Physicochemical characteristics and immunoregulatory activities of polysaccharides from five cultivars of Chrysanthemi Flos

Abstract This study compared the physicochemical characteristics and immunomodulatory activities of chrysanthemums’ polysaccharides (JPs) from five cultivars. Significant differences were found in the molecular weights, the ratios of monosaccharide compositions, and morphological properties. Polysaccharides of Gongju (GJP) had the lowest molecular weight populations and polysaccharides of Boju (BJP) had the highest. SEM showed that GJP and polysaccharides of Qiju had looser and uniform surface structures, which are beneficial for being developed into instant products. Immunoregulatory assay revealed that JPs enhanced the phagocytosis and proliferation of RAW264.7 cells without obvious cytotoxicity, and upregulated the release level of TNF‐α, IFN‐γ, and NO. Immune‐enhancing activity correlated with their molecular weights, the contents of glucuronic acid and arabinose, and microstructure, which performed differently according to different cultivars. The results suggested that BJP and polysaccharides of Hangbaiju are more suitable to be developed as new functional foods for enhancing immunity, and provided a reference for selection based on the immunization requirements.


| INTRODUC TI ON
Tea is the second largest consumed drink after water worldwide.
The flower head of the Chrysanthemum morifolium Ramat. is widely consumed as tea owing to the wonderful flavor, color, and acclaimed health benefits . Chrysanthemum is a functional food and herbal medicine to cure fever, sore throats, and headache (Liang et al., 2014;Lin et al., 2015). The small molecules, such as phenols, flavonoids, alkanes, terpenoids, and unsaturated fatty acids, are often in charge of its bioactivities, and the compositions are varied obviously from different cultivars, geographic variations, and harvest time of chrysanthemum Yuan et al., 2015). So far, present researches on chrysanthemum mainly focused on these small-molecule compounds and their bioactivities as well as extraction technology (Yuan et al., 2015). Five major cultivars in market circulation, including Hangbaiju (Zhejiang province), Boju and Gongju (Anhui province), Huaiju (Henan province), and Qiju (Hebei province), classified by their origins, are well known in Asia and Europe for more than two thousand years (Yuan et al., 2015).
Meanwhile, chrysanthemum is one of the most important economic crops in China which is currently the world's largest producer and exporter of chrysanthemum. It is widely consumed by producing tea, wine, cakes, porridge, condiment, and is used as a dietary supplement.
Polysaccharides are one of the dominant substances in hot water extracts, and the water-soluble polysaccharides are a group of active components promising immunocompetence with little toxicity (Yang et al., 2017). Natural carbohydrates are abundant in chrysanthemum, The inhibitory effects on α-amylase and α-glucosidase were 2.7 and 17.9 times that of acarbose, respectively. The study of digestion in vitro and fecal fermentation suggested that snow chrysanthemum polysaccharides (JHP) could be partially degraded under the gastrointestinal digestion and further consumed by gut microbiota. JHP could increase beneficial bacteria significantly (Wu et al., 2021). The primary therapeutic effect of chrysanthemum used in herbal medicine is heat-clearing and detoxifying, specifically anti-influenza virus, involving macrophage immunodeficiency. Previous reports showed that polysaccharides from Huaiju and Hangbaiju possess antitumor effect (Zheng et al., 2015), as well as polysaccharides from Boju can stimulate the proliferation of T and B lymphocyte via inducing the production of mitogen (Zheng et al., 2006). The bioactivities of polysaccharides can be significantly affected by their physicochemical properties, including molecular size, ratios of constituent monosaccharides, glycosidic linkages, and the chain conformations (Gong et al., 2015;Su et al., 2016). The physicochemical characteristics and the immune bioactivity of chrysanthemum's polysaccharides (JPs) may be influenced by cultivars and geographical environment, which are worthy of systematic comparative research. Meanwhile, selecting chrysanthemum plantations for better application in food and drug industry from the perspective of immunization requirements is very useful.
In this study, physicochemical properties and immunostimulatory activities of polysaccharides from five cultivars of chrysanthemum were compared. The effects of the five polysaccharides' water solution on RAW264.7 cells' reproductive capacity, phagocytosis, and release levels of inflammatory cytokines were tested.
These results provide detailed information for polysaccharides in chrysanthemums from different cultivars, including the variation in polysaccharides' physicochemical properties and immunostimulatory activities. This study would supply chrysanthemum's polysaccharides with high immunocompetence that can be potentially used in the manufacture of a new functional food and pharmaceutical.

| Plants and reagents
The fresh flower of Qiju was gathered from Anguo, Hebei province; Hangbaiju was gathered from Jiaxing, Zhejiang province; Boju was collected from Bozhou and Gongju was gathered from Huangshan, Anhui province; and Huaiju was collected from Jiaozuo, Henan province. The specimens had been identified by Professor Wenyuan Gao

| Extraction of polysaccharides from chrysanthemum
The polysaccharides were acquired on the basis of the previous method  with minor modification. The dried samples (50 g) were cut into pieces and refluxed with ethanol (500 ml, 75°C) twice for 4 h to remove liposoluble constituents and pigments, and then filtered. Each residue was refluxed for 4 h with ten times of deionized water at 75°C for twice. The filter liquor was obtained by suction filtration through a filter paper on a Buchner funnel and concentrated with a vacuum rotary evaporator at 60°C to 200 ml, followed by removing of the free proteins from the extracts using the Sevage reagent (chloroform:n-butanol = 4:1, v/v). After the organic solvent was removed, polysaccharides were collected by precipitating with 800 ml anhydrous ethanol and maintained overnight at 4°C. The precipitate was gathered by centrifugation (3,000 g, 10 min) and was redissolved and dialyzed against distilled water for 48 h with water change every 4 h (molecular weight cut-off 8-14 kDa). The solution was lyophilized to acquire samples of polysaccharides. The polysaccharides obtained from "Boju," "Gongju," "Huaiju," "Qiju," and "Hangbaiju" were named "BJP," "GJP," "HJP," "QJP," and "HBJP," respectively.

| Determination of polysaccharides and proteins
The total polysaccharides (TP) were measured using phenol-sulfuric acid method and glucose acted as standard substance to establish the standard curve. The protein content in the polysaccharides was measured using Coomassie brilliant blue method and BSA was used as standard (Zhao et al., 2017).

| UV-vis spectroscopy
UV-vis spectra of polysaccharides water solution were obtained via a Cary 60 UV-visible spectrophotometer (Agilent, USA) within 190-400 nm.

| Molecular weight evaluation
Molecular weights (Mw) were measured on the basis of the previous method with minor modification . An HPLC (Shimadzu, Kyoto, Japan) system with a gel-filtration chromatography column of TSK gel GMPW XL column (7.8 mm × 300 mm, Tosoh Corp., Japan) and an evaporative light scattering detector (ELSD) was used. The flow rate of carrier gas was controlled at 2.2 L/min and the temperature of drift tube was 70°C. The polysaccharides' water solution (5 mg/ml) were filtered through a 0.22μm filter film and 30 μl of samples was injected. The column temperature was controlled at 30°C, and each sample was eluted with ultrapure water (0.5 ml/min). The specification curve was constructed with sequentially increased Mw of dextrans (5, 12, 50, 150, 210, and 410 kDa).

| Monosaccharide constitution
Each sample (4 mg) was first dissolved in trifluoroacetic acid (2 ml, 2 M) and then hydrolyzation was carried out at 120°C for 6 h in a sealed ampoule, and dried by a vacuum rotary evaporator at 60°C. Methanol (2 ml) was added and codistillated to eliminate the unreacted trifluoroacetic acid, and this treatment was repeated three times. Subsequently, the hydrolysates (200 μl) and monosaccharide standards (200 μl, 2 mg/ml) were performed with derivatization procedure. NaOH (50 μl, 0.5 M) and methanolic solutions of PMP (50 μl, 0.5 M) were added, and maintained for 30 min at 70°C reducing sugar to sugar alcohol. After cooling, 50 μl of HCl (0.3 M) was added to neutralize the residual NaOH and ultrapure water of 1 ml was added. Chloroform (1 ml) was added to extract and abandon the excess PMP. Multiplicating this process, the water solution was passed through a 0.22μm syringe filter to analyze monosaccharide constitution, and a Shimadzu HPLC equipped with a photodiode array detector (PAD) was used. PMP-derived samples (10 μl) were applied to a kromasil 100-5 C18 column (4.6 mm × 250 mm, 5 μm) at 30°C and the UV monitor was set at 250 nm. Acetonitrile and ammonium acetate buffer (20 mM, pH 5.0) with a volume ratio of 22:78 were used as mobile phase and the flow rate was controlled at 1.0 ml/min. The monosaccharides were confirmed by the standards ( Figure S1) and the ratio was calculated by their peak areas.

| FTIR spectral analysis
The dry powder samples (2 mg) were ground and tablet with KBr (200 mg) was prepared. The FTIR spectrum of each sample was detected on a Tensor 27 spectrometer (Bruker Optics Inc.) and recorded from 4000 to 400 cm −1 .

| Morphological properties
The shape, size, and surface properties of JPs were recorded using a field emission SEM (NOVA Nanosem 430, FEI Company). The JPs were adhered to a specimen holder and sputtered with gold for 70 s at 20 mA. Each sample was scanned with an accelerating voltage at 20 kV.

| RAW264.7 cell viability assay
The MTT assay was performed to evaluate the RAW264.7 cell proliferation when treated with three concentrations of JPs water solution (12.5, 50, and 200 µg/ml) . RAW264.7 cells were preincubated in a 96-well microplate with 1 × 10 5 cells per well (100 µl) for 24 h and JPs water solution (100 µl) with three concentrations were used to treat. The positive control was treated with lipopolysaccharide (LPS, 50 µg/ml). After continuous culturing for 48 h, 50 µl of 2 mg/ml MTT was appended followed by another cultivation for 4 h. Dimethyl sulfoxide (100 µl) was appended to dissolve the precipitate after removing the medium. Cell viability was evaluated by determining the absorption at 570 nm using an Infinite M200 PRO spectrophotometer (Tecan Co.).

| Phagocytosis assay
The determination of cells' phagocytic ability was performed following the method of Neutral red uptake reported by Bi et al. (2018).
The positive control was treated with LPS (50 µg/ml) and the solvent control was treated with double-distilled water. The NO content in the supernatants was detected by the Griess reagent.

| Statistics
All experiments were carried out for three times. One-way analysis of variance (ANOVA) was carried out to test significant differences (p < .05) with S-N-K and LSD post hoc test was carried out to analyze univariate comparisons by IBM SPSS 20.0.

| RE SULTS AND D ISCUSS I ON
3.1 | The yield of the crude polysaccharides content, polysaccharides, and protein content It turned out that the yield of crude polysaccharides ranged from 6.43% (QJP) to 11.05% (HBJP), as shown in Table 1, which was higher than that of Yuan et al.'s (2019) study (3.2%-7.3%). The content of polysaccharides from JPs ranged from 51.45% to 60.47%, which was lower than 78.8%-89.2% (Yuan et al., 2019). BJP contained markedly higher level than others (p < .05). The existence of protein was confirmed by UV absorption peaks at 280 nm ( Figure 1a) and the content was determined by Coomassie brilliant blue, as well as the peak value was detected in GJP (7.48%). The absorption at 260 nm is characteristic for nucleic acid, implying that the JPs containing nucleic acid and the peak value were detected in GJP. Based on the yield of the crude polysaccharides content, total polysaccharides, and protein contents, HBJP and BJP can be recommended for chrysanthemum polysaccharide resource in the industrial manufacture.

| Molecular weights
The standard curve of Mw is shown in Figure S2. The polysaccharides extracted from chrysanthemums presented wide and dispersive molecular size distribution, as shown in Table 1 and Figure S3.

TA B L E 1
The yield, total polysaccharides, protein, molecular weight, and monosaccharide composition of BJP, GJP, HJP, QJP, and HBJP

| Monosaccharide analysis
The monosaccharide components in polysaccharides from diffident chrysanthemums were similar, which comprised Rha, Ara, GalA, Man, Glc, and Gal with various ratios (Figure 1b). The results showed that HJP and BJP were abundant in Glc (60.08% and 66.63%) ( a higher content of uronic acid might possess a higher bioactivities according to previous reports (Ma et al., 2013;Wang et al., 2019;Zhao et al., 2017). In addition to the gene, precipitation, light radiation, and temperature of the planting areas were different according to the geographic variations, which would influence the content of secondary metabolites and the polysaccharide accumulation . In this study, it was speculated that the monosaccharide compositions were influenced by environmental characteristics.

| FTIR
The FTIR spectra of five polysaccharides are shown in Figure 2, and they seem similar. The absorption peak centered at 3426 cm −1 corresponded to hydroxyl groups. The band centered at 2923 cm −1 corresponded to the C-H asymmetric extensional vibration of the CH 2 or CH 3 . The peak at 1749 cm −1 was assigned to the absorption of C = O, which indicated the presence of uronic acids (Cao et al., 2018). The peak at 1616 cm −1 was characteristic for the protein secondary structural components, meaning that conjugated proteins showed the presence of α-glycosidic linkages (Zhao et al., 2017).

| SEM
The morphological properties of JPs exhibited differences in shape, cracks. GJP owns the smallest particle size (1000×) and is made up of a uniform collection of particles, which was similar to the apparent structure of QJP. BJP possessed accumulated thin layer structure and small globular particles attach. The morphological properties of polysaccharides are important for the reconstitution of the powder products, which affect the solubility of the final products . Therefore, a smaller particle size, more uniform, and loose structure of GJP and QJP were beneficial when used in food and medicine industry than the compact configuration of HJP and HBJP.

| Immune-enhancing activity of JPs
Previous studies observed that polysaccharides administered orally can activate the immune system in the small intestine . The immunomodulatory effect of polysaccharides is mainly through the stimulation of multi-effector cells .

| RAW264.7 cell viability
Macrophages are considered to be of vital importance to innate immune response and mediate host defense (Du et al., 2018). In order to estimate the impact of five sources of JPs on the viability of RAW264.7 macrophages, MTT assays were performed at three different concentrations (12.5, 50, and 200 µg/ml). JPs at low concentration did not affect the cell viability remarkably as shown in

| Phagocytic capacity of RAW264.7 cell
Phagocytosis is one of the most basic defense systems in innate immunity, involved in interfering with the viral replication, distinguishing and phagocytosing foreign bodies, such as bacteria, viruses, damaged cells . The results of neutral red uptake test indicated that JPs ( and binding to various membrane receptors . Ara, Gal, and Rha contained in polysaccharides from Colocasia esculenta can be recognized by toll-like receptor-2 (TLR-2) and TLR-4 in previous study . Therefore, the high content of the sum of the three monosaccharides in HBJP made it own the best phagocytic capacity. Previous studies also reported that glucose contained in polysaccharides was recognized in scavenger receptor, glucocorticoid receptor, TLR-2, and TLR-4 (Chen et al., 2017). Therefore, a higher content of glucose in BJP and HJP was the dominant factor that led to a higher immune activity.

| Cytokine secretion of RAW264.7 cell
Cytokines, low molecular weight and soluble pleiotropic peptides or glycoproteins, can mediate the unleashing of an effective immune response, link innate and adaptive immunity, and influence the macrophage's microenvironment . TNFα, IFNγ, and NO are major proinflammatory cytokines that participate in inflammatory response and are in charge of antineoplastic, antiviral, and cytolytic functions . JPs can remarkably upregulate the secretion level of TNFα and IFNγ (p < .001), exhibiting an effective proinflammatory activity and the cytokine release level depending on the concentration of JPs (Figure 4c,d). The concentration of TNFα in RAW264.7 cells was determined as 0.40 ng/ml approximately at the low concentration of JPs and the highest reached 0.84 ng/ml at 200 µg/ml of HBJP. The peak value was higher than that of LPS. A higher released level of TNFα stimulated by HBJP was correlated to lower Mw, higher ratios of GalA and Ara (Cao et al., 2018;Fimbres et al., 2018). Compared with blank group, the secretion of IFNγ was also remarkably improved in RAW264.7 cells when treated with JPs (p < .001), and the peak value was obtained at 200 µg/ml of BJP. The others were in the following order roughly: NO is a signaling molecule related to macrophage cytolytic function, inflammatory response, and signal transduction regulation in the immune response (Yang et al., 2018;Zhang et al., 2012). The production level of NO in cells handled with JPs was also measured in this study. Results showed that treatment with a moderate and Previous studies showed that plant polysaccharides containing uronic acids were valid in activating macrophages, and improving the release of immune-related cytokines, as well as increasing the expression of immune-related genes (Cao et al., 2018;Tang et al., 2018).
According to the present results, the immune-enhancing activity of BJP and HBJP is more intensified than others. The immune activity is closely related to polysaccharides' physicochemical characteristics, for instance, Mw, water solubility, chemical compositions,  Wu et al., 2022). It was reported that arabinose contained in polysaccharides showed an immunomodulatory activity . developed into immune foods and pharmaceuticals, especially HBJP and BJP. This study provided an option of consumption based on the need of immunization. GJP and QJP are good candidates for exploring as a functional food additive due to the loose and uniform structure. In short, the present study has supplied the essential information about the variances in physicochemical characteristics and immunization of polysaccharides from five edible and medicinal chrysanthemums.

ACK N OWLED G M ENTS
We are grateful for supports from the Special Project for

CO N FLI C T O F I NTE R E S T
The authors confirm that they have no conflicts of interest with respect to the study described in this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.