Extraction, Structural Characterization, and Anti-Hepatocellular Carcinoma Activity of Polysaccharides From Panax ginseng Meyer

Polysaccharides are the main active ingredients of ginseng. To extract the most effective polysaccharides against hepatocellular carcinoma (HCC), we isolated and characterized the polysaccharides from the mountain cultivated ginseng (MCG) and compared their composition and cytotoxic effect with cultivated ginseng (CG) polysaccharide against HepG2 cell lines for the first time. MCG polysaccharides and CG polysaccharides were fractionated into two fractions such as MTPS-1, MTPS-2 and CTPS-1, CTPS-2 by salting out, respectively. Compared to CG, MCG possessed appreciable cytotoxic effect against HepG2 cells among that MTPS-1 possess fortified effect. Then, MTPS-1 was selected for further isolation process and seven acidic polysaccharides (MCGP-1–MCGP-7) were obtained using ethanol precipitation, ion-exchange, and gel permeation chromatography techniques. Structural characteristics of the polysaccharides (MCGP-1–MCGP-7) were done by adapting methylation/GC-MS and NMR analysis. Overall, MCGP-3 polysaccharide was found to possess significant cytotoxic effect against HepG2 cells with the IC50 value.


INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the malignant tumors with poor prognosis and lower survival rate (1,2). Among them, HCC accounts for more than 90% of liver cancer and has the characteristics of uncertain growth, invasion, and anti-apoptosis, which leads to drug resistance and treatment failure (3,4). Therefore, it is still necessary to discover new and effective therapeutic drugs.
Panax ginseng Meyer is a well-known traditional Chinese medicine where the main components of this species are saponins and polysaccharides. The research on ginsenosides is relatively in-depth, however due to the molecular weight limitation, the studies on ginseng polysaccharides were limited. Nowadays, natural polysaccharides seek much attention among researchers due to their effectiveness and non-toxic nature (5)(6)(7)(8)(9). Recently polysaccharides from natural sources have been utilized as a drug to treat specific cancer owing to their substantial proliferation, migration, and apoptosis effect (10,11).
Ginseng in the market is mainly classified into two grades: cultivated ginseng (CG) and mountain cultivated ginseng (MCG) (12). It has been pointed out in the literature that butanol-extracted wild ginseng (BX-MG) shows high anti-tumor activity by inhibiting the proliferation of HCC cells and inducing their apoptosis, which may be related to the presence of more saponins (13). However, there are no reports on the extraction and separation of MCG polysaccharides, structural analysis, and screening for anti-tumor activity. In this study, the monosaccharide composition and structure analysis of the polysaccharides from MCG were studied for the first time, and the anti-HCC activity of MCG polysaccharides was screened. In addition, the anti-HCC activity of MCG polysaccharide and CG polysaccharide were compared, and the relationship between the activity and structure of ginseng polysaccharide were briefly discussed.
The seven fractions were further purified by DEAE-52 column chromatography and gel column chromatography ( Figure 1). The MCGP-1-MCGP-7 homogeneous polysaccharides were obtained from the seven fractions (MTPS-40 (I-II) to MTPS-80). Cultivated ginseng powder (200 g) was prepared in the same way as mountain-cultivated ginseng to obtain crude cultivated ginseng polysaccharide (CTPS), cultivated ginseng salt-soluble polysaccharides (CTPS-1), and cultivated ginseng salt-insoluble polysaccharides (CTPS-2). The total sugar content was determined by the phenol-sulfuric acid method at 490 nm with the standard of D-glucose (15). The protein content was determined by Bradford method at 595 nm with the standard of bovine serum albumin (BSA) (16).

Homogeneity, Molecular Weight and Monosaccharide Composition Determination
The homogeneity, molecular weight and monosaccharide composition analysis were performed according to the previous method (17).

Methylation and GC-MS Analysis
The method of Ciucanu and Kerek (19) was used for methylation analysis and the other details were referenced to the previous report (11). For the uronic acid-containing polysaccharide, the reduction of uronic acid was carried out according to the Feng method (20). Finally, after compared the linkages of native polysaccharide and reduced polysaccharide, the linkage of every residue was concluded.

NMR Spectroscopic Analysis
MCGP-3 and MCGP-4 were exchanged with deuterium by freezedrying D 2 O (99.9 atom%) three times. The samples (20 mg) were finally dissolved in D 2 O. The 13 C NMR (150 MHz) and heteronuclear single quantum coherence (HSQC) spectra were obtained on a Brucker Avance III HD 600 spectrometer (Germany).

Cell Viability Assay
HepG2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and placed in a 37°C incubator filled with 5% CO 2 gas (21).
The activity screening of polysaccharides were determined using Sulforhodamine B (SRB) experiment (22). HepG2 cells was seeded into 96-well plates and treated with different concentrations of monomer compound/mixture for 48 h. After drug treatment, Cells were fixed with 50% TCA solution. The cells were stained with 0.4% SRB staining solution, and the optical density (OD) value was measured at 540 nm using a microplate reader (Elx 800 Bio-Tek, USA). Experiments were repeated at least three times.

Statistical Analysis
The data were displayed as means ± SEM. Statistical analysis was performed using GraphPad Prism 7.0 (SanDiego, CA, USA). To compare the differences between multiple groups, one-way analysis of variance (ANOVA) was performed in this software. P <0.05 indicated statistically significant.

Extraction, Separation and Purification of P. ginseng Polysaccharide
The roots of MCG was decocted with distilled water and precipitated by alcohol, de-starch with a-amylase and deproteinized by Sevag method to obtain crude mountaincultivated ginseng polysaccharides (MTPS). The MTPS was divided into salt soluble polysaccharide (MTPS-1) and salt insoluble polysaccharide (MTPS-2) by salting out. CG polysaccharides (CTPS) was prepared in the same way as MTPS, CTPS was divided into cultivated ginseng salt-soluble polysaccharides (CTPS-1) and cultivated ginseng salt-insoluble polysaccharides (CTPS-2) by salting out. MTPS-I was redissolved with distilled water then fractionated by graded precipitation at final ethanol concentrations of 40, 50, 60, 70, and 80%, respectively. The five fractions were further purified by DEAE-52 column chromatography and gel column chromatography to obtain seven homogeneous polysaccharides MCGP-1-MCGP-7. MCGP-1-MCGP-7 showed a single symmetric peak by HPGPC ( Figure 2).

Monosaccharide Composition and Molecular Weight Distribution Analysis
The molecular weight, protein content, total sugar content and monosaccharide compositions of all the fractions from the CG and MCG are shown in Table 1 Table 2. Compared to MCGP-3 and MCGP-4, the carboxyl-reduced MCGP-3-R and MCGP-4-R showed an increase ratio of 1, 4-linked galactose, indicating that the galacturonic acid in MCGP-3 and MCGP-4 was 1, 4-linked.

Partial Acid Hydrolysis of MCGP-3 and MCGP-4
The hydrolysates of MCGP-3 and MCGP-4 from different hydrolysis times were analyzed by HPGPC. MCGP-3 was  gradually hydrolyzed with different times, and the molecular weight of the remaining polysaccharide hydrolyzed at 8 and 6 h resembles same pattern, indicating that the core structures of MCGP-3 were not broken by acid hydrolysis, however, the side chains were gradually cleaved.
To investigate the core structures of MCGP-3 and MCGP-4, the hydrolysates present after 1, 2, 4, 6, and 8 h hydrolysis reactions were collected and separated via dialysis into two fractions. Fraction I contained the intact core, found in the dialysis membrane, and fraction II contained oligosaccharides and monosaccharides, found in the dialysate. In the remaining polymer of MCGP-3, Rha and GalA increased from 6.005 and 15.67% (MCGP-3-0 h) to 9.848 and 42.751% (MCGP-3-8 h-I), respectively; Ara and Gal decreased from 20.956 and 22.877 to 0.901% and 9.573, respectively. In the remaining polymer of MCGP-4, Rha and GalA increased from 4.53 and 26.74% (MCGP-4-0 h) to 6.54 and 62.43% (MCGP-4-8 h-I), respectively; Ara and Gal decreased from 20.11 and 39.74 to 1.73 and 17.65%, respectively. The changes in monosaccharide composition of the released monosaccharides and oligosaccharides were found to be consistent with those observed for the remaining polymer. These changes in monosaccharide composition indicated that after 8 h of hydrolysis, Ara was completely cleaved, Gal was partially hydrolyzed, and little of Rha and GalA were hydrolyzed, indicating that Ara was located on the surface of the molecules, Rha and GalA probably exist in the core of MCGP-3 or MCGP-4.   In order to explore the structure-activity relationship of polysaccharides, MTPS-1 were further fractionated into seven acidic fractions (MCGP-1-MCGP-7) by a combination of ethanol precipitation, ion-exchange and gel permeation chromatography. To further evaluate the inhibitory activity of 7 monomer compounds on HepG2 cells, the cells were treated with MCGP-1-MCGP-7 at different concentrations (3.125-100 mg/ml) for 24, 48, and 72 h, respectively. The result is shown in Figure 6 and Table 3, MCGP-1-MCGP-7 at the concentration range of 3.125-50.0 µg/ml, significantly decreased the viability in HepG2 cells, and the inhibitory effect appears in a time and concentration dependent manner. After treatment for 72 h, MCGP-2 and MCGP-3 showed maximum cytotoxicity to HepG2 cells than others, with the IC 50 value of 13.87 ± 0.54 and 13.02 ± 0.36 mg/ml, respectively. MCGP-4 showed low cytotoxicity to HepG2 cells, with the IC 50 value of 35.67 ± 0.35 mg/ml.

DISCUSSION
Among HCC cases, the proportion of patients accounts for approximately 88% (31), it is necessary to discover and develop highly effective and low-toxic drugs for the treatment of HCC. Since synthetic drugs have their inevitable shortcomings, the natural products isolated from traditional Chinese medicine have attracted the attention of researchers in recent years.
In this study, the anti-tumor activity of MCG polysaccharides (MTPS, MTPS-1, and MTPS-2) and CG polysaccharides (CTPS, CTPS-1, and CTPS-2) was analyzed and compared, and it was found that MTPS-1 could significantly inhibit proliferation on HepG2 cells. Subsequently, MTPS-1 was further tracked and separated to obtain seven pectin polysaccharides MCGP-1-MCGP-7, and screened for anti HepG2 cells activity. The results Results of the structural characterization of MCGP-3-MCGP-4 indicated that the characteristic compositions of RG-I pectin, that is Rha, GalA, Ara, and Gal (17). The monosaccharide composition of MCGP-3 and MCGP-4 was similar, mainly containing Gal, GalA, Ara, Glc, and Rha, and a small amount of GlcA and Man. The content of Glc in MCGP-3 is higher than that of MCGP-4, and the content of GalA in MCGP-4 is higher than that of MCGP-3. The composition of main chains and branches of MCGP-3 and MCGP-4 was analyzed by partial acid hydrolysis. The study found that the main chain of MCGP-3 was mainly composed of GalA, Gal, Glc, and Rha, the content is 42.75, 9.57, 34.11, and 9.85%, respectively; the main chain of MCGP-4 was mainly composed of GalA, Gal, Glc, and Rha, the content is 62.43, 17.65, 4.28, and 6.54%, respectively. The branch chain of MCGP-3 was mainly composed of Gal, Ara, Glc, Rha, and GalA, the content is 31.98, 34.62, 15.90, 8.23, and 26.78%, respectively; the branch chain of MCGP-4 was mainly composed of Ara, Gal, Glc, Rha, and GalA, the content is 44.05, 48.07, 2.16, 2.78, and 1.90%, respectively. These results confirmed that MCGP-3 and MCGP-4 were RG-Itype pectin branched with a-1, 5/1,3,5-arabinan and b-1,4galactan side chains. The biological activities of pectin might be related to the RG-I domains (28,32). Several recent literatures have reported that some RG-I domains of the pectin have great immuno-modulating activities (33). The Ara residues linked to the surface of the molecules played an important role in  HCC is usually asymptomatic or atypical in its early stages. When the patient feels obvious discomfort, most of the disease has entered the advanced stage. HCC is not sensitive to chemotherapy and radiotherapy. Common treatment strategies include surgical resection, liver transplantation, vascular intervention, and radiofrequency ablation. Missed the early surgical resection treatment period, and can only choose drug treatment. It is used for the treatment of hepatocellular carcinoma where cancer cells have specific receptors, mainly including epidermal growth factor receptor (EGFR) inhibitors, vascular endothelial growth factor receptor (VEGFR) antagonists, etc. Sorafenib was the only molecularly targeted drug approved for the treatment of advanced hepatocellular carcinoma in China. Traditional Chinese medicine treatment of hepatocellular carcinoma has attracted much attention, and many active ingredients of traditional Chinese medicine can also be used for targeted therapy of HCC.

CONCLUSION
In this study, the polysaccharides from mountain cultivated ginseng (MCG) were extracted, isolated and structurally characterized for the first time. Moreover, the isolated polysaccharides from MCG were tested for their cytotoxic effect against HepG2 cells compared with polysaccharides from CG. Results revealed that the homogeneous polysaccharides MCGP-1-MCGP-7 isolated from MTPS-1 have a good inhibitory effect on HepG2 cells, and they are time-and concentration-dependent. Therefore, MCG polysaccharide is expected to become a potential new drug for the treatment of HCC