Endogenous calcium attenuates the immunomodulatory activity of a polysaccharide from Lycium barbarum L. leaves by altering the global molecular conformation

Accepted Manuscript Endogenous calcium attenuates the immunomodulatory activity of a polysaccharide from Lycium barbarum L. leaves by altering the global molecular conformation

Bo Zhang, Mengze Wang, Chu Wang, Tongtong Yu, Qiang Wu, Yao Li, Zhaolin Lv, Junfeng Fan, Liying Wang, Bolin Zhang PII: DOI: Reference:

S0141-8130(18)34361-7 https://doi.org/10.1016/j.ijbiomac.2018.11.067 BIOMAC 10944

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

19 August 2018 3 November 2018 12 November 2018

Please cite this article as: Bo Zhang, Mengze Wang, Chu Wang, Tongtong Yu, Qiang Wu, Yao Li, Zhaolin Lv, Junfeng Fan, Liying Wang, Bolin Zhang , Endogenous calcium attenuates the immunomodulatory activity of a polysaccharide from Lycium barbarum L. leaves by altering the global molecular conformation. Biomac (2018), https://doi.org/ 10.1016/j.ijbiomac.2018.11.067

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Endogenous calcium attenuates the immunomodulatory activity of a polysaccharide from Lycium barbarum L. leaves by altering the global molecular conformation

a,1

, Mengze Wang a,1, Chu Wang a, Tongtong Yu a, Qiang Wu a, Yao Li a,

Department of Food Science and Engineering, College of Biological Sciences and

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Zhaolin Lv a, Junfeng Fan a,*, Liying Wang b, Bolin Zhang a

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Bo Zhang

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Technology, Beijing Forestry University, Beijing 100083, China Ningxia Senmiao Co. Ltd, Yinchuan, Ningxia 750004, China

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These authors contributed equally to this work.

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*Correspondence: Junfeng Fan, Associate Professor, Department of Food Science and

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Engineering, College of Bioscience and Biotechnology, Beijing Forestry University, P.O.112, 35Qinghua East Road, Haidian District, Beijing, 100083, China. Phone: (8610)6233-6700, Fax: (8610) 6233-8221, E-mail: [email protected]

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Abstract A heteropolysaccharide, LP5, was purified from Lycium barbarum L. leaves. It was identified as a calcium-rich polysaccharide (8.6mg calcium/g) with a molecular weight of 2.50×105Da. The polysaccharide was composed of six kinds of

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monosaccharides, of which mannose and xylose are the main components. And it

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contains 16.37% glucuronic acid. Studies on RAW264.7 cells demonstrated that this

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polysaccharide exhibited potent immunomodulatory activity, including an increase in

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phagocytic activity, as well as the release of both nitric oxide and cytokines. However, after the depletion of calcium, the polysaccharide exhibited greater

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immunomodulatory activity (p<0.05). Further conformation analysis confirmed that the polysaccharide changed from a compact spherical conformation to a random coil

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structure in aqueous solution after the depletion of calcium, which resulted in

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increased immunomodulatory activity by LP5.

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Keywords: Goji, Mannose, Uronic acid, FTIR, Macrophage, Conformation

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1. Introduction

Stress, and the sedentary lifestyle and fast pace of urban life, have reduced immunity among the general population, increasing the probability of infection and illness.

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Studies of the immunomodulatory effects of plant polysaccharides have garnered

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interest from researchers and the molecular weight, monosaccharide composition, and

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glycosidic linkages of immunomodulatory polysaccharides from Cyclocarya paliurus,

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Chenopodium quinoa seeds, and Dendrobium huoshanense have been investigated thoroughly [1-3]. The effects of mineral incorporation on the functional properties

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of polysaccharides are also being widely studied. One investigation showed that the addition of exogenous iron (III) to a polysaccharide improved its iron uptake and

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functional activity [4]. Plant-derived polysaccharides are rich in minerals, such as calcium, iron, and selenium. Recently, the physical properties of a calcium-rich

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polysaccharide from Plantain seeds (Plantago sp.), and the antioxidant effects of a selenium-rich polysaccharide from Grifola frondosa, were investigated [5, 6].

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However, the influence of endogenous minerals on the immunomodulatory activity of polysaccharides is still unclear. Goji (Lycium barbarum L.) is a medicinal shrub belonging to the Solanaceae. In China, it is cultivated on an area exceeding 1,220 km2 [7]. Goji leaves (LL) are a folk tonic and traditional Chinese medicine. The Compendium of Materia Medica, a famous Chinese pharmacopoeia written in the Ming Dynasty, discusses how LL can

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strengthen tendons, remove toxic heat, tonify the kidney, and improve eyesight. LL is currently used to improve hypoxia and immune-regulating activities, reduce blood glucose levels, and promote splenocyte proliferation [8, 9]. LL has equal value to goji berries as a medicinal and tonic ingredient, and contains many nutrients and bioactive

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substances including polyphenols, flavonoids, alkaloids, minerals, and vitamins [7].

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The LL polysaccharide (LP) content (~10.8%) is roughly equal to that of goji berries

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(~10%). Furthermore, LP contains significant amounts of calcium, as determined by

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the ionic interaction of uronic acids and calcium [10]. To our knowledge, the role of calcium in the conformational and functional properties of LP remains unclear.

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We hypothesized that endogenous calcium could influence the immunomodulatory activity of LP. Therefore, we first extracted and screened LPs

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showing immunomodulatory activity. The structure of LP was characterized by gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared

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spectroscopy (FTIR), and nuclear magnetic resonance (NMR). After treatment with EDTA, the immunomodulatory activities of LP with and without calcium were

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compared by analyzing phagocytosis, NO production, and the release of cytokines by RAW264.7 macrophages. Finally, the influence of calcium on the structural conformation of LP was analyzed using advanced polymer chromatography (APC) with a multi-angle laser light scattering detector (APC-MALLS), to clarify the mechanism of action of calcium on the immunomodulatory activity of LP. 2. Materials and methods 4

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2.1. Materials and chemicals Lycium barbarum L. leaves (LL) were harvested in July 2015 in Yinchuan, China. After harvest, LL were dried for 8 h at 60°C and crushed into a powder (40 mesh). RAW264.7 mouse macrophage cells were purchased from the National

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Infrastructure of Cell Line Resource (Beijing, China). Dulbecco’s Modified Eagle

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Medium (DMEM) was purchased from BioDee Biotechnology (Beijing, China). Fetal

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bovine serum (FBS) was purchased from Tianhang Biotechnology (Zhejiang, China).

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Enzyme-linked immunosorbent assay (ELISA) kits used for the analyses of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) were purchased from

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Heyuan Biotechnology (Shanghai, China), and nitric oxide (NO) kits were purchased from Beyotime Biotechnology Co. Ltd. (Nanjing, China). Bovine serum albumin

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(BSA), the galactose uronic acid standard, and lipopolysaccharide (LPS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The glucose standard

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was purchased from Beijing Chemical Works (Beijing, China). All other monosaccharides standards were purchased from Solarbio (Beijing, China).

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2.2. Proximate composition of LL The LL powder was analyzed for moisture, protein, lipid, ash and carbohydrate according to the Association of Analytical Chemists (AOAC) [11]. The moisture content was determined by the gravimetric method at 105 °C to constant weight; the crude protein (N×6.25) was estimated by the macro-Kjeldahl method [12]; the lipids was evaluated by the Soxhlet method using petroleum ether as the extraction solvent; 5

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the ash content was estimated via incineration in a muffle at 550 ± 15 °C. The total carbohydrates were obtained by difference, subtracting the moisture, ash, protein and lipid values from 100. Calcium content was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES) [5].

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In order to determine the total sugar, LL suspension (1 g LL in 15 mL distilled

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water) was added to 10 mL HCl (6 M), and the mixture was incubated in a water bath

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(98 ± 2°C) for 30 min to extract soluble sugar. After filtration, the filtrate was

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neutralized with 6 M NaOH, and the concentration of total sugar was determined by phenol-sulfuric acid method [13]. A standard curve of glucose was used as reference.

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2.3 LP preparation and biochemical analysis

We used a previous method [10] with some modifications for the preparation of

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polysaccharides. Briefly, 50 g powder of LL was extracted with 2,500 mL water in an autoclave (121°C, 0.1 MPa) for 1 h, precipitated with EtOH (99%), and further

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deproteined with Sevage reagent (chloroform:butanol = 4:1, v/v), followed by decolorization with AD-8 macroporous resin. The resulting polysaccharides were

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purified using a DEAE-52 cellulose column (26 × 600 mm) eluted with deionized water and step gradient of NaCl solutions (0, 0.05, 0.1, 0.2, and 0.4 M) at a flow rate of 1 mL/min. Because the fraction eluted by 0.4 M NaCl had a symmetric peak and the highest polysaccharide content, which was determined with the phenol-sulfuric acid method [13], this fraction was further purified using a Sephadex G-100 column

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(16 mm × 100 cm) eluted by deionized water at a flow rate of 1 mL/min. The eluent was lyophilized and the powder was named LP5. The protein concentration of LP5 was determined by the Folin-phenol method [14]. The content of uronic acid and polyphenol were analyzed by the sulfuric acid

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carbazole [15] and Folin-Ciocalteu method [16], respectively. Calcium content was

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determined using the ICP-AES [5].

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2.4. LP5 characterization

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2.4.1. Molecular mass

Advanced polymer chromatography (Acquity APC System; Waters, Milford, MA,

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USA) with refractive index detector (RID) and MALLS (DAWN HELEOS-II) was applied to determine molecular weight (MW) distribution. LP5 (2 mg) was fully

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dissolved in deionized water (1 mL) and filtered through a 0.22-μm membrane and injected (50 μL) into the APC system: column of Acquity APC AQ 450 (2.5 μm),

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column temperature of 38°C, mobile phase of 0.1M NaNO3, flow rate of 0.38 mL/min, and run time of 30 min. A calibration curve was produced using Dextran T- series

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standards. The MW of LP5 was estimated with reference to the calibration curve by means of the RID (35°C). 2.4.2. Monosaccharide composition analysis LP5 (5 mg) was dissolved in 1 mL trifluoroacetic acid (TFA; 2 M) and hydrolyzed at 120°C for 2 h. After completely removing the excess TFA, the monosaccharides were treated with silylation as described previously [17] and analyzed by GC. The molar 7

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ratio of monosaccharide was obtained by calculating its molar concentration based on the peak area and the concentration of monosaccharide standard. 2.4.3. FTIR spectroscopy The infrared spectrum of LP5 was analyzed with a KBr pellet on a FTIR

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4,00 and 4,000 cm-1 according to our previous method [10].

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2.4.4. NMR spectra

H and 13C NMR spectra of LP5 were obtained with an NMR spectrometer (Bruker

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spectrophotometer (Bruker Vertex 70, Bruker Optics, Ettlingen, Germany) between

Avance-500) using a sample of 10 mg/mL polysaccharide in D2O (99.9%). Chemical

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shifts were reported in ppm.

2.5. Cell culture and immunomodulatory activity

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2.5.1. Depletion of minerals from LP5 To deplete calcium, LP5 was treated with EDTA (8.0%, pH 8.0) for 6 h and dialyzed

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against ultrapure water (18.2 MΩ) from a Milli-Q water purification system (Millipore, Bedford, MA, USA). The dialyzed LP5 was freeze-dried and the powder

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was named as LP5E. 2.5.2. Cell culture

RAW264.7 cells were cultured in a cell incubator at 5% CO2 and 37°C with complete DMEM media (DMEM supplemented with 10% FBS). The cells were passaged every 2 days, and the excess cells were cryopreserved (DMEM with 20% FBS). Cells from passages 20–25 were used for our experiments. 8

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2.5.3. Phagocytosis assay Cell proliferation was determined by an MTT reduction assay [18]. RAW264.7 cells were incubated at 2 × 104 cells/well in a 96-well plate for 24 h with complete media and exposed to serial concentrations of LP5, LP5E (10, 20, 40, 80, and 160 μg/mL),

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or LPS (0.25, 0.5, 1, and 2 μg/mL). Complete medium was used as a normal control

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in the assay.

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Phagocytosis was assayed using a method previously reported [19]. RAW264.7

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cells were cultured at 2 × 104 cells/well in a 96-well plate for 24 h, and treated with LP5 and LP5E (10, 20, 40, 80, and 160 μg/mL) or LPS (1 μg/mL) for another 24 h.

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Next, 0.075% neutral red was added and the plate was incubated with 5% CO2 at 37°C for 30 min. The plate was gently washed three times to remove the remaining

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pigment. Next, 150 μL of the cell lysates (99% ethanol : 0.1 mol/L acetic acid = 1:1, v/v) were added and the plate was cultured for 2 h at room temperature. Absorbance

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was measured at 540 nm. 2.5.4. NO production

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NO production was determined using Griess reagent [20]. Cells were cultured at 2 × 104 cells/well in a 96-well plate for 24 h, and treated with LP5, LP5E (10, 20, 40, 80, and 160 μg/mL), or LPS (1 μg/mL) for another 24 h. Supernatant (50 μL) from the cell culture was extracted and mixed with 50 μL Griess Reagent I and then 50 μL Griess Reagent II. Absorbance was measured with a microplate reader at 540 nm. The

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amount of NO was calculated using the generated sodium nitrite standard curve (y = 221.25x – 10.512, r2 = 0.9933). 2.5.5. Determination of TNF-α and IL-6 RAW264.7 cells were diluted to a density of 2 × 104 cells/well and incubated in a

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96-well plate for 24 h. The media was discarded and various concentrations of LP5

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and LP5E (10, 20, 40, 80 and 160 μg/mL) were added. After incubation for 24 h, the

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culture solution was collected and assayed using cytokine secretion assay kits

2.6. Molecular properties

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2.6.1. Mw and chain conformation

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according to the manufacturer’s instructions [20].

The MW of LP5E was determined using APC as mentioned in Section 2.3.1. The LP

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chain conformation was also tested using APC-MALLS, which had a conformation that showed molecular weight (MW), radius of gyration (Rg) and the slope of the

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curve representing the dependence of Rg on Mw. 2.6.2. Determination of intrinsic viscosity

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The LP5 and LP5E solutions (2–10 mg/mL) were prepared by dispersing the samples in deionized water. Viscosity measurements were performed using a rheometer (MCR 302, Anton Paar, Shanghai, China) with cone plate (50 mm) and the entire solution was studied at a shear rate of 300 s-1 and temperature of 25°C [21]. Intrinsic viscosity ([η], IV) was calculated through extrapolation to infinite dilution according to the Huggins empirical expression: 10

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2.7. Statistical analysis

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All data was analyzed by SPSS11.5 software, and the differences between the two

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groups were analyzed by the Duncan method (p < 0.05).

3.1. Proximate composition of LL

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3. Results and discussion

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LL has high nutritional and medicinal value and commonly consumed as vegetable in the northwest region and Guangdong province of China. Analysis on the proximate

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composition shows that LL contains high content of crude protein, ash and carbohydrate, accounting for 14.7%, 25.8% and 49.5% of the dry leaves, respectively

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(Table 1). These results are consistent with those reported previously [22]. The total sugar of LL was 16.1%, which suggested that LL was rich in soluble sugar.

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Interestingly, the content of calcium reached to 5.1% in dried LL, which was 50 and 10 times higher than that of goji berry and green tea, respectively [22, 23]. Our previous study on the polysaccharide from LL showed that minerals such as calcium linked with polysaccharide through ionic interactions [10]. 3.2. Purification and Composition of LP5

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The crude hot-water extracts were 8.25% dry weight of the LL. Five major fractions were isolated from the crude extract by a DEAE-Cellulose-52 column (Fig. 1A). The polysaccharide amount of LPs ranged from 88.34 to 96.20%, with LP5 being the most pure (96.20%). Following purification by Sephadex G-100, there was a single,

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symmetrical peak for LP5, and its carbohydrate content increased slightly to 97.6%.

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We also measured that LP5 contained only 0.07% protein and no polyphenols were

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detected, further proving that LP5 is a highly purified polysaccharide. APC showed

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that LP5 was a homogenous polysaccharide with a MW of 2.50 × 105 Da. LP5 contained a high amount of calcium (8.6 mg/g), which is consistent with our previous

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study [10]. The uronic acid content of LP5 was 18.23%, which is less than the 47.68%

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calcium with uronic acids.

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reported in a previous study [8]. These results suggested that LP5 binds abundant

GC spectroscopic analysis revealed that LP5 was composed of six kinds of

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monosaccharides, namely ribose, xylose, mannose, galactose, glucose, and glucuronic acid in the ratio of 1.0: 3.38: 4.60: 2.48: 1.75: 2.59 (Fig. 1B). Mannose and xylose

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were the major monosaccharide constituents. This result differed from that of the LP eluted with 0.1 N NaCl, which was mainly consisted of glucose and ribose [10]. Glucuronic acid accounted for 16.37% of the total monosaccharides, which is consistent with the uronic acid results in LP5. 3.3. FTIR spectra and NMR analysis

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LP5 possesses the characteristic FTIR spectra of a carbohydrate (Fig. 1C), showing a typical major broad stretching peak at 3436 cm−1 for the -OH group. The weak bands at 2936 cm−1 indicated the C-H stretching vibration of -CH2-. The bands at 1729 cm−1 may be attributed to the absorption of the C=O groups, and the bands at 1626 cm−1

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may also be C=O groups which are caused by C=O stretching vibration of

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-NHCOCH3- in polysaccharides [19, 24]. The bands at 1413 cm−1 were caused by a

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COO- stretching vibration, indicating the presence of uronic acids [25]. The

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absorptions at 1094 cm−1 were attributed to the asymmetric vibration of C-O-C glycosidic rings, indicating the presence of pyranose [8]. The band at 964 was also

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ascribed to α-D-glucopyranose [6]. And bands at 800 cm−1 indicated the presence of mannose [26]. There is a small absorption peak at 715 cm−1, indicating the existence

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of xylose [24].

Figure 2 showed the main chemical shifts of NMR spectra from LP5. The 1H

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NMR spectrum (Fig. 2A) showed six anomeric proton signals that appeared at δ 5.30, 5.15, 5.00, 4.94, 4.45, and 4.42 ppm in a relative integral of nearly 1.00: 3.25: 2.98 :

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1.70 : 3.43 : 5.13. This ratio is consistent with the results of the GC analysis. The anmoeric proton signals at δ 4.45, and 4.42 ppm were attributed to the β-pyranose unit, whereas others were accredited to α-pyranose forms. The proton H1-H6 of each sugar residue had chemical shifts of δ 4.10–3.30 ppm. The 13C NMR spectrum signals of LP5 were in the regions ranging from δ 60–110 ppm (Fig. 2B), which represent the typical distribution of NMR signals in polysaccharides [27]. The six major signals at δ 13

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96.96, 100.92, 103.38, 107.67, 108.10 and 109.45 for six anomeric carbons supported the conculsion that the polysaccharide consisted of six monosaccharides. It is worth noting that in the 13C spectrum an obvious peak at δ 175.41 ppm was the typical carbon signal of –COOH from the glucuronic acid residue [5, 28]. This result is

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consistent with the FTIR analysis.

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3.4. Immunomodulatory activity of polysaccharide treated with EDTA

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Innate immunity serves as an essential first-line defense against microbial pathogens

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and uses macrophages as a type of phagocytic cells [29]. The effect of LP5 on innate immunity was determined using RAW264.7 macrophages; LPS was used as a positive

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control to indicate activation of the immune system [30]. To determine the effect of calcium on immunomodulatory activity, EDTA was used to remove calcium from

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LP5. The contents of calcium and uronic acid in LP5E were 450 μg/g and 30.45%, respectively. The MTT assay showed that the concentrations of LP5 and LP5E were

macrophages.

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less than 200 μg/mL, and that LPS at 1 μg/mL was not cytotoxic to RAW264.7

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Phagocytosis is an important function of macrophages in immune-surveillance against malignant cells and pathogen. Compared with control, LP5 enhanced phagocytic function in a dose-dependent manner (Fig. 3A). Low concentrations (10 and 20 μg/mL) of LP5 had no influence on phagocytosis, while concentrations ≥ 40 μg/mL significantly increased the phagocytosis of RAW264.7 cells (p < 0.05). LP5 was effective at a concentration range of 40‒1600 μg/mL, consistent with a previous 14

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the phagocytic activity of RAW267.4 cells.

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NO appears to be a major mediator of macrophages by acting as a destroyer of

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bacteria and tumor cells [20]. At a concentration of 1 μg/mL, LPS caused an NO

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release of 37.93 μM (Fig. 3B). As the concentration increased to 10 μg/mL (low concentration), LP5 caused an increase in NO release, with the highest release of

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43.14 μg/mL observed at a high concentration of 160 μg/mL. Interestingly, LP5E had a significantly enhanced effect on NO release at 20‒160 μg/mL than LP5 (p < 0.05).

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These results indicated that LP5 could regulate the primary innate immune response of RAW264.7 macrophages, and that the depletion of endogenous calcium from LP5

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enhanced the immunomodulatory activity of the cells. Pathogen-activated macrophages also release cytokines, including TNF-α and

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IL-6, which are major immune and inflammatory mediators [20]. In the cytokine assay, the release of IL-6 and TNF-α were all enhanced in the cells as LP5 concentration increased (Fig. 3C and D). The release of IL-6 in the cells treated with LP5 was significantly lower than those treated with LP5E at 80 and 160 μg/mL (p < 0.05). The release of TNF-α in the cells treated with LP5 was significantly lower than those treated with LP5E at 20‒160 μg/mL (p < 0.05). These results further suggested 15

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that LP5 could increase the immunomodulatory activity of macrophages and that endogenous calcium had a negative effect on cytokine release of the cells. 3.5. Chain conformation and viscosity The Mw and radius of gyration (Rg) of LP5 and LP5E were determined to understand

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why endogenous calcium intensely affected the immunomodulatory activities of

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polysaccharides. There was no significant difference between the Mw of LP5E (2.51

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× 105 Da) and LP5 (2.50 × 105 Da), indicating that calcium was integrated into a

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single polysaccharide chain as an intra-chain component. However, the Rg of LP5E (54.3 ± 3.9 nm) was much higher than LP5 (44.7 ± 3.8 nm). This result suggested that

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LP5 exhibits a globular conformation while LP5E has a loose-and-stretched chain conformation.

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We also studied the slope of the curve representing the dependence of Rg on Mw to determine the conformation of the polysaccharide. The slopes of LP5 and LP5E

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were 0.30 and 0.42 (Fig. 4A and B), respectively, indicating that LP5 had an almost compact globular conformation, and LP5E an irregularly loose coil conformation [31].

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Correspondingly, the intrinsic viscosity increased from 0.71 to 0.88 mL/mg after calcium was depleted from LP5 (Fig. 4C and D). These results suggested that the depletion of calcium led to the change in LP5 conformation from compact globule to loose coil. Macrophages respond to the stimuli of infectious agents mainly through the NF-κB pathway, producing NO and pro-inflammatory cytokines [26]. 16

ACCEPTED MANUSCRIPT Polysaccharides have been shown to induce NF-κB signaling pathways by binding to TLR2 receptors in macrophages, further upregulating NO, TNF-α, and phagocytosis [32, 33]. In this study, LP5 may have enhanced phagocytic activity and promoted the release of both NO and cytokines in macrophages. Therefore, LP5 was presumed to

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interact with TLR2 receptors and thus activate NF-κB signaling pathways. However,

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LP5E exhibited a higher immunomodulating effect than LP5. This result may be

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explained by its loose coil conformation, which facilitated the interaction of the

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polysaccharide with the Toll-like receptor (TLR) 2, in comparison with the compact globular shape of other molecules. Therefore, endogenous calcium may act to

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decrease the immunomodulating effect of polysaccharides in this study. However, further studies are required to elucidate the exact interaction of polysaccharide

4. Conclusion

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molecules and the TLR2 receptor.

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This study demonstrated that the endogenous calcium strongly affects the physical configuration and thus immune modulating activity of the polysaccharides from

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Lyciumbarbarum L. leaves. LL was a calcium-rich heteropolysaccharide, mainly composed of mannose and xylose. Studies on RAW264.4 cells have demonstrated that this polysaccharide exhibits potent immunomodulatory activity, which increases following the depletion of calcium. Conformational analysis confirmed that the polysaccharide changed from a compact spherical conformation to a random coil structure in aqueous solution following depletion of calcium, which may increase its 17

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chance of contact with the macrophage. This study will promote further research on the structure-activity relationships of LL polysaccharides. And LL could be utilized as an excellent source of polysaccharide with potent immunomodulatory activity.

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Acknowledgements

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We extend special thanks to the Key Research and Development Program of Ningxia

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Hui Autonomous Region (the East-West China Science and Technology Cooperation

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Project: Bioactive activities and product development of minerals and polysaccharides from Lycium barbarum L. leaves), and the Beijing Forestry University training

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program for undergraduates (Grant no. X201810022071 and X201810022077).

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The authors declare that they have no conflict of interest.

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Figure Legends

Figure 1. LL polysaccharides purification, FTIR and monosaccharide analysis: separation profile of LP on DEAE-Cellulose-52 column eluted with water and NaCl

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solutions (step gradient), including purification of LP5 fraction on Sephadex G-100

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column with water as eluent (A); FTIR spectrum of purified LP5 (B); gas

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chromatogram of monosaccharide standards (C) and monosaccharide analysis of LP5

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(D)

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Figure 2. NMR spectra of LP5 in D2O. (A) 1H, (B) 13C spectra were included.

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Figure 3. Effects of LP5 and LP5E on (A) phagocytic activity, (B) NO production, and (C) IL-6 and (D) TNF-α release in RAW264.7 cells. Different letters represent

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significant difference among groups.

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Figure 4. The conformation plot slope of LP5 (A) and LP5E (B) and the viscosity of LP5 (C) and LP5E (D). (p<0.05).

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Table 1. Proximate compositions of L. barbarum L. leaves (g/100 g dry weight) Moisture

Crude proteins

Crude lipids Ash

Calcium

Carbohydrates Total sugar

7.9 ± 0.6

14.7 ± 0.3

2.1 ± 1.1

5.1 ± 0.6

49.5 ± 4.0

25.8 ± 0.8

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Values are means ± SD (n = 3).

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16.1 ± 0.7

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The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see:

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Highlights

Polysaccharide from Lycium barbarum L. leaves (LP) was characterized.

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LP is a Ca-rich polysaccharide mainly composed of mannose and xylose.

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Endogenous calcium attenuates LP’s immunomodulatory activity.

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Depletion of Ca leads to changes in LP’s globular conformation.

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