Immunomodulatory effect of polysaccharides from the extraction of Codonopsis javanica (Blume) Hook. f. et Thomson (Campanulaceae) roots in female rats

Abstract In this study, the crude polysaccharides of C. javanica (CJP) was obtained from Codonopsis javanica (Blume) Hook. f. et Thomson using hot water extraction method, which was separated and purified by DEAE-cellulose column and Sepharose CL-6B column. The structure of the purified component was preliminary characterized by gas chromatography-mass spectrometer (GC-MS), high performance gel permeation chromatography (HPGPC) and infrared spectroscopy (IR). By examining the degree of delayed-type hypersensitivity (DTH) in mice and carbon particle clearance index, the immunomodulatory activity was clarified. The results showed that the extraction rate of CJP was 24.9 ± 0.5%. After purification, the refined polysaccharides component (CJP-2) was obtained. The structural characterization results indicated that CJP-2 was mainly composed of mannose, glucose, and galactose, and its molecular weight was 790 Da. Immunomodulation results showed that the low and medium levels of CJP significantly enhanced the degree of DTH in mice (P < 0.05). CJP can improve the clearance index of mice and enhance their charcoal removal function. The study indicates that C. javanica is a good source of polysaccharides, and CJP may be a new type of immunomodulator. Graphical Abstract


Materials and reagents
The materials were collected from Guizhou, China and identified as Codonopsis javanica 1.2. Purity and molecular weight determinations CJP was dissolved in distilled water (25 mg/mL), and purified with DEAE-cellulose column (35 cm× 60 cm). The polysaccharides were fractionated from the column, eluting with gradient NaCl aqueous solution (0.05, 0.1, 0.2, 0.5 and 1.0 M). Each fraction was monitored by the phenol-sulfuric acid method. The eluted fractions were further purified by Sepharose CL-6B column (2.6×100 cm). The single peak was collected and lyophilized, and the purity and molecular weights were evaluated by gel permeation chromatography (GPC) on an Agilent PL Aquagel-OH MIXED column (7.5mm×300 mm, 8 μm). Samples were eluted with 0.05 M Na 2 SO 4 at a flow rate of 1.0 mL/min, maintained at a temperature of 35 °C and detected by a refractive index detector (Deng et al. 2015). Preliminary calibration of the column was performed using standard dextrans (F-2, T-10, T-40, T-70 and T-500) and glucose. All data provided by the GPC system were collected and analyzed using the Workstation software package.

Analysis of monosaccharide composition by GC-MS
CJP-2 was hydrolyzed with 2 M TFA (2 mL) at 120 °C for 4 h, and the excess amount of acid was completely removed by ethanol. The hydrolyzed product was reduced with NaBH 4 (20 mg, 2 h, vibrated every 30 min), and then acidified with acetic acid. Excess boric acid was removed by co-distillation with methanol (1-2 mL), the product was completely dried at 100 °C for 15 min.
Then 1:1 pyridine-acetic anhydride were added and reacted in oven for 1 h at 100 ℃.
Subsequently, the same volume of chloroform was added to extract for 3 times, then the chloroform extracts was combined and washed with distilled water, and dried under reduced pressure, the residue was sugar alcohol acetate derivative, which was then analyzed by GC-MS on an Agilent Chromatograph (Agilent Technologies, 5975C United States of America) equipped with an HP-5MS quartz capillary column (30m×0.25mm×0.25 μm).
The GC analysis was carried out under the following conditions: The temperature of column was increased from 50 °C to 300 °C at a rate of 8 °C/min, Helium was injected at a speed of 1.0 mL/min, injection temperature was 250 °C, split ratio was 20:1. The standard monosaccharides (rhamnose, arabinose, xylose, mannose, glucose and galactose) were prepared and subjected to GC analysis respectively in the same way (Du et al. 2019;Zhi et al. 2019).

Infrared spectral analysis
The IR spectra of the polysaccharides were obtained using a Fourier transform IR spectrophotometer (FT-IR, Vector 22 Bruker). Before performing FT-IR measurement in the frequency range of 4000-500 cm -1 , the purified polysaccharides were ground with KBr powder and then pressed into pellets.

Animal administration and grouping
48 female mice were randomly divided into 4 groups (n=12 each) after feeding for 7 days, including blank group, the polysaccharides groups of high, medium and low of CJP. The drug was prepared with 0.5% CMCNa solution, 3 days before the model was administered, intragastric administration according to 20 mL/kg body weight for 30 days, the blank group was treated with physiological saline. (2, 4-dinitrofluorobenzene) treated mice 10 days before the end of the experiment, the hair was removed by 6% NaS from the abdomen of each mouse to achieve the scope (3 cm×3 cm), and then the mice were sensitized with 50 μL of 5% 2,4-dinitrofluorobenzene DNFB dissolved in acetone-sesame oil (1:1, 10 mg/mL) on each shaved abdomen (Wang et al. 2017;. After 5 days, mice were challenged with 10 μL 5% DNFB on both sides of their right ear. The mice were sacrificed by cervical dislocation 24h later, and both ears were cut to obtain 8 mm diameter pieces and weighed ).

Mice carbon clearance model establishment 1.5.2.1. Animal administration and grouping
A total of 60 female mice were randomly divided into 6 groups (n=10 each): blank group (CT), Chueun oral liquid group (CY), polysaccharides group of Lentinus edodes (Berk.) Sing. (XGDT), polysaccharides groups of high, medium and low of CJP. The CY group were given Chueun oral liquid according to body weight of 200 mL/kg, while XGDT group were given Lentinus edodes polysaccharides of 45 mg/kg, 1 times/d, fasting for 1 hour before but water.

Determination of the carbon clearance index
After 30 consecutive days, the mice were given tail-vein injections of India ink (100 mL/kg) which were diluted at a ratio of 1: 4 in physiological saline, 2 and 10 min after injection, 20 μL blood were drawn from the orbital venous plexus using a sterile syringe and then immediately dispensed into 2 mL 0.1% Na 2 CO 3 solution. And then OD 680nm was measured by microplate where OD 1 , OD 2 represent the OD 680nm value from the 2 blood samples, respectively; t 2 and t 1 represent the time when the 2 blood samples were taken.

Statistical analysis
Data were expressed as the mean standard deviation of the means (S.D.) and statistical analysis was performed by paired samples t-test using SPSS 17.0 to evaluate the significance of differences between groups. Note: compared with CT group, *P < 0.05, **P < 0.01; compared with CY group, ΔP < 0.05, ΔΔP < 0.01