functions Cell culture RAW 264.7 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Dulbecco’s modified eagle medium supplemented with 10% FBS, 1% P/S at 37°C in a humidified atmosphere of 5% CO 2 . Cytotoxicity assay RAW 264.7 cells (5×10 3 cells/well) were cultured in 96-well microplates overnight, and then treated with LPS (0.4 µg/mL) and a series of concentrations of PCP for 24 h, respectively. Equal volume of culture medium was used as blank control. Subsequently, the original culture medium was discarded and stained with 100 µL of culture medium containing 10% CCK8 for 4 h in dark. The absorbance values were read at 450 nm and the cell viability was calculated as the ratio of absorbance values between sample and vehicle control group. Nitric oxide determination RAW 264.7 cells (5×10 4 cells/well) were seeded in 96-well microplates overnight, and then cells were treated with a series of concentrations of PCP and LPS (0.4 µg/mL) for 24 h, respectively. Equal volume of culture medium was used as vehicle control. Subsequently, 75 µL of supernatants were collected and mixed with an equal volume of modified Griess reagent at room temperature for 15 min. The absorbance was measured at 540 nm. NO production was expressed as ratio of absorbance values between sample and LPS treated group. Phagocytic activity test FITC-dextran was used for phagocytic assay. RAW 264.7 cells (1×10 5 cells/well) were cultured in 24-well plates overnight and then incubated with culture medium, LPS (0.4 µg/mL) and a series of concentrations of PCP for 18 h, respectively. Then the cells were treated with FITC-dextran (0.1 mg/mL in culture medium) and incubated at 37°C for additional 1 h in dark. After incubation, the cells were collected with cold PBS after washed for three times. BD Accuri™ C6 Cytometer (BD Biosciences, San Jose, CA, USA) was used to analyze. The percentage of phagocytosis was expressed as ratio of phagocytic rate between treatment and control cells. Determination of endotoxin contamination Endotoxin detection specific limulus test kit was used for avoiding endotoxin contamination. The results showed that PCP0-PCP5 had no endotoxin contamination in the samples. Statistical analysis GraphPad Prism 8.0.2 was used to analyze and process the data. Data were presented as mean ± SEM from at least three independent experiments for each sample. Statistical significance between the experimental groups was determined by Student’s t-test, and p values less than 0.05 were considered as statistically significant. Results And Discussion Appearance of P. cyrtonema and its polysaccharides After steam treatment, the color of P. cyrtonema slices deepened gradually. The raw material (PC0) was milky white, after first steam the color turned reddish brown with lighter color in center (PC1). Then, for the second time of steam treatment, slice PC2 changed to dark brown. Slice PC3 changed into black and began to appear luster, the color of PC4 and PC5 almost no change. The color of the polysaccharide powders was also changed accordingly, similar to the raw materials (Fig. 1 ). Traditionally, the number of steam treatment according to the color of materials. The color of PC5, same to those of PC3 and PC4, was black. Therefore, 5 times were selected for the steam of P. cyrtonema . Molecular weight distribution of PCP HPSEC-MALLS-RID is a useful method for determining the absolute molecular weights (Mw), the dispersibility index (DPI) and radius of gyrations (Rg) of polysaccharides without standards [ 31 ]. Figure 2 showed that HPSEC-MALLS-RID chromatograms of PCP in 0.9% NaCl aqueous solution at 35°C, and molecular mass distribution results of PCP0-PCP5 were summarized in Table 1 . To facilitate the comparison of the effect of steaming treatment on the molecular weight distribution of PCP, the same peak division was performed on PCP1-PCP5 according to that of PCP0, as shown in Fig. 2 A, which was divided into three peaks. In PCP0, Peak3 had the highest content and a molecular weight of ~ 7.85×10 3 Da, with Mw/Mn of 1.16. After the first steaming treatment, Mw of PCP1 increased dramatically to 1.01×10 7 Da, and gradually decreased with the increase of steaming times (Fig. 2 B). This indicated that steam could change the extraction and/or molecular weight distribution of PCP. In addition, the detection at UV 280 nm showed that PCP0 had a very low signal, but steamed PCP showed obviously high signals. With the increase of steaming times, the peak height of protein signal increased firstly, PCP2 to the highest, and then decreased gradually (Fig. 2 D). This implied that steaming treatment induced Maillard reaction of polysaccharides in P. cyrtonema . And it gradually degraded since the third steaming treatment. Table 1 The molecular weight, polydispersity index (Mw/Mn) and contents of PCP0-PCP5 Sample Peak1 Peak2 Peak3 Total Content % Mw, kDa (error%) Mw/Mn (error%) Rz, nm Content% Mw, kDa (error%) Mw/Mn (error%) Rz, nm Content% Mw, kDa (error%) Mw/Mn (error%) Rz, nm Content% PCP0 1.08×10 4 (± 2.8%) 1.43(± 5.1%) 56.7 (± 4.2%) 0.1 70.4 (± 2.4%) 3.64(± 5.6%) 21.9 (± 18.5%) 20.7 7.85 (± 2.6%) 1.16(± 6.4%) 17.9 (± 29.3%) 49.5 70.3 PCP1 1.01×10 4 (± 3.9%) 1.70(± 6.2%) 65.8 (± 4.2%) 1.4 173 (± 2.8%) 3.55(± 6.4%) 34.4 (± 9.1%) 48.6 102 (± 3.3%) 1.18(± 7.2%) 39.2 (± 8.7%) 12.5 62.5 PCP2 5.87×10 4 (± 4.0%) 1.44(± 5.7%) 52.0 (± 6.4%) 0.7 381 (± 3.6%) 3.15(± 4.8%) 29.0 (± 15.1%) 54.0 146 (± 4.2%) 1.18(± 5.3%) 31.5 (± 15.3%) 23.9 78.6 PCP 3 9.28×10 4 (± 4.5%) 1.88(± 5.4%) 42.3 (± 10.4%) 0.9 788 (± 3.3%) 2.15(± 5.3%) 37.2 (± 9.2%) 41.3 169 (± 3.7%) 1.18(± 6.0%) 39.7 (± 9.2%) 28.4 70.6 PCP 4 7.93×10 4 (± 4.6%) 3.84(± 4.9%) 50.3 (± 7.6%) 0.5 408 (± 4.2%) 1.85(± 4.1%) 30.7 (± 16.0%) 40.3 798 (± 5.1%) 1.22(± 4.8%) 33.4 (± 16.6%) 34.5 75.3 PCP 5 2.92×10 4 (± 3.8%) 1.16(± 4.0%) 44.7 (± 7.9%) 0.3 206 (± 3.6%) 2.59(± 3.5%) 23.0 (± 24.7%) 32.4 364 (± 4.5%) 1.06(± 3.5%) 22.2 (± 32.8%) 36.5 69.2 Monosaccharide composition After complete acid hydrolysis, monosaccharide composition of PCP was determined by HPAEC-PAD (Supplementary Fig. S2). Results showed that PCP without steam treatment was mainly composed of GalA, Man and Glc. After steaming, all PCP were mainly composed of Gal, Man and GalA, with small amount of Ara, Rha and Glc. The molar ratio of Gal, GalA, Man, Rha, Ara and Glc in PCP0 was 3:6:16:1:3:8, and PCP1-PCP5 were 7.3:5:6:1:2.4:1, 7.9:3.1:4.3:1:1.6:0.6, 7.4:2.6:2.7:1:0.8:0.4, 6:2.1:2.6:1:0.6:0.5 and 5.9:1.7:2.6:1:0.3:0.5, respectively (Supplementary Table 2). Obviously, the monosaccharide composition of PCPs changed dramatically after the first steaming. The content of Man and Glc decreased significantly while the content of Gal increased obviously. As the steaming times increased, the content of Man and Glc remained stable starting from PCP3 while Gal decreased in PCP4 and PCP5. At the same time, GalA and Ara also decreased. According to previous studies [ 32 ], PCP contained mainly Man, Glc, Gal and Ara, which is consistent with our results. Interestingly, according to the study by Li et al.[ 18 ], Glc and Ara increased with the steam treatment which was inconsistent with our research results, which might attribute to different samples and/or steaming conditions. Besides, previous studies have shown that PCP contain a lot of fructose [ 33 ], while the traditional complete acid hydrolysis method can cause fructose loss. Therefore, we also degraded the samples with pectinase and observed changes in monosaccharide composition. The results showed that the molar ratio of Gal, GalA, Man, Rha, Ara, Glc and Fru in PCP0-PCP5 was 7.5:35:7.9:1:7.9:7.7:55.4, 11.5:16.9:2.6:1:3.8:0.6:0.8, 14:10.7:2.1:1:2.7:0.6:0.3, 11.1:7.8:1.5:1:1.1:0.6:0.2, 9.6:6.2:1.7:1:0.7:0.6:0.2 and 8.3:4.6:1.8:1:0.4:0.3:0.1, respectively (Supplementary Table 3). The result of pectinase hydrolysis was similar to that of complete acid hydrolysis, except the content of Fru which was very different. The content of Fru was decreased dramatically after the first steam treatment, and almost disappear in PCP1-PCP5. This indicated that the steam processing changes the polysaccharide structure and lose a large amount of Fru. At the same time, we examined changes in Fru and Glc in 75% ethanol supernatant during ethanol precipitation, and the contents of Fru and Glc in the supernatant increased with steaming. The results showed that the composition of polysaccharide changed with the increase of steaming times, and the contents of monosaccharides such as fructose and glucose or oligosaccharides in free state increased. As the polysaccharide is relatively stable under the condition of normal temperature, however, under the steaming condition of high pressure and high temperature, various monosaccharide components can undergo dehydration and degradation, and Maillard reaction [ 34 ] can occur under the common existence of other components such as amino acids. Polysaccharides are composed of various monosaccharides that are degraded during processing and undergo further reaction changes that alter the monosaccharide composition of polysaccharides. PACE profiles of PCP Saccharide mapping based on PACE has been proven to be one of powerful methods for the routine analysis of oligosaccharides derived from polysaccharides [ 30 ]. Therefore, both partial acid hydrolysates and enzymatic hydrolysates of PCP were compared using saccharide mapping based on PACE analysis (Fig. 3 A) and their similarity was conducted using Quantity-One software and Similarity Evaluation System. The HPSEC-MALLS-RID chromatogram and ion chromatogram of PCP0-PCP5 after treat with pectinase, β-1,4-Galactanase, β-1,4-Mannanase or TFA were used to detect glycoside bond variations and sugar composition to determine glycoside bonds with significant differences in the steaming process (Supplementary Figs. 3 & 4) Their hydrolysates had high similarity except the partial acid hydrolysates of PCP0 and the β-1,4-manannase hydrolysates of PCP1 and PCP2 (Table 2 ). In addition, the hydrolysates obtained by different hydrolysis methods showed different similarity after steaming, especially the β-1,4-galactanase hydrolysates of PCP. PACE fingerprints of pectinase hydrolysates from PCP showed that the content of GalA in PCP0 was lower than in other PCP. The fingerprints of β-1,4-galactanase hydrolysates indicated that no β-1,4-Gal p were detected in PCP0. However, after steaming treatment, β-1,4-Gal p appeared in PCP1-PCP5. In the case of β-1,4-manannase hydrolysates, it indicated that PCP contained small number of polysaccharides with β-1,4-Man p , and after steaming, they could be easily extracted, and the level of β-1,4-Man p in PCP increased. For the partial acid hydrolysates analysis, PCP0 showed an obviously different PACE fingerprints compared with other PCP. All these results indicated that steaming treatment had a huge impact on PCP. Table 2 The correlation coefficient of PCPs to their simulative mean chromatogram. Samples The simulative mean chromatograms PACE HPTLC SMC-PAH SMC-GA SMC-MA SMC-PE SMC-PE SMC-CAH PCP0 100.00 100.00 100.00 100.00 100.00 100.00 PCP1 0.75 0.00 0.81 0.96 0.76 0.51 PCP2 0.76 0.00 0.77 0.96 0.77 0.51 PCP3 0.76 0.00 0.88 0.96 0.77 0.51 PCP4 0.75 0.00 0.89 0.96 0.77 0.51 PCP5 0.72 0.00 0.88 0.96 0.76 0.50 HPTLC fingerprints of PCP HPTLC showed the samples of complete acid hydrolysis and pectin-decomposing PCP had verified the experimental results of monosaccharide composition to some extent. According to the results of phenylamine-phosphoric acid coloration, pectin enzymatic samples contain more oligosaccharides than completely hydrolyzed samples (Fig. 3 B), and according to 10% ethanol sulfuric acid colorization, the complete acid hydrolysis sample of PCP0 might contain little of small molecular substances. In other samples, bands of monosaccharides and oligosaccharides can be clearly observed. Colorization of ninhydrin coloration showed that there almost were no amino acids (Fig. 3 B & 3 C). HPTLC profiles similarity was shown in Table 2 , which indicated that no significant difference between pectinase and complete acid hydrolysates of PCP. Morphology of PCP The biological activity of natural polysaccharide is also closely related to its chain conformation besides molecular weight [ 35 ]. Therefore, it is very important to study the chain conformation of PCP in aqueous solution to understand the effect of steam treatment on its structure and biological activity. The conformation of polysaccharide can be analyzed according to the theory of dilute polymer solution. Generally, the chain conformation of polysaccharides in aqueous solution is determined by the double logarithmic plot of Rg vs the molecular mass of polysaccharides according to Mark-Houwink equation Rg = kMw ν [ 36 ]. According to the polymer solution theory, the exponent ( v ) is 0.2–0.4 for branched polymers with a compact helical chain conformation, 0.3 for spheres, 0.5–0.6 for flexible polymers in good solvents and 0.6-1.0 for semi-flexible chains [ 37 ]. According to the calculation results of HPSEC-MALLS/RI, the v index of PCP0-PCP5 was concentrated between 0 and 0.3 (Supplementary Fig. S1). The results showed that PCP0-PCP5 appeared as irregular monodisperse spheres in 0.9% aqueous sodium chloride solution. Atomic force microscopy (AFM) has become a powerful tool to directly characterize the structure and properties of polymers [ 38 ]. The planar images with height and diameter (scanned at 3×3 µm) of PCP0-PCP5 in aqueous solution obtained by AFM were shown in Fig. 4 . An irregular monodisperse spherical shape of all PCP were observed, consistent with the results of HPSEC-MALLS-RID. Their molecular height was in the range of 1 to 1.5 nm, and the diameter was ranged from 15 to 20 nm. Specifically, with the steaming times increased, the height of PCP increased, and the diameter decreased gradually. There was almost no significant difference after the second steaming, except PCP4 with the height and diameter of 1.4 nm and 20.0 nm, respectively. This is likely to be related to the change of polysaccharides structure and/or fractions caused by steaming. ABTS scavenge ability of PCP ABTS scavenge ability of PCP was shown in Fig. 6A, and all PCP showed scavenge ability against ABTS radicals in different extents, and PCP0 showed the lowest capacity. Generally, steamed PCP had higher dose-dependent free radical scavenge ability, and IC 50 values of PCP1-PCP5 were 4.89, 1.81, 1.79, 2.21, 3.04 mg/mL, respectively. Change of steamed PCP in antioxidant capacity may attribute to Maillard reaction of polysaccharides during steam processing [ 39 ], which was supported by UV 280nm absorption and molecular weights increased after steaming (Fig. 2 ). Though the ability of PCP scavenging ABTS radicals increased with the number of steaming times [ 18 ], IC 50 showed that antioxidant activity of PCP3 reached to the strongest, and then decreased with the following steam treatment. The variation might be due to the different steam treatment conditions. Anyway, steaming significantly enhances antioxidant capacity of PCP, which is beneficial to its efficacy in delaying aging [ 8 ], lowering blood sugar [ 6 , 40 , 41 ] and regulating blood lipids [ 2 ]. The significance of steaming to health beneficial effects P. cyrtonema should be further well investigated. Immunostimulatory activity of PCP Macrophages play an indispensable role in the innate and adaptive immunity of the human body [ 42 ]. Studies have shown that high levels of NO are associated with immune responses during antitumor and antiviral processes, which can trigger cell proliferation, apoptosis, signal transduction, immune defense and other physiological processes [ 43 ]. Phagocytosis is a basic cellular process that plays an important role in the immune system [ 44 ]. In this study, RAW 264.7 cells were treated with a series of concentrations of PCP and their effects on NO production and phagocytic activity were investigated. Effects of PCP on NO production of macrophages were shown in Fig. 6C, though viability of RAW 264.7 cells was not significantly affected in the ranges of investigated concentration (Fig. 6B). Steaming reduced the effect of PCP on NO production of macrophages. As a result, effect of PCP0 was the best, while steamed PCP was reduced with increasing steam times, PCP3-PCP4 were only effective at the highest concentration (200 µg/mL), and PCP5 showed no such effect. Flow cytometry was used to determine the fluorescence intensity in cells after RAW 264.7 cells devoured FITC-dextran. The results showed that LPS (0.4 µg/mL) and PCP could promote the phagocytic activity of macrophages in a dose-dependent manner (Fig. 6D). With the increase of steam times, their ability on phagocytosis was weakened, and after the third steam treatment (PCP3-PCP5), they showed no effect on FITC-dextran phagocytose compared with that of blank control group. Conclusions Steaming treatment significantly influenced the physicochemical properties and bioactivities of polysaccharides from P. cyrtonema , one of the origins of HuangJing considered as well-known tonic herb. In brief, steaming could significantly increase the molecular weights, UV absorption and antioxidant activity of polysaccharides from P. cyrtonema . Polysaccharides with glycosidic linkages such as β-1,4-Gal p and β-1,4-man p obviously increased after steaming, but fructan could be completely degraded. In addition, steaming could signification decrease the immunopotentiation activity, such as NO release and phagocytosis of RAW 264.7 cells of polysaccharides from P. cyrtonema . Considering the conflict results of steaming on antioxidant and immunopotentiation activities of PCP, 2 times of continuously steam is the optimal choice. However, further study is still necessary to well understand the beneficial effect of steaming on HuangJing . Abbreviations AFM Atomic Force Microscopy ABTS potassium persulfate and ascorbic acid ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid Ara Arabinose CAH Complete Acid Hydrolysates CCK8 Cell counting kit 8 CMM Chinese Materia Medica DEX Dextran DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl Sulfoxide dn/dc refractive index increment DPI Dispersibility Index FBS Fetal Bovine Serum FBS Fetal Bovine Serum FITC-Dextran Fluorescein Isothiocyanate-Dextran FT-IR Fourier Transform Infrared Spectroscopy Fuc Fucose Gal Galactose GA Galacturonic Acid GC Gas Chromatography Glc Glucose GlcA Glucoronic Acid HPAEC High Performance Anion Exchange Chromatography HPLC High Performance Liquid Chromatography HPSEC High Performance Size Exclusion Chromatography HPTLC High Performance Thin Layer Chromatography KG Konjac Glucomannan LPS Lipopolysaccharides MALLS Multi-angle Laser Light Scattering Man Mannose Mw Molecular Weights Mw/Mn Polydispersity Index NaOH Sodium Hydroxide NO Nitric Oxide P/S Penicillin/Streptomycin PACE Carbohydrate Gel Electrophoresis PAH Partial Acid Hydrolysates PBS Phosphate-buffered Saline PC Polygonatum cyrtonema PCP Polygonatum cyrtonema polysaccharides PCS the 75% ethanol supernatant of Polygonatum cyrtonema polysaccharides PGA Polygalacturonic Acid Rg Radius of Gyration Rha Rhamnose RID Refractive Index Detector SEC Size Exclusion Chromatography SEM Standard Error of Mean TCM Traditional Chinese Medicine TFA Trifluoroacetic Acid TLC Thin Layer Chromatography UV Ultraviolet Vc Ascorbic acid Xyl Xylose. Declarations Ethical approval and consent to participate This article is compliance with ethical standard and does not contain any studies with human participants or animals performed by any of the authors. Consent for publication Not applicable. Availability of data and materials Data available on request from the authors. The data that support the findings of this study are available from the corresponding author, Shao-ping Li, upon reasonable request. Conflicts of interest The authors declare that they have no conflict of interest. Funding The research was partially funded by grants from the National Key R&D Program of China (2019YFC1711300), the Science and Technology Development Fund, Macau SAR (File no. 0017/2019/AKP), the Key-Area Research and Development Program of Guangdong Province (File no. 2020B1111110006) and the University of Macau (File no. MYRG2018-00083-ICMS /MYRG2019-00128-ICMS /CPG2021-00009-ICMS). Author contributions SPL and JZ designed the study and offered supervision, project administration and funding acquisition. ZRC conducted the experiments and statistical analyses, drafted the manuscript. BJZ, ZXC and WC helped carry out experiments. SPL and JQW reviewed and revised the manuscript. All authors read and approved the final manuscript. Acknowledgements All herbal materials were provided by Mr. Lei Li of Jinzhai Senfeng Agricultural Technology Development Co., Ltd., Anhui, China. The research was partially funded by grants from the National Key R&D Program of China (2019YFC1711300), the Science and Technology Development Fund, Macau SAR (File no. 0017/2019/AKP), the Key-Area Research and Development Program of Guangdong Province (File no. 2020B1111110006) and the University of Macau (File no. MYRG2018-00083-ICMS /MYRG2019-00128-ICMS /CPG2021-00009-ICMS). And the authors would like to thank the reviewers and also the authors of all references. CRediT author statement All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Author statement Zhe-rui Chen: Investigation, Formal analysis, Writing - Original Draft Bao-jie Zhu: Investigation, Formal analysis Zhi-xin Chen : Investigation Wen Cao: Investigation Jun-Qiao Wang: Writing - Review & Revision Jing Zhao: Conceptualization, Resources, Writing - Review & Revision, Supervision, Project administration, Funding acquisition Shao-ping Li: Conceptualization, Resources, Writing - Review & Revision, Supervision, Project administration, Funding acquisition Declaration of interests ☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: References Deng Y, He K, Ye X, Chen X, Huang J, Li X, et al. Saponin rich fractions from Polygonatum odoratum (Mill.) Druce with more potential hypoglycemic effects. J Ethnopharmacol. 2012;141(1):228 – 33. Li JX, Shang YX, Wang Y, Gao J, Xue N, Huang C, et al. Hypoglycemic and Hypolipidemic Activity of Polygonatum sibiricum Fermented with Lactobacillus brevis YM 1301 in Diabetic C57BL/6 Mice. J Med Food. 2021;24(7):720–31. Chen ZB, Liu JJ, Kong X, Li H. Characterization and Immunological Activities of Polysaccharides from Polygonatum sibiricum . Biol Pharm Bull. 2020;43(6):959–67. Cui XW, Wang SY, Cao H, Guo H, Li YJ, Xu FX, et al. A Review: The Bioactivities and Pharmacological Applications of Polygonatum sibiricum polysaccharides. Molecules. 2018;23(5):12. doi: 10.3390/molecules23051170 . Wang WJ, Li S, Song MX. Polygonatum sibiricum polysaccharide inhibits high glucose-induced oxidative stress, inflammatory response, and apoptosis in RPE cells. J Recept Signal Transduct Res. 2021. doi: 10.1080/1079989320211883061 . Luo JY, Chai YY, Zhao M, Guo QQ, Bao YH. Hypoglycemic effects and modulation of gut microbiota of diabetic mice by saponin from Polygonatum sibiricum . Food Funct. 2020;11(5):4327–38. Cai JL, Zhu YL, Zuo YJ, Tong QZ, Zhang ZG, Yang L, et al. Polygonatum sibiricum polysaccharide alleviates inflammatory cytokines and promotes glucose uptake in high-glucose- and high-insulin-induced 3T3-L1 adipocytes by promoting Nrf2 expression. Mol Med Rep. 2019;20(4):3951–8. Zheng SY. Protective effect of Polygonatum sibiricum Polysaccharide on D-galactose-induced aging rats model. Sci Rep. 2020;10(1):2246. doi: 10.1038/s41598020590557 . Wang YJ, Liu N, Xue X, Li Q, Sun DQ, Zhao ZX. Purification, structural characterization and in vivo immunoregulatory activity of a novel polysaccharide from Polygonatum sibiricum . Int J Biol Macromol. 2020;160:688–94. Sun TT, Zhang H, Li Y, Liu Y, Dai W, Fang J, et al. Physicochemical properties and immunological activities of polysaccharides from both crude and wine-processed Polygonatum sibiricum . Int J Biol Macromol. 2020;143:255–64. Liu ZG, Ni HY, Yu L, Xu SW, Bo RN, Qiu TX, et al. Adjuvant activities of CTAB-modified Polygonatum sibiricum polysaccharide cubosomes on immune responses to ovalbumin in mice. Int J Biol Macromol. 2020;148:793–801. Zhang JZ, Liu N, Sun C, Sun DQ, Wang YJ. Polysaccharides from Polygonatum sibiricum Delar. ex Redoute induce an immune response in the RAW264.7 cell line via an NF-kappa B/MAPK pathway. RSC Adv. 2019;9(31):17988–94. Yelithao K, Surayot U, Park W, Lee S, Lee DH, You S. Effect of sulfation and partial hydrolysis of polysaccharides from Polygonatum sibiricum on immune-enhancement. Int J Biol Macromol. 2019;122:10–8. Li B, Wu PP, Fu WW, Xiong Y, Zhang L, Gao YB, et al. The Role and Mechanism of miRNA-1224 in the Polygonatum sibiricum Polysaccharide Regulation of Bone Marrow-Derived Macrophages to Osteoclast Differentiation. Rejuvenation Res. 2019;22(5):420–30. Zhao Z, Liang Z, Chan K, Lu G, Lee EL, Chen H, et al. A unique issue in the standardization of Chinese materia medica: processing. Planta Med. 2010;76(17):1975–86. Cheng X, Ji H, Cheng X, Wang D, Li T, Ren K, et al. Characterization, Classification, and Authentication of Polygonatum sibiricum Samples by Volatile Profiles and Flavor Properties. Molecules. 2021;27(1):18. doi: 10.3390/molecules27010025 . Wu WJ, Huang NW, Huang JP, Wang LL, Wu LL, Wang Q, et al. Effects of the steaming process on the structural properties and immunological activities of polysaccharides from Polygonatum cyrtonema . J Funct Foods. 2022;88:104866. Li Q, Zeng J, Gong P, Wu Y, Li H. Effect of steaming process on the structural characteristics and antioxidant activities of polysaccharides from Polygonatum sibiricum rhizomes. Glycoconj J. 2021;38(5):561–72. Fan BL, Wei GL, Gan XF, Li TT, Qu ZY, Xu S, et al. Study on the varied content of Polygonatum cyrtonema polysaccharides in the processing of steaming and shining for nine times based on HPLC-MS/MS and chemometrics. Microchem J. 2020;159:105352. Jin J, Lao J, Zhou RR, He W, Qin Y, Zhong C, et al. Simultaneous Identification and Dynamic Analysis of Saccharides during Steam Processing of Rhizomes of Polygonatum cyrtonema by HPLC-QTOF-MS/MS. Molecules. 2018;23(11):14. doi: 10.3390/molecules23112855 . Zhang CH, Yun YH, Fan W, Liang YZ, Yu Y, Tang WX. Rapid analysis of polysaccharides contents in Glycyrrhiza by near infrared spectroscopy and chemometrics. Int J Biol Macromol. 2015;79:983–7. Lv Y, Yang X, Zhao Y, Ruan Y, Yang Y, Wang Z. Separation and quantification of component monosaccharides of the tea polysaccharides from Gynostemma pentaphyllum by HPLC with indirect UV detection. Food Chem. 2009;112(3):742–6. Xie PS, Leung AY. Understanding the traditional aspect of Chinese medicine in order to achieve meaningful quality control of Chinese materia medica. J Chromatogr A. 2009;1216(11):1933–40. Guan J, Li SP. Discrimination of polysaccharides from traditional Chinese medicines using saccharide mapping-enzymatic digestion followed by chromatographic analysis. J Pharm Biomed Anal. 2010;51(3):590–8. Cheong KL, Wu DT, Zhao J, Li SP. A rapid and accurate method for the quantitative estimation of natural polysaccharides and their fractions using high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector. J Chromatogr A. 2015;1400:98–106. Wu DT, Xie J, Hu DJ, Zhao J, Li SP. Characterization of polysaccharides from Ganoderma spp. using saccharide mapping. Carbohydr Polym. 2013;97(2):398–405. Wu DT, Cheong KL, Wang LY, Lv GP, Ju YJ, Feng K, et al. Characterization and discrimination of polysaccharides from different species of Cordyceps using saccharide mapping based on PACE and HPTLC. Carbohydr Polym. 2014;103:100–9. Wu DT, Meng LZ, Wang LY, Lv GP, Cheong KL, Hu DJ, et al. Chain conformation and immunomodulatory activity of a hyperbranched polysaccharide from Cordyceps sinensis. Carbohydr Polym. 2014;110:405–14. Zhu BJ, Yan ZY, Hong L, Li SP, Zhao J. Quality evaluation of Salvia miltiorrhiza from different geographical origins in China based on qualitative and quantitative saccharide mapping and chemometrics. J Pharm Biomed Anal. 2020;191:113583. Wu DT, Cheong KL, Deng Y, Lin PC, Wei F, Lv XJ, et al. Characterization and comparison of polysaccharides from Lycium barbarum in China using saccharide mapping based on PACE and HPTLC. Carbohydr Polym. 2015;134:12–9. Deng Y, Li M, Chen LX, Chen XQ, Lu JH, Zhao J, et al. Chemical characterization and immunomodulatory activity of acetylated polysaccharides from Dendrobium devonianum . Carbohydr Polym. 2018;180:238–45. Yelithao K, Surayot U, Lee JH, You S. RAW264.7 Cell Activating Glucomannans Extracted from Rhizome of Polygonatum sibiricum . Prev Nutr Food Sci. 2016;21(3):245–54. Fan BL, Wei GL, Gan XF, Li TT, Qu ZY, Xu S, et al Study on the varied content of P olygonatum cyrtonema polysaccharides in the processing of steaming and shining for nine times based on HPLC-MS/MS and chemometrics. Microchemical Journal. 2020;159. Ellis GP. The Maillard Reaction , in Advances in Carbohydrate Chemistry , M.L. Wolfrom, Editor. 1959, Academic Press. pp. 63–134. Casu B, Lindahl U, Structure and biological interactions of heparin and heparan sulfate , in Advances in Carbohydrate Chemistry and Biochemistry . 2001, Academic Press. p. 159–206. Liu W, Wang H, Yu J, Liu Y, Lu W, Chai Y, et al. Structure, chain conformation, and immunomodulatory activity of the polysaccharide purified from Bacillus Calmette Guerin formulation. Carbohydr Polym. 2016;150:149–58. Wang J, Ma Z, Zhang L, Fang Y, Jiang F, Phillips GO. Structure and chain conformation of water-soluble heteropolysaccharides from Ganoderma lucidum . Carbohydr Polym. 2011;86(2):844–51. Wang J, Nie S. Application of atomic force microscopy in microscopic analysis of polysaccharide. Trends Food Sci Technol. 2019;87:35–46. Brands CMJ, Alink GM, van Boekel M, Jongen WMF. Mutagenicity of heated sugar-Casein systems: Effect of the Maillard reaction. J Agric Food Chem. 2000;48(6):2271–5. Wang Y, Lan CJ, Liao X, Chen D, Song WG, Zhang QL. Polygonatum sibiricum polysaccharide potentially attenuates diabetic retinal injury in a diabetic rat model. J Diabetes Investig. 2019;10(4):915–24. Wang Y, Qin SC, Pen GQ, Chen D, Han C, Miao CR, et al. Potential ocular protection and dynamic observation of Polygonatum sibiricum polysaccharide against streptozocin-induced diabetic rats' model. Exp Biol Med (Maywood). 2017;242(1):92–101. Hao NB, Lü MH, Fan YH, Cao YL, Zhang ZR, Yang SM. Macrophages in tumor microenvironments and the progression of tumors. Clin Exp Immunol. 2012;2012:11. Boscá L, Zeini M, Través PG, Hortelano S. Nitric oxide and cell viability in inflammatory cells: a role for NO in macrophage function and fate. Toxicology. 2005;208(2):249–58. Greenberg S, Grinstein S. Phagocytosis and innate immunity. Curr Opin Immunol. 2002;14(1):136–45. Supplementary Files ChenCM2022Supplementarydata.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 Jun, 2022 Reviewers invited by journal 18 Jun, 2022 Editor assigned by journal 16 May, 2022 First submitted to journal 11 May, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1645421","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":114588530,"identity":"415c5833-182c-4e79-be37-18f285dea18b","order_by":0,"name":"Zherui 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17:01:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":170592,"visible":true,"origin":"","legend":"\u003cp\u003eHPSEC-MALLS-RID chromatograms (\u003cstrong\u003eA\u003c/strong\u003e), comparative chromatograms on RI (\u003cstrong\u003eB\u003c/strong\u003e), LS (\u003cstrong\u003eC) \u003c/strong\u003eand UV\u003cstrong\u003e (D) \u003c/strong\u003echromatograms of PCP0-PCP5.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-1645421/v1/11f9bd842975f7a8bc477ab7.png"},{"id":23011911,"identity":"ca671dd7-824f-4a16-9182-5d1cf464ee4a","added_by":"auto","created_at":"2022-06-23 16:56:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":936040,"visible":true,"origin":"","legend":"\u003cp\u003ePACE profiles of partial acid hydrolysis (PAH) and enzymatic digestion of PCP (\u003cstrong\u003eA\u003c/strong\u003e), HPTLC profiles of complete acid hydrolysis (CAH) (\u003cstrong\u003eB\u003c/strong\u003e) and pectinase hydrolysis (\u003cstrong\u003eC\u003c/strong\u003e) of PCPs, colorized with aniline-diphenylamine-phosphoric acid solution, sulfuric acid ethanol solution and ninhydrin solution from left to right.\u003c/p\u003e\u003cp\u003e\t\u003cstrong\u003ePGA\u003c/strong\u003e is polygalacturonic acid, \u003cstrong\u003eGA\u003c/strong\u003e is galacturonic acid, \u003cstrong\u003eKG\u003c/strong\u003e is konjac glucomannan,\u003cstrong\u003e DEX\u003c/strong\u003e is dextran.\u003cstrong\u003e S1\u003c/strong\u003e is Rha, Man and Fru from top to bottom, \u003cstrong\u003eS2\u003c/strong\u003e is Glc, Gal and GalA from top to bottom respectively, \u003cstrong\u003ePE\u003c/strong\u003e is the blank control of pectinase.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-1645421/v1/1c3ebed47fab5a1fec36836d.png"},{"id":23012489,"identity":"8c1c8232-9223-4a86-a0e8-83ab9f1dd643","added_by":"auto","created_at":"2022-06-23 17:01:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":340184,"visible":true,"origin":"","legend":"\u003cp\u003ePlanar view of 0.01 μg/mL PCP0-PCP5 observed under atomic force microscope.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-1645421/v1/acc41a54f0231a4edbc93c2a.png"},{"id":23011914,"identity":"3005f5f0-9127-42b6-8733-3d13b638be3f","added_by":"auto","created_at":"2022-06-23 16:56:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":69741,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of PCP0-PCP5 on ABTS free radical scavenging activity (\u003cstrong\u003eA\u003c/strong\u003e), as well as cell viability \u003cstrong\u003e(B)\u003c/strong\u003e, NO production \u003cstrong\u003e(C)\u003c/strong\u003e and phagocytosis \u003cstrong\u003e(D)\u003c/strong\u003e of RAW 264.7 macrophages.\u003cstrong\u003e \u003c/strong\u003eAll values were expressed as mean ± SEM of three independent experiments. * \u003cem\u003ep \u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.001 \u003cem\u003evs\u003c/em\u003e control group, unmarked results indicate no significant difference \u003cem\u003evs\u003c/em\u003e control group.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-1645421/v1/f54b29934c6bfc64b870b639.png"},{"id":23012491,"identity":"6c59ed15-f9bf-4bc4-8728-79e36fa7e95e","added_by":"auto","created_at":"2022-06-23 17:01:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1309303,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1645421/v1/4411d345-27e9-48fc-8450-0f505fe1c9b1.pdf"},{"id":23011916,"identity":"2e988865-c93c-40fd-8e03-6fe4059bb152","added_by":"auto","created_at":"2022-06-23 16:56:11","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":897520,"visible":true,"origin":"","legend":"","description":"","filename":"ChenCM2022Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-1645421/v1/8582e0b6e7928489cd56e1d2.docx"}],"financialInterests":"","formattedTitle":"Effects of steam on polysaccharides from Polygonatum cyrtonema based on saccharide mapping analysis and pharmacological activity assays","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e Hua is one of origins of Polygonata Rhizoma (\u003cem\u003eHuangJing\u003c/em\u003e in Chinese), which has a long history of use in China as a tonic. It has high medicinal and edible value in medicine and food, and is beneficial to lowering blood sugar [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], blood lipids [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and immune potentiation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Polysaccharides are considered as its major active components [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which showed the effects of anti-oxidation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], anti-aging [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and immunomodulation [\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. According to the theory of traditional Chinese medicine, the processing of Chinese raw materials is usually essential which can improve the efficacy or reduce the toxicity of crude drugs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. According to \u003cem\u003eBen Cao Gang Mu\u003c/em\u003e, \u003cem\u003eHuangJing\u003c/em\u003e should be processed with \u0026ldquo;nine steaming and nine drying\u0026rdquo; (\u003cem\u003eJiu-Zheng-Jiu-shai\u003c/em\u003e in Chinese), which means repeatedly steaming and drying until the herb turns black inside before being used as medicine. \u003cem\u003eYi Lin Zhuan Yao\u003c/em\u003e, a medical book published in Qing dynasty, recorded that \u003cem\u003eHuangJing\u003c/em\u003e had the adverse effect of \"stinging the throat\". The process of \u0026ldquo;nine steaming and nine drying\u0026rdquo; was always used to eliminate or alleviate the adverse reactions, reduce irritation to throat, and improve its efficacy.\u003c/p\u003e \u003cp\u003eHowever, there had no standardization of steaming of \u003cem\u003eHuangJing\u003c/em\u003e. In ancient China, people believed the number of steaming times is judged based on its color and shape.\u003c/p\u003e \u003cp\u003eRecently, small molecular components in \u003cem\u003eP. sibiricum\u003c/em\u003e (another plant origin of \u003cem\u003eHuangJing\u003c/em\u003e), including aldehydes and alkanes decreased and ketones, nitrogen heterocycles increased during steaming process [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Though effect of steam on compositional monosaccharides, molecular weights, antioxidant and immunomodulatory activities of polysaccharides from \u003cem\u003eHuangJing\u003c/em\u003e has been investigated [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], comprehensive study for effects of steam on polysaccharide from \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e based on saccharide mapping, a powerful method developed for polysaccharides analysis, and pharmacological activity are still necessary.\u003c/p\u003e \u003cp\u003eConsider to the complexity of polysaccharides, the establishment of simple and fast qualitative and quantitative methods with good accuracy and high specificity has been the key and bottleneck for the quality control of polysaccharides from traditional Chinese medicines. The analytical methods of polysaccharides, including conventional quantitative methods (i.e., colorimetric assays) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] or recent qualitative methods (i.e., HPLC, GC, FT-IR) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], show low specificity and poor accuracy for quantification or barely revealed limited structural information. In view of this, our research group proposed the strategy of saccharide mapping for qualitative analysis and quantitative detection of polysaccharides [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For this method, high performance size exclusion chromatography coupled with multi angle laser light scattering and refractive index detection (HPSEC-MALLS-RID) as one of the most powerful techniques can quickly and accurately determine the content and relative molecular weight of natural polysaccharides and their different components based on their universal refractive index increment (dn/dc) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In addition, saccharides mapping based on polysaccharide analysis using carbohydrate gel-electrophoresis (PACE) is simple, reproducible, high resolution and high throughput. It has been proved to be one of the most effective methods for the quality control of natural resources polysaccharides [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, six polysaccharides were extracted and obtained from \u003cem\u003eP. cyrtonema\u003c/em\u003e consecutively steamed for 0 to 5 times, respectively, and their molecular weights and distributions, UV absorption, saccharide mapping based on polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and HPTLC, as well as antioxidant and immunostimulatory activities were investigated and compared.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials and chemicals\u003c/h2\u003e \u003cp\u003eFresh cultivated rhizomes of \u003cem\u003eP. cyrtonema\u003c/em\u003e were collected from Jinzhai Senfeng Agricultural Technology Development Co., Ltd., Anhui, China. After removing fibrous root, the rhizomes of \u003cem\u003eP. cyrtonema\u003c/em\u003e were cleaned, dried and then cut into thin slices (3 mm\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mm). Species identification was performed by Professor SP Li, one of corresponding authors. Voucher specimens of these samples were deposited at the Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China.\u003c/p\u003e \u003cp\u003eABTS, potassium persulfate and ascorbic acid (\u0026ge;\u0026thinsp;99%) were purchased from International Laboratory (San Bruno, CA), Fluka (Selzer, Germany) and Aladdin (Shanghai, China), respectively. Polygalacturonic acid (PGA), galacturonic acid (GA), konjac glucomannan (KG), dextran (DEX), pectinase (EC 3.2.1.15), endo-1,4-β-D-mannanase (EC 3.2.1.78) and endo-1,4-β-D-galactanase (EC 3.2.1.89) were purchased from Megazyme (Wicklow, Ireland). 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Griess reagent, lipopolysaccharides (LPS) and fluorescein isothiocyanate-dextran (FITC-Dextran) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Endotoxin detection specific limulus test kit was purchased from Bioendo TEShnology (Xiamen, China). Cell counting kit 8 (CCK8) was purchased from MedChemExpress. Phosphate-buffered saline (PBS), Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin (P/S) were purchased from Gibco-Invitrogen (Paisley, Scotland, UK). Nylon membrane filters (0.22/0.45 \u0026micro;m) were purchased from Millipore (Billerica, MA). Deionized water was prepared using a Millipore MilliQ-Plus system (Millipore, Billerica, MA). All the other reagents were of analytical grade.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eP. cyrtonema\u003c/span\u003e \u003cb\u003eand polysaccharides\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eSteam of P. cyrtonema\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. cyrtonema\u003c/em\u003e slices were steamed in an autoclave at 115 ℃ for 2 h (0.07 MPa) each time, followed by vacuum drying to be collected as the sample named as PC1. Afterwards, PC1 was further steamed and dried under the same condition to obtain the second sample named as PC2. Similarly, PC3, PC4 and PC5 were prepared, respectively. All those samples included the raw material without steaming (PC0) were grounded and sieved.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eExtraction of the polysaccharides\u003c/h2\u003e \u003cp\u003eEach sample (PC0-PC5) of 50.0 g was soaked at room temperature for 2 h, followed by hot water extraction (95 \u0026ordm;C) for further 2 h, material-to-liquid ratio was 1:15. The water extracts were precipitated with ethanol at final concentration of 75% to harvest polysaccharides. The supernatant (PCS) and precipitate were collected. Then small molecular weight substances (less than 3 KDa) were removed after ultrafiltration. The polysaccharides were dried with freeze-drier to obtain PCP0, PCP1, PCP2, PCP3, PCP4 and PCP5, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSample pretreatment of PCP\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003ePartial and complete acid hydrolysis of PCP\u003c/h2\u003e \u003cp\u003eAccording to a previously reported method with minor modification [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], PCP solution (2 mg/mL) was treated with trifluoroacetic acid (TFA) at a final concentration of 1.0 mol/L and incubated at 80\u0026deg;C for 2 h to gather partial acid hydrolysates (PAH). At the same time, the PCP solution (4 mg/mL) of each sample was mixed with equal volume of 4 mol/L TFA for complete acid hydrolysis at 105 \u0026ordm;C for 4 h to gather complete acid hydrolysates (CAH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eEnzymatic hydrolysis of PCP\u003c/h2\u003e \u003cp\u003eThree enzymes of pectinase, β-1,4-galactanase and β-1,4-mannanase were selected to depolymerize PCP (2 mg/mL) at 40\u0026deg;C for 12 h. After incubation, enzymes were inactivated at 80\u0026deg;C for 20 min. As for PACE analysis, the enzymatic PCP hydrolysates should be freeze-dried and derivatized with ANTS. While no additional treatment is required for HPTLC and monosaccharides analysis. Polysaccharide standards including PGA, GA and KG were treated with those enzymes, respectively, under the same conditions. PCP solution without TFA or enzymes treatment was used as blank control.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePhysicochemical characterization of polysaccharides\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eMolecular weights and chain conformation analysis\u003c/h2\u003e \u003cp\u003eThe molecular weights and their distribution of PCP were determined by HPSEC-MALLS-RID according to our previous report [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. HPSEC-MALLS-RID detection method is composed of multi-angle light scattering (MALS) detectors (Wyatt Technology Co., Santa Barbara, CA, USA), Agilent 1260 series LC/DAD system (Agilent Technologies, Palo Alto, CA, USA) and a refractometer (RID, Optilab rEX, Wyatt Technology Co.) in series at 35 \u0026ordm;C. The chromatographic columns are TOSOH gel columns TSK-GEL G5000PWXL (300 mm \u0026times; 7.8 mm) and TSK-GEL G3000PWXL (300 mm \u0026times; 7.8 mm), and the mobile phase is 0.9% NaCl solution. The flow rate is 0.5 mL/min with 100 \u0026micro;L injection volume. All the PCP were dissolved in mobile phase at 2 mg/mL and filtered through 0.45 \u0026micro;m filter membrane before injection. ASTRA 7.3.2 software was used to process the data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eCompositional monosaccharides analysis\u003c/h2\u003e \u003cp\u003eThe complete acid and pectinase hydrolyzed samples as well as PCS were analyzed by HPAEC-PAD system (Thermo Scientific\u0026trade; Dionex\u0026trade; ICS-5000\u003csup\u003e+\u003c/sup\u003e, Dionex, USA). Ten monosaccharide standards including Fuc, Ara, Rha, Gal, Glc, Xyl, Man, Fru, GalA and GlcA were used to calculate the content of each monosaccharide in the samples. All the samples were filtered through 0.45 \u0026micro;m membrane before analysis. The mobile phase consisted of 88% deionized water and 12% 10mM NaOH, running for 22 minutes at a flow rate of 0.4mL/min under a CarboPac PA200 (3 mm \u0026times; 250 mm) analytical column with a system temperature of 25\u0026deg;C (Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003ePACE analysis of partial acid and enzymatic hydrolysates\u003c/h2\u003e \u003cp\u003ePACE was performed according to previous report [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Briefly, all lyophilized derivatized hydrolysates of PCP were redissolved in isovolumetric urea (6 mol/L), and separated by Mini-Protean Tetra System, a vertical slab gel electrophoresis apparatus from Bio-Rad. Gels were imaged using an In-Genius LHR CCD camera system (Syngene, Cambridge, UK) under UV 365 nm. Quantity-One software (Ver4.6.2, BioRad) and Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Matlab version, Ver1.315, developed by the Research Center of Modernization of Chinese Herbal Medicine, Central South University and the Hong Kong Polytechnic University) were used for similarity analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eHPTLC analysis of complete acid and pectinase hydrolysates\u003c/h2\u003e \u003cp\u003eMerck silica gel 60 plates pre-washed with methanol were used for HPTLC analysis. The method was modified according to previous report [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In brief, the PCP0-PCP5 samples after complete acid and pectinase hydrolysis were prepared into 8 mg/mL, respectively. SP-III electric thin-layer strip sampler (KEZHE SHANGHAI, China) was used for semi-automatic sampling. The bands were 7 mm wide, 5 mm distance, and 10 mm from the bottom edge. Then the plate was firstly developed to 90 mm with 1-butanol/isopropanol/acedic acid/water, 7:5:1:2 (v/v/v/v) as developing reagent at room temperature. Then the plate was dried and placed in the same chamber to develop 95 mm with the same developing reagent as described above. Finally, the developed plates were dried and colorized with aniline-diphenylamine-phosphoric acid solution, 10% sulfuric acid ethanol solution and 0.2% ninhydrin solution, respectively, then heated at 105 \u0026ordm;C and photographed under white light.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eAFM analysis\u003c/h2\u003e \u003cp\u003ePCP0-PCP5 (1 mg/mL) were fully dissolved in ultrapure water and diluted to a concentration of 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e \u0026micro;g/mL. Using droplet deposition method, pipette 5\u0026micro;L of solution onto the surface of newly cut mica sheet and dry it at room temperature. After the sample was dry, used BioScope Resolve AFM (Bruker Co., Santa Barbara, USA) for AFM measurement. NanoscopeAnalysis 1.8 software was used for image analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidation of PCP against ABTS radicals\u003c/h2\u003e \u003cp\u003eIn brief, 7 mmol/L ABTS aqueous solution and 2.5 mmol/L potassium persulfate aqueous solution were mixed in a ratio of 1:1, and then stand in the dark for 12 hours. This solution was diluted with deionized water to reach a 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 absorbance value at 734 nm and obtained the ABTS working solution. PCP0-PCP5 solution (0.5, 1, 2, 4, 8 mg/mL) and ascorbic acid (0.015, 0.03, 0.06, 0.12, 0.24, 0.5, 1, 2, 4, 8 mg/mL) were prepared. In a 96-well plate, 200 \u0026micro;L of ABTS working solution and 10 \u0026micro;L of the sample solution were added to each well, and the reaction was kept in the dark for 6 min. After the reaction, the absorbance was measured, and the ABTS clearance rate formula was as followed:\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003eC (%) = [1-(A\u003csub\u003e1\u003c/sub\u003e-A\u003csub\u003e2\u003c/sub\u003e)/A\u003csub\u003e0\u003c/sub\u003e] \u0026times;100\u003c/h2\u003e \u003cp\u003eC was the clearance rate, A\u003csub\u003e0\u003c/sub\u003e is the control absorbance, A\u003csub\u003e1\u003c/sub\u003e is the sample absorbance, and A\u003csub\u003e2\u003c/sub\u003e was the background absorbance, which was to eliminate the interference of tested solution.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffects of PCP on macrophage functions\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eRAW 264.7 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Dulbecco\u0026rsquo;s modified eagle medium supplemented with 10% FBS, 1% P/S at 37\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eCytotoxicity assay\u003c/h2\u003e \u003cp\u003eRAW 264.7 cells (5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well) were cultured in 96-well microplates overnight, and then treated with LPS (0.4 \u0026micro;g/mL) and a series of concentrations of PCP for 24 h, respectively. Equal volume of culture medium was used as blank control. Subsequently, the original culture medium was discarded and stained with 100 \u0026micro;L of culture medium containing 10% CCK8 for 4 h in dark. The absorbance values were read at 450 nm and the cell viability was calculated as the ratio of absorbance values between sample and vehicle control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003eNitric oxide determination\u003c/h2\u003e \u003cp\u003eRAW 264.7 cells (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in 96-well microplates overnight, and then cells were treated with a series of concentrations of PCP and LPS (0.4 \u0026micro;g/mL) for 24 h, respectively. Equal volume of culture medium was used as vehicle control. Subsequently, 75 \u0026micro;L of supernatants were collected and mixed with an equal volume of modified Griess reagent at room temperature for 15 min. The absorbance was measured at 540 nm. NO production was expressed as ratio of absorbance values between sample and LPS treated group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003ePhagocytic activity test\u003c/h2\u003e \u003cp\u003eFITC-dextran was used for phagocytic assay. RAW 264.7 cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured in 24-well plates overnight and then incubated with culture medium, LPS (0.4 \u0026micro;g/mL) and a series of concentrations of PCP for 18 h, respectively. Then the cells were treated with FITC-dextran (0.1 mg/mL in culture medium) and incubated at 37\u0026deg;C for additional 1 h in dark. After incubation, the cells were collected with cold PBS after washed for three times. BD Accuri\u0026trade; C6 Cytometer (BD Biosciences, San Jose, CA, USA) was used to analyze. The percentage of phagocytosis was expressed as ratio of phagocytic rate between treatment and control cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of endotoxin contamination\u003c/h2\u003e \u003cp\u003eEndotoxin detection specific limulus test kit was used for avoiding endotoxin contamination. The results showed that PCP0-PCP5 had no endotoxin contamination in the samples.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8.0.2 was used to analyze and process the data. Data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from at least three independent experiments for each sample. Statistical significance between the experimental groups was determined by Student\u0026rsquo;s t-test, and \u003cem\u003ep\u003c/em\u003e values less than 0.05 were considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e \u003cb\u003eAppearance of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eP. cyrtonema\u003c/span\u003e \u003cb\u003eand its polysaccharides\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter steam treatment, the color of \u003cem\u003eP. cyrtonema\u003c/em\u003e slices deepened gradually. The raw material (PC0) was milky white, after first steam the color turned reddish brown with lighter color in center (PC1). Then, for the second time of steam treatment, slice PC2 changed to dark brown. Slice PC3 changed into black and began to appear luster, the color of PC4 and PC5 almost no change. The color of the polysaccharide powders was also changed accordingly, similar to the raw materials (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Traditionally, the number of steam treatment according to the color of materials. The color of PC5, same to those of PC3 and PC4, was black. Therefore, 5 times were selected for the steam of \u003cem\u003eP. cyrtonema\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eMolecular weight distribution of PCP\u003c/h2\u003e \u003cp\u003eHPSEC-MALLS-RID is a useful method for determining the absolute molecular weights (Mw), the dispersibility index (DPI) and radius of gyrations (Rg) of polysaccharides without standards [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e showed that HPSEC-MALLS-RID chromatograms of PCP in 0.9% NaCl aqueous solution at 35\u0026deg;C, and molecular mass distribution results of PCP0-PCP5 were summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. To facilitate the comparison of the effect of steaming treatment on the molecular weight distribution of PCP, the same peak division was performed on PCP1-PCP5 according to that of PCP0, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, which was divided into three peaks. In PCP0, Peak3 had the highest content and a molecular weight of ~\u0026thinsp;7.85\u0026times;10\u003csup\u003e3\u003c/sup\u003e Da, with Mw/Mn of 1.16. After the first steaming treatment, Mw of PCP1 increased dramatically to 1.01\u0026times;10\u003csup\u003e7\u003c/sup\u003e Da, and gradually decreased with the increase of steaming times (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This indicated that steam could change the extraction and/or molecular weight distribution of PCP. In addition, the detection at UV 280 nm showed that PCP0 had a very low signal, but steamed PCP showed obviously high signals. With the increase of steaming times, the peak height of protein signal increased firstly, PCP2 to the highest, and then decreased gradually (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This implied that steaming treatment induced Maillard reaction of polysaccharides in \u003cem\u003eP. cyrtonema\u003c/em\u003e. And it gradually degraded since the third steaming treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe molecular weight, polydispersity index (Mw/Mn) and contents of PCP0-PCP5\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"14\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003ePeak1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003ePeak2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c13\" namest=\"c10\"\u003e \u003cp\u003ePeak3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal Content\u003c/p\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMw, kDa\u003c/p\u003e \u003cp\u003e(error%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMw/Mn (error%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRz, nm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eContent%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMw, kDa\u003c/p\u003e \u003cp\u003e(error%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMw/Mn (error%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRz, nm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eContent%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMw, kDa\u003c/p\u003e \u003cp\u003e(error%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eMw/Mn (error%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eRz, nm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eContent%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCP0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.08\u0026times;10\u003csup\u003e4\u003c/sup\u003e(\u0026plusmn;\u0026thinsp;2.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.43(\u0026plusmn;\u0026thinsp;5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e56.7 (\u0026plusmn;\u0026thinsp;4.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e70.4 (\u0026plusmn;\u0026thinsp;2.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e3.64(\u0026plusmn;\u0026thinsp;5.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e21.9 (\u0026plusmn;\u0026thinsp;18.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e20.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e7.85 (\u0026plusmn;\u0026thinsp;2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e1.16(\u0026plusmn;\u0026thinsp;6.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e17.9 (\u0026plusmn;\u0026thinsp;29.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e49.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e70.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCP1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.01\u0026times;10\u003csup\u003e4\u003c/sup\u003e(\u0026plusmn;\u0026thinsp;3.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.70(\u0026plusmn;\u0026thinsp;6.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e65.8 (\u0026plusmn;\u0026thinsp;4.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e173 (\u0026plusmn;\u0026thinsp;2.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e3.55(\u0026plusmn;\u0026thinsp;6.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e34.4 (\u0026plusmn;\u0026thinsp;9.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e48.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e102 (\u0026plusmn;\u0026thinsp;3.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e1.18(\u0026plusmn;\u0026thinsp;7.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e39.2 (\u0026plusmn;\u0026thinsp;8.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e62.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCP2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e5.87\u0026times;10\u003csup\u003e4\u003c/sup\u003e(\u0026plusmn;\u0026thinsp;4.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.44(\u0026plusmn;\u0026thinsp;5.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e52.0 (\u0026plusmn;\u0026thinsp;6.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e381 (\u0026plusmn;\u0026thinsp;3.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e3.15(\u0026plusmn;\u0026thinsp;4.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e29.0 (\u0026plusmn;\u0026thinsp;15.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e54.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e146 (\u0026plusmn;\u0026thinsp;4.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e1.18(\u0026plusmn;\u0026thinsp;5.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e31.5 (\u0026plusmn;\u0026thinsp;15.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e23.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e78.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCP 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e9.28\u0026times;10\u003csup\u003e4\u003c/sup\u003e(\u0026plusmn;\u0026thinsp;4.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.88(\u0026plusmn;\u0026thinsp;5.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e42.3 (\u0026plusmn;\u0026thinsp;10.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e788 (\u0026plusmn;\u0026thinsp;3.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e2.15(\u0026plusmn;\u0026thinsp;5.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e37.2 (\u0026plusmn;\u0026thinsp;9.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e41.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e169 (\u0026plusmn;\u0026thinsp;3.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e1.18(\u0026plusmn;\u0026thinsp;6.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e39.7 (\u0026plusmn;\u0026thinsp;9.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e28.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e70.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCP 4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e7.93\u0026times;10\u003csup\u003e4\u003c/sup\u003e(\u0026plusmn;\u0026thinsp;4.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.84(\u0026plusmn;\u0026thinsp;4.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e50.3 (\u0026plusmn;\u0026thinsp;7.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e408 (\u0026plusmn;\u0026thinsp;4.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.85(\u0026plusmn;\u0026thinsp;4.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e30.7 (\u0026plusmn;\u0026thinsp;16.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e40.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e798 (\u0026plusmn;\u0026thinsp;5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e1.22(\u0026plusmn;\u0026thinsp;4.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e33.4 (\u0026plusmn;\u0026thinsp;16.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e34.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e75.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCP 5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e2.92\u0026times;10\u003csup\u003e4\u003c/sup\u003e(\u0026plusmn;\u0026thinsp;3.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.16(\u0026plusmn;\u0026thinsp;4.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e44.7 (\u0026plusmn;\u0026thinsp;7.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e206 (\u0026plusmn;\u0026thinsp;3.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e2.59(\u0026plusmn;\u0026thinsp;3.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e23.0 (\u0026plusmn;\u0026thinsp;24.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e32.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e364 (\u0026plusmn;\u0026thinsp;4.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e1.06(\u0026plusmn;\u0026thinsp;3.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e22.2 (\u0026plusmn;\u0026thinsp;32.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e36.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e69.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eMonosaccharide composition\u003c/h2\u003e \u003cp\u003eAfter complete acid hydrolysis, monosaccharide composition of PCP was determined by HPAEC-PAD (Supplementary Fig. S2). Results showed that PCP without steam treatment was mainly composed of GalA, Man and Glc. After steaming, all PCP were mainly composed of Gal, Man and GalA, with small amount of Ara, Rha and Glc. The molar ratio of Gal, GalA, Man, Rha, Ara and Glc in PCP0 was 3:6:16:1:3:8, and PCP1-PCP5 were 7.3:5:6:1:2.4:1, 7.9:3.1:4.3:1:1.6:0.6, 7.4:2.6:2.7:1:0.8:0.4, 6:2.1:2.6:1:0.6:0.5 and 5.9:1.7:2.6:1:0.3:0.5, respectively (Supplementary Table\u0026nbsp;2). Obviously, the monosaccharide composition of PCPs changed dramatically after the first steaming. The content of Man and Glc decreased significantly while the content of Gal increased obviously. As the steaming times increased, the content of Man and Glc remained stable starting from PCP3 while Gal decreased in PCP4 and PCP5. At the same time, GalA and Ara also decreased. According to previous studies [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], PCP contained mainly Man, Glc, Gal and Ara, which is consistent with our results. Interestingly, according to the study by Li et al.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Glc and Ara increased with the steam treatment which was inconsistent with our research results, which might attribute to different samples and/or steaming conditions.\u003c/p\u003e \u003cp\u003eBesides, previous studies have shown that PCP contain a lot of fructose [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], while the traditional complete acid hydrolysis method can cause fructose loss. Therefore, we also degraded the samples with pectinase and observed changes in monosaccharide composition. The results showed that the molar ratio of Gal, GalA, Man, Rha, Ara, Glc and Fru in PCP0-PCP5 was 7.5:35:7.9:1:7.9:7.7:55.4, 11.5:16.9:2.6:1:3.8:0.6:0.8, 14:10.7:2.1:1:2.7:0.6:0.3, 11.1:7.8:1.5:1:1.1:0.6:0.2, 9.6:6.2:1.7:1:0.7:0.6:0.2 and 8.3:4.6:1.8:1:0.4:0.3:0.1, respectively (Supplementary Table\u0026nbsp;3). The result of pectinase hydrolysis was similar to that of complete acid hydrolysis, except the content of Fru which was very different. The content of Fru was decreased dramatically after the first steam treatment, and almost disappear in PCP1-PCP5. This indicated that the steam processing changes the polysaccharide structure and lose a large amount of Fru.\u003c/p\u003e \u003cp\u003eAt the same time, we examined changes in Fru and Glc in 75% ethanol supernatant during ethanol precipitation, and the contents of Fru and Glc in the supernatant increased with steaming. The results showed that the composition of polysaccharide changed with the increase of steaming times, and the contents of monosaccharides such as fructose and glucose or oligosaccharides in free state increased. As the polysaccharide is relatively stable under the condition of normal temperature, however, under the steaming condition of high pressure and high temperature, various monosaccharide components can undergo dehydration and degradation, and Maillard reaction [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] can occur under the common existence of other components such as amino acids. Polysaccharides are composed of various monosaccharides that are degraded during processing and undergo further reaction changes that alter the monosaccharide composition of polysaccharides.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003ePACE profiles of PCP\u003c/h2\u003e \u003cp\u003eSaccharide mapping based on PACE has been proven to be one of powerful methods for the routine analysis of oligosaccharides derived from polysaccharides [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, both partial acid hydrolysates and enzymatic hydrolysates of PCP were compared using saccharide mapping based on PACE analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and their similarity was conducted using Quantity-One software and Similarity Evaluation System. The HPSEC-MALLS-RID chromatogram and ion chromatogram of PCP0-PCP5 after treat with pectinase, β-1,4-Galactanase, β-1,4-Mannanase or TFA were used to detect glycoside bond variations and sugar composition to determine glycoside bonds with significant differences in the steaming process (Supplementary Figs.\u0026nbsp;3 \u0026amp; 4) Their hydrolysates had high similarity except the partial acid hydrolysates of PCP0 and the β-1,4-manannase hydrolysates of PCP1 and PCP2 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, the hydrolysates obtained by different hydrolysis methods showed different similarity after steaming, especially the β-1,4-galactanase hydrolysates of PCP. PACE fingerprints of pectinase hydrolysates from PCP showed that the content of GalA in PCP0 was lower than in other PCP. The fingerprints of β-1,4-galactanase hydrolysates indicated that no β-1,4-Gal\u003cem\u003ep\u003c/em\u003e were detected in PCP0. However, after steaming treatment, β-1,4-Gal\u003cem\u003ep\u003c/em\u003e appeared in PCP1-PCP5. In the case of β-1,4-manannase hydrolysates, it indicated that PCP contained small number of polysaccharides with β-1,4-Man\u003cem\u003ep\u003c/em\u003e, and after steaming, they could be easily extracted, and the level of β-1,4-Man\u003cem\u003ep\u003c/em\u003e in PCP increased. For the partial acid hydrolysates analysis, PCP0 showed an obviously different PACE fingerprints compared with other PCP. All these results indicated that steaming treatment had a huge impact on PCP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe correlation coefficient of PCPs to their simulative mean chromatogram.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003eThe simulative mean chromatograms\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003ePACE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eHPTLC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSMC-PAH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSMC-GA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSMC-MA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSMC-PE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSMC-PE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSMC-CAH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCP0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eHPTLC fingerprints of PCP\u003c/h2\u003e \u003cp\u003eHPTLC showed the samples of complete acid hydrolysis and pectin-decomposing PCP had verified the experimental results of monosaccharide composition to some extent. According to the results of phenylamine-phosphoric acid coloration, pectin enzymatic samples contain more oligosaccharides than completely hydrolyzed samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and according to 10% ethanol sulfuric acid colorization, the complete acid hydrolysis sample of PCP0 might contain little of small molecular substances. In other samples, bands of monosaccharides and oligosaccharides can be clearly observed. Colorization of ninhydrin coloration showed that there almost were no amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). HPTLC profiles similarity was shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, which indicated that no significant difference between pectinase and complete acid hydrolysates of PCP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eMorphology of PCP\u003c/h2\u003e \u003cp\u003eThe biological activity of natural polysaccharide is also closely related to its chain conformation besides molecular weight [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, it is very important to study the chain conformation of PCP in aqueous solution to understand the effect of steam treatment on its structure and biological activity. The conformation of polysaccharide can be analyzed according to the theory of dilute polymer solution. Generally, the chain conformation of polysaccharides in aqueous solution is determined by the double logarithmic plot of Rg \u003cem\u003evs\u003c/em\u003e the molecular mass of polysaccharides according to Mark-Houwink equation Rg\u0026thinsp;=\u0026thinsp;kMw\u003csup\u003eν\u003c/sup\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. According to the polymer solution theory, the exponent (\u003cem\u003ev\u003c/em\u003e) is 0.2\u0026ndash;0.4 for branched polymers with a compact helical chain conformation, 0.3 for spheres, 0.5\u0026ndash;0.6 for flexible polymers in good solvents and 0.6-1.0 for semi-flexible chains [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. According to the calculation results of HPSEC-MALLS/RI, the \u003cem\u003ev\u003c/em\u003e index of PCP0-PCP5 was concentrated between 0 and 0.3 (Supplementary Fig. S1). The results showed that PCP0-PCP5 appeared as irregular monodisperse spheres in 0.9% aqueous sodium chloride solution.\u003c/p\u003e \u003cp\u003eAtomic force microscopy (AFM) has become a powerful tool to directly characterize the structure and properties of polymers [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The planar images with height and diameter (scanned at 3\u0026times;3 \u0026micro;m) of PCP0-PCP5 in aqueous solution obtained by AFM were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. An irregular monodisperse spherical shape of all PCP were observed, consistent with the results of HPSEC-MALLS-RID. Their molecular height was in the range of 1 to 1.5 nm, and the diameter was ranged from 15 to 20 nm. Specifically, with the steaming times increased, the height of PCP increased, and the diameter decreased gradually. There was almost no significant difference after the second steaming, except PCP4 with the height and diameter of 1.4 nm and 20.0 nm, respectively. This is likely to be related to the change of polysaccharides structure and/or fractions caused by steaming.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003eABTS scavenge ability of PCP\u003c/h2\u003e \u003cp\u003eABTS scavenge ability of PCP was shown in Fig.\u0026nbsp;6A, and all PCP showed scavenge ability against ABTS radicals in different extents, and PCP0 showed the lowest capacity. Generally, steamed PCP had higher dose-dependent free radical scavenge ability, and IC\u003csub\u003e50\u003c/sub\u003e values of PCP1-PCP5 were 4.89, 1.81, 1.79, 2.21, 3.04 mg/mL, respectively. Change of steamed PCP in antioxidant capacity may attribute to Maillard reaction of polysaccharides during steam processing [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], which was supported by UV 280nm absorption and molecular weights increased after steaming (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Though the ability of PCP scavenging ABTS radicals increased with the number of steaming times [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], IC\u003csub\u003e50\u003c/sub\u003e showed that antioxidant activity of PCP3 reached to the strongest, and then decreased with the following steam treatment. The variation might be due to the different steam treatment conditions. Anyway, steaming significantly enhances antioxidant capacity of PCP, which is beneficial to its efficacy in delaying aging [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], lowering blood sugar [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and regulating blood lipids [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The significance of steaming to health beneficial effects \u003cem\u003eP. cyrtonema\u003c/em\u003e should be further well investigated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eImmunostimulatory activity of PCP\u003c/h2\u003e \u003cp\u003eMacrophages play an indispensable role in the innate and adaptive immunity of the human body [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Studies have shown that high levels of NO are associated with immune responses during antitumor and antiviral processes, which can trigger cell proliferation, apoptosis, signal transduction, immune defense and other physiological processes [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Phagocytosis is a basic cellular process that plays an important role in the immune system [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In this study, RAW 264.7 cells were treated with a series of concentrations of PCP and their effects on NO production and phagocytic activity were investigated. Effects of PCP on NO production of macrophages were shown in Fig.\u0026nbsp;6C, though viability of RAW 264.7 cells was not significantly affected in the ranges of investigated concentration (Fig.\u0026nbsp;6B). Steaming reduced the effect of PCP on NO production of macrophages. As a result, effect of PCP0 was the best, while steamed PCP was reduced with increasing steam times, PCP3-PCP4 were only effective at the highest concentration (200 \u0026micro;g/mL), and PCP5 showed no such effect.\u003c/p\u003e \u003cp\u003eFlow cytometry was used to determine the fluorescence intensity in cells after RAW 264.7 cells devoured FITC-dextran. The results showed that LPS (0.4 \u0026micro;g/mL) and PCP could promote the phagocytic activity of macrophages in a dose-dependent manner (Fig.\u0026nbsp;6D). With the increase of steam times, their ability on phagocytosis was weakened, and after the third steam treatment (PCP3-PCP5), they showed no effect on FITC-dextran phagocytose compared with that of blank control group.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eSteaming treatment significantly influenced the physicochemical properties and bioactivities of polysaccharides from \u003cem\u003eP. cyrtonema\u003c/em\u003e, one of the origins of \u003cem\u003eHuangJing\u003c/em\u003e considered as well-known tonic herb. In brief, steaming could significantly increase the molecular weights, UV absorption and antioxidant activity of polysaccharides from \u003cem\u003eP. cyrtonema\u003c/em\u003e. Polysaccharides with glycosidic linkages such as β-1,4-Gal\u003cem\u003ep\u003c/em\u003e and β-1,4-man\u003cem\u003ep\u003c/em\u003e obviously increased after steaming, but fructan could be completely degraded. In addition, steaming could signification decrease the immunopotentiation activity, such as NO release and phagocytosis of RAW 264.7 cells of polysaccharides from \u003cem\u003eP. cyrtonema\u003c/em\u003e. Considering the conflict results of steaming on antioxidant and immunopotentiation activities of PCP, 2 times of continuously steam is the optimal choice. However, further study is still necessary to well understand the beneficial effect of steaming on \u003cem\u003eHuangJing\u003c/em\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAFM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAtomic Force Microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eABTS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epotassium persulfate and ascorbic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANTS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e8-aminonaphthalene-1,3,6-trisulfonic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAra\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArabinose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eComplete Acid Hydrolysates\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCK8\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCell counting kit 8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChinese Materia Medica\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDextran\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco's Modified Eagle Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDimethyl Sulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003edn/dc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erefractive index increment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDispersibility Index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal Bovine Serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal Bovine Serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFITC-Dextran\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluorescein Isothiocyanate-Dextran\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFT-IR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFourier Transform Infrared Spectroscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFuc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFucose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGal\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGalactose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGalacturonic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGas Chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGlc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGlcA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucoronic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPAEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh Performance Anion Exchange Chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh Performance Liquid Chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPSEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh Performance Size Exclusion Chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPTLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh Performance Thin Layer Chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKonjac Glucomannan\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipopolysaccharides\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMALLS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMulti-angle Laser Light Scattering\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMan\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMannose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMw\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMolecular Weights\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMw/Mn\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolydispersity Index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNaOH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium Hydroxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNitric Oxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP/S\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePenicillin/Streptomycin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePACE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarbohydrate Gel Electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePartial Acid Hydrolysates\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered Saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e polysaccharides\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethe 75% ethanol supernatant of \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e polysaccharides\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolygalacturonic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRg\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRadius of Gyration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRha\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRhamnose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRID\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRefractive Index Detector\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSize Exclusion Chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard Error of Mean\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTraditional Chinese Medicine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTrifluoroacetic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThin Layer Chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUltraviolet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAscorbic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eXyl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eXylose.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article is compliance with ethical standard and does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available on request from the authors. The data that support the findings of this study are available from the corresponding author, Shao-ping Li, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConflicts of interest\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was partially funded by grants from the National Key R\u0026amp;D Program of China (2019YFC1711300), the Science and Technology Development Fund, Macau SAR (File no. 0017/2019/AKP), the Key-Area Research and Development Program of Guangdong Province (File no. 2020B1111110006) and the University of Macau (File no. MYRG2018-00083-ICMS /MYRG2019-00128-ICMS /CPG2021-00009-ICMS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPL and JZ designed the study and offered supervision, project administration and funding acquisition. ZRC conducted the experiments and statistical analyses, drafted the manuscript. BJZ, ZXC and WC helped carry out experiments. SPL and JQW reviewed and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll herbal materials were provided by Mr. Lei Li of Jinzhai Senfeng Agricultural Technology Development Co., Ltd., Anhui, China. The research was partially funded by grants from the National Key R\u0026amp;D Program of China (2019YFC1711300), the Science and Technology Development Fund, Macau SAR (File no. 0017/2019/AKP), the Key-Area Research and Development Program of Guangdong Province (File no. 2020B1111110006) and the University of Macau (File no. MYRG2018-00083-ICMS /MYRG2019-00128-ICMS /CPG2021-00009-ICMS). And the authors would like to thank the reviewers and also the authors of all references.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT author statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZhe-rui Chen:\u0026nbsp;\u003c/strong\u003eInvestigation, Formal analysis, Writing - Original Draft\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBao-jie Zhu:\u003c/strong\u003e Investigation, Formal analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZhi-xin Chen\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eInvestigation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWen Cao:\u0026nbsp;\u003c/strong\u003eInvestigation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJun-Qiao Wang:\u0026nbsp;\u003c/strong\u003eWriting - Review \u0026amp; Revision\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJing Zhao:\u0026nbsp;\u003c/strong\u003eConceptualization, Resources, Writing - Review \u0026amp; Revision, Supervision, Project administration, Funding acquisition\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShao-ping Li:\u0026nbsp;\u003c/strong\u003eConceptualization, Resources, Writing - Review \u0026amp; Revision, Supervision, Project administration, Funding acquisition\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:\u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDeng Y, He K, Ye X, Chen X, Huang J, Li X, \u003cem\u003eet al.\u003c/em\u003e Saponin rich fractions from \u003cem\u003ePolygonatum odoratum\u003c/em\u003e (Mill.) Druce with more potential hypoglycemic effects. J Ethnopharmacol. 2012;141(1):228 \u0026ndash; 33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi JX, Shang YX, Wang Y, Gao J, Xue N, Huang C, et al. Hypoglycemic and Hypolipidemic Activity of \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e Fermented with Lactobacillus brevis YM 1301 in Diabetic C57BL/6 Mice. J Med Food. 2021;24(7):720\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen ZB, Liu JJ, Kong X, Li H. Characterization and Immunological Activities of Polysaccharides from \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e. Biol Pharm Bull. 2020;43(6):959\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui XW, Wang SY, Cao H, Guo H, Li YJ, Xu FX, et al. A Review: The Bioactivities and Pharmacological Applications of \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e polysaccharides. Molecules. 2018;23(5):12. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/molecules23051170\u003c/span\u003e\u003cspan address=\"10.3390/molecules23051170\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang WJ, Li S, Song MX. \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e polysaccharide inhibits high glucose-induced oxidative stress, inflammatory response, and apoptosis in RPE cells. J Recept Signal Transduct Res. 2021. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/1079989320211883061\u003c/span\u003e\u003cspan address=\"10.1080/1079989320211883061\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo JY, Chai YY, Zhao M, Guo QQ, Bao YH. Hypoglycemic effects and modulation of gut microbiota of diabetic mice by saponin from \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e. Food Funct. 2020;11(5):4327\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai JL, Zhu YL, Zuo YJ, Tong QZ, Zhang ZG, Yang L, et al. \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e polysaccharide alleviates inflammatory cytokines and promotes glucose uptake in high-glucose- and high-insulin-induced 3T3-L1 adipocytes by promoting Nrf2 expression. Mol Med Rep. 2019;20(4):3951\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng SY. Protective effect of \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e Polysaccharide on D-galactose-induced aging rats model. Sci Rep. 2020;10(1):2246. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598020590557\u003c/span\u003e\u003cspan address=\"10.1038/s41598020590557\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang YJ, Liu N, Xue X, Li Q, Sun DQ, Zhao ZX. Purification, structural characterization and in vivo immunoregulatory activity of a novel polysaccharide from \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e. Int J Biol Macromol. 2020;160:688\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun TT, Zhang H, Li Y, Liu Y, Dai W, Fang J, et al. Physicochemical properties and immunological activities of polysaccharides from both crude and wine-processed \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e. Int J Biol Macromol. 2020;143:255\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu ZG, Ni HY, Yu L, Xu SW, Bo RN, Qiu TX, et al. Adjuvant activities of CTAB-modified \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e polysaccharide cubosomes on immune responses to ovalbumin in mice. Int J Biol Macromol. 2020;148:793\u0026ndash;801.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang JZ, Liu N, Sun C, Sun DQ, Wang YJ. Polysaccharides from \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e Delar. ex Redoute induce an immune response in the RAW264.7 cell line via an NF-kappa B/MAPK pathway. RSC Adv. 2019;9(31):17988\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYelithao K, Surayot U, Park W, Lee S, Lee DH, You S. Effect of sulfation and partial hydrolysis of polysaccharides from \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e on immune-enhancement. Int J Biol Macromol. 2019;122:10\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi B, Wu PP, Fu WW, Xiong Y, Zhang L, Gao YB, et al. The Role and Mechanism of miRNA-1224 in the \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e Polysaccharide Regulation of Bone Marrow-Derived Macrophages to Osteoclast Differentiation. Rejuvenation Res. 2019;22(5):420\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Z, Liang Z, Chan K, Lu G, Lee EL, Chen H, et al. A unique issue in the standardization of Chinese materia medica: processing. Planta Med. 2010;76(17):1975\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng X, Ji H, Cheng X, Wang D, Li T, Ren K, et al. Characterization, Classification, and Authentication of \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e Samples by Volatile Profiles and Flavor Properties. Molecules. 2021;27(1):18. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/molecules27010025\u003c/span\u003e\u003cspan address=\"10.3390/molecules27010025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu WJ, Huang NW, Huang JP, Wang LL, Wu LL, Wang Q, et al. Effects of the steaming process on the structural properties and immunological activities of polysaccharides from \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e. J Funct Foods. 2022;88:104866.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Zeng J, Gong P, Wu Y, Li H. Effect of steaming process on the structural characteristics and antioxidant activities of polysaccharides from \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e rhizomes. Glycoconj J. 2021;38(5):561\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan BL, Wei GL, Gan XF, Li TT, Qu ZY, Xu S, et al. Study on the varied content of \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e polysaccharides in the processing of steaming and shining for nine times based on HPLC-MS/MS and chemometrics. Microchem J. 2020;159:105352.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin J, Lao J, Zhou RR, He W, Qin Y, Zhong C, et al. Simultaneous Identification and Dynamic Analysis of Saccharides during Steam Processing of Rhizomes of \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e by HPLC-QTOF-MS/MS. Molecules. 2018;23(11):14. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/molecules23112855\u003c/span\u003e\u003cspan address=\"10.3390/molecules23112855\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang CH, Yun YH, Fan W, Liang YZ, Yu Y, Tang WX. Rapid analysis of polysaccharides contents in Glycyrrhiza by near infrared spectroscopy and chemometrics. Int J Biol Macromol. 2015;79:983\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLv Y, Yang X, Zhao Y, Ruan Y, Yang Y, Wang Z. Separation and quantification of component monosaccharides of the tea polysaccharides from \u003cem\u003eGynostemma pentaphyllum\u003c/em\u003e by HPLC with indirect UV detection. Food Chem. 2009;112(3):742\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie PS, Leung AY. Understanding the traditional aspect of Chinese medicine in order to achieve meaningful quality control of Chinese materia medica. J Chromatogr A. 2009;1216(11):1933\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan J, Li SP. Discrimination of polysaccharides from traditional Chinese medicines using saccharide mapping-enzymatic digestion followed by chromatographic analysis. J Pharm Biomed Anal. 2010;51(3):590\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheong KL, Wu DT, Zhao J, Li SP. A rapid and accurate method for the quantitative estimation of natural polysaccharides and their fractions using high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector. J Chromatogr A. 2015;1400:98\u0026ndash;106.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu DT, Xie J, Hu DJ, Zhao J, Li SP. Characterization of polysaccharides from \u003cem\u003eGanoderma\u003c/em\u003e spp. using saccharide mapping. Carbohydr Polym. 2013;97(2):398\u0026ndash;405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu DT, Cheong KL, Wang LY, Lv GP, Ju YJ, Feng K, et al. Characterization and discrimination of polysaccharides from different species of \u003cem\u003eCordyceps\u003c/em\u003e using saccharide mapping based on PACE and HPTLC. Carbohydr Polym. 2014;103:100\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu DT, Meng LZ, Wang LY, Lv GP, Cheong KL, Hu DJ, et al. Chain conformation and immunomodulatory activity of a hyperbranched polysaccharide from \u003cem\u003eCordyceps\u003c/em\u003e sinensis. Carbohydr Polym. 2014;110:405\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu BJ, Yan ZY, Hong L, Li SP, Zhao J. Quality evaluation of \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e from different geographical origins in China based on qualitative and quantitative saccharide mapping and chemometrics. J Pharm Biomed Anal. 2020;191:113583.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu DT, Cheong KL, Deng Y, Lin PC, Wei F, Lv XJ, et al. Characterization and comparison of polysaccharides from \u003cem\u003eLycium barbarum\u003c/em\u003e in China using saccharide mapping based on PACE and HPTLC. Carbohydr Polym. 2015;134:12\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng Y, Li M, Chen LX, Chen XQ, Lu JH, Zhao J, et al. Chemical characterization and immunomodulatory activity of acetylated polysaccharides from \u003cem\u003eDendrobium devonianum\u003c/em\u003e. Carbohydr Polym. 2018;180:238\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYelithao K, Surayot U, Lee JH, You S. RAW264.7 Cell Activating Glucomannans Extracted from Rhizome of \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e. Prev Nutr Food Sci. 2016;21(3):245\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan BL, Wei GL, Gan XF, Li TT, Qu ZY, Xu S, et al Study on the varied content of P\u003cem\u003eolygonatum cyrtonema\u003c/em\u003e polysaccharides in the processing of steaming and shining for nine times based on HPLC-MS/MS and chemometrics. Microchemical Journal. 2020;159.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllis GP. \u003cem\u003eThe Maillard Reaction\u003c/em\u003e, in \u003cem\u003eAdvances in Carbohydrate Chemistry\u003c/em\u003e, M.L. Wolfrom, Editor. 1959, Academic Press. pp.\u0026nbsp;63\u0026ndash;134.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCasu B, Lindahl U, \u003cem\u003eStructure and biological interactions of heparin and heparan sulfate\u003c/em\u003e, in \u003cem\u003eAdvances in Carbohydrate Chemistry and Biochemistry\u003c/em\u003e. 2001, Academic Press. p.\u0026nbsp;159\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu W, Wang H, Yu J, Liu Y, Lu W, Chai Y, et al. Structure, chain conformation, and immunomodulatory activity of the polysaccharide purified from Bacillus Calmette Guerin formulation. Carbohydr Polym. 2016;150:149\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Ma Z, Zhang L, Fang Y, Jiang F, Phillips GO. Structure and chain conformation of water-soluble heteropolysaccharides from \u003cem\u003eGanoderma lucidum\u003c/em\u003e. Carbohydr Polym. 2011;86(2):844\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Nie S. Application of atomic force microscopy in microscopic analysis of polysaccharide. Trends Food Sci Technol. 2019;87:35\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrands CMJ, Alink GM, van Boekel M, Jongen WMF. Mutagenicity of heated sugar-Casein systems: Effect of the Maillard reaction. J Agric Food Chem. 2000;48(6):2271\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Lan CJ, Liao X, Chen D, Song WG, Zhang QL. \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e polysaccharide potentially attenuates diabetic retinal injury in a diabetic rat model. J Diabetes Investig. 2019;10(4):915\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Qin SC, Pen GQ, Chen D, Han C, Miao CR, et al. Potential ocular protection and dynamic observation of \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e polysaccharide against streptozocin-induced diabetic rats' model. Exp Biol Med (Maywood). 2017;242(1):92\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHao NB, L\u0026uuml; MH, Fan YH, Cao YL, Zhang ZR, Yang SM. Macrophages in tumor microenvironments and the progression of tumors. Clin Exp Immunol. 2012;2012:11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBosc\u0026aacute; L, Zeini M, Trav\u0026eacute;s PG, Hortelano S. Nitric oxide and cell viability in inflammatory cells: a role for NO in macrophage function and fate. Toxicology. 2005;208(2):249\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreenberg S, Grinstein S. Phagocytosis and innate immunity. Curr Opin Immunol. 2002;14(1):136\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Polygonatum cyrtonema, Polysaccharides, Steam, Chemical composition, Bioactivity","lastPublishedDoi":"10.21203/rs.3.rs-1645421/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1645421/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003ePolygonatum cyrtonema, \u003c/em\u003eone of origins of Polygonata Rhizoma (\u003cem\u003eHuangJing\u003c/em\u003e in Chinese),\u003cem\u003e \u003c/em\u003eis traditionally steamed repeatedly before being used as herbal medicine in China. However, there had no standardization of steaming of \u003cem\u003eHuangJing\u003c/em\u003e. Therefore, comprehensive study for effects of steam on polysaccharide from \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e based on saccharide mapping, a powerful method developed for polysaccharides analysis, and pharmacological activity are still necessary in order to explore the effect of steam on the physiochemical and biological activities of its polysaccharides and determine the standard for \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eTo explore the effect of steam on the physiochemical and biological activities of \u003cem\u003eP. cyrtonema\u003c/em\u003e polysaccharides (PCP), six polysaccharides named PCP0, PCP1, PCP2, PCP3, PCP4 and PCP5 were extracted from the herb consecutively steamed for 0 to 5 times, respectively. Their molecular weight distribution, monosaccharide composition and PACE fingerprints were investigated through HPSEC-MALLS-RID, HPAEC-PAD and saccharide mapping based on polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and HPTLC, respectively. In addition, their antioxidant ability and immunostimulatory activities on RAW 264.7 cells in term of NO production and phagocytosis were compared.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eResults suggested that molecular weights could be changed during steam, which increased by first steaming and then decreased with further steaming though all polysaccharides molecular weight were 10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e Da. They all showed irregularly spherical conformation in aqueous solution based on AFM imaging. Monosaccharide composition and PACE fingerprints was significantly different after steaming, i.e., galactose increased while glucose and mannose decreased, and β-1,4-Gal\u003cem\u003ep\u003c/em\u003e appeared while β-1,4-man\u003cem\u003ep\u003c/em\u003e increased after steaming. Steamed PCP significantly increased scavenging activity against ABTS radicals, while PCP0 had better immunostimulatory effect on RAW 264.7 in terms of NO production and phagocytosis.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eIn summary, steam significantly affected the chemical composition and bioactivities of polysaccharides from \u003cem\u003eP. cyrtonema\u003c/em\u003e. Considering the conflict results of steaming on antioxidant and immunopotentiation activities of PCP, 2 times of continuously steam is the optimal choice under the modern process condition.\u003c/p\u003e","manuscriptTitle":"Effects of steam on polysaccharides from Polygonatum cyrtonema based on saccharide mapping analysis and pharmacological activity assays","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-23 16:56:08","doi":"10.21203/rs.3.rs-1645421/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-06-19T00:19:06+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-06-18T23:50:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-05-16T09:38:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2022-05-11T06:43:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b85eea4d-5e94-4413-bdb1-4466e1e6783f","owner":[],"postedDate":"June 23rd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-08-01T01:39:33+00:00","versionOfRecord":[],"versionCreatedAt":"2022-06-23 16:56:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1645421","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1645421","identity":"rs-1645421","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}