This article is mentioned in:

Introduction

Bee pollen is the male gametophyte of gymnosperms and angiosperms, which contains all the necessary nutrients for plant growth and development. Bee pollen is known to be a natural and healthy food that improves human immunity (1). Bee pollen polysaccharides exhibit antitumor (2) and immunomodulating activities (3,4). Previous studies have demonstrated that pollen significantly inhibits tumor growth and enhances immunomodulating activity, while alleviating the distress of chemotherapy in cancer patients (5,6).

Polysaccharides constitute one group of the main components of plants, and have complex structural features including type I (RG-I) and type II rhamnogalacturonan (RG-II), homogalacturonan (HG) and arabinogalactan (AG). Polysaccharides have been reported to possess a broad spectrum of biological activities, particularly antitumor (7–10) and immunomodulating activities (11–16). Rosa rugosa is a common type of plants in China, and its bee pollen has been used as an immunological food for a long time. Polysaccharides are the main active components in bee pollen. However, there is limited information regarding the fractionation and antitumor activity of bee pollen polysaccharides. To the best of our knowledge, this is the first study to investigate the total fractionation and antitumor activity of bee pollen polysaccharides from Rosa rugosa.

Materials and methods

Materials

Bee pollen from Rosa rugosa was kindly provided by the Feed Research Institute Chinese Academy of Agricultural Sciences (Beijing, China). Sepharose® CL-6B was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium:Nutrient Mixture F12 (DMEM/F12) medium and calf serum were purchased from Gibco (Carlsbad, CA, USA). Trypsin was obtained from Amersco (Framingham, MA, USA), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) from Sigma (St. Louis, MO, USA), and penicillin/streptomycin from the Tianjin Hao Yang Biological Manufacture Co., Ltd. (Tianjin, China). All the additional analytical grade chemicals and reagents were produced in China.

Analytical methods

Total carbohydrate contents were determined using the phenol-sulphuric acid method (16,17). Uronic acid contents were determined using the m-hydroxydiphenyl method (16,18). All the gel permeation and anion exchange chromatographies were monitored by assaying total sugar and uronic acid contents. Sugar composition analysis was performed as described by Zhang et al(15) and Yu et al(19). Each polysaccharide sample (2 mg) was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120°C for 2 h. The monosaccharide derivatives were released using 1-phenyl-3-methyl-5-pyrazolone and analyzed on a DIKMA Inertsil ODS-3 column (4.6×150 mm) connected to a Shimadzu high performance liquid chromatography (HPLC) system (LC-10ATvp pump and SPD-10AVD UV-VIS detector).

Cell culture

The cell lines HT-29 and HCT116 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). HT-29 cells were cultured in DMEM/F12 medium supplemented with 10% calf serum and 100 IU/ml penicillin and streptomycin. HCT116 cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% calf serum and 100 IU/ml penicillin and streptomycin. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.

Cell proliferation assay

HT-29 and HCT116 cells were seeded in a 96-well plate (Costar®) at a density of 1×104 cells/well, adhered for 24 h, and then treated for 72 h with the polysaccharide fractions at varying concentrations (7). Control cells were similarly treated without the polysaccharides. The media were removed and 100 μl/well of MTT solution (0.5 mg/ml) were added. The supernatants were discarded after a 4-h incubation. The resulting formazan was dissolved in 100 μl DMSO and the absorbance at 570 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). All the experiments were performed in triplicate and repeated at least three times. Cell proliferation under all the conditions was expressed as a percentage of the control, which was set at 100%.

Statistical analysis

Data were presented as the means ± standard deviation (SD). Statistical analysis was performed using SPSS version 17.0 software. One-way analysis of variance (ANOVA) test was used to perform a statistical comparison between the treatment and control groups. P<0.05 and <0.01 were considered to indicate a statistically significant difference.

Results

Isolation and fractionation of bee pollen polysaccharides

The water-soluble polysaccharides were extracted from the bee pollen of Rosa rugosa with hot water. Following precipitation by the addition of 4 volumes of 95% ethanol, a total polysaccharides fraction, referred to as WRPP (water-soluble Rosa rugosa bee pollen polysaccharides), was obtained with a yield of 4.6% (w/w). WRPP contained 58.1% total sugar and 13.3% uronic acid. WRPP was a water-soluble light yellow powder. Sugar composition analysis by HPLC indicated that WRPP consisted of galactose (Gal) (21.4%), arabinose (Ara) (47.9%), rhamnose (Rha) (3.4%), galacturonic acid (GalA) (12.1%), glucose (Glc) (11.6%), mannose (Man) (2.6%) and glucoronic acid (GlcA) (1.0%).

WRPP contain neutral and acidic polysaccharides and showed a wide molecular weight distribution using Sepharose CL-6B (Fig. 1). To fractionate WRPP, it was applied on anion-exchange chromatography (Fig. 2). The elution profiles on DEAE-Cellulose column are shown in Fig. 3. Three fractions of WRPP-N, WRPP-1 and WRPP-2 were eluted with distilled water, 0.2 and 0.4 M NaCl, respectively. The molecular distribution on Sepharose CL-6B was analyzed (Fig. 1). WRPP-N exhibited one main peak near the total volume, while small curves prior to the main peak also existed, suggesting a mixture of polysaccharides with different molecular weights. WRPP-1 showed wide distribution from the out volume to the total volume of Sepharose CL-6B, while uronic acid showed a small peak. WRPP-2 exhibited a main peak near the out volume, while an additional peak of rich uronic acid appeared near the total volume.

Figure 1 Molecular weight distributions of polysaccharide fractions using Sepharose CL-6B. ■, total sugars; ○, uronic acid. Vo, out volume; Vt, total volume.

Figure 2 Fractionation scheme of bee pollen polysaccharides from Rosa rugosa.

Figure 3 Elution profiles of WRPP on DEAE-Cellulose by a stepwise gradient of NaCl. ■, total sugars; ○, uronic acid; dashed line, concentration of NaCl.

Composition analysis of the polysaccharide fractions

The monosaccharide compositions and yields of the collected fractions are listed in Table I. WRPP-N was composed of Glc (34.3%), Gal (25.0%), Ara (36.7%) and Man (4.0%), while uronic acid was not detected. The iodine test suggested that no starch was present, while WRPP-N was composed of glucan, arabinogalactans (AGs) and few mannoglucans.

Table I Yields and structral features of the collected fractions.

Table I

Yields and structral features of the collected fractions.

		Monosaccharide composition (%)
Fraction	Yield (%)	Rha	GalA	Gal	Ara	Man	Glc	GlcA
WRPP	4.6a	3.4	12.1	21.4	47.9	2.6	11.6	1.0
WRPP-N	26.9b	0	0	25.0	36.7	4.0	34.3	0
WRPP-1	21.4b	3.0	12.4	24.7	53.9	1.9	3.0	1.1
WRPP-2	13.1b	7.8	23.0	15.0	48.7	1.7	2.8	0.9

a Yield as % of bee pollen dry weight;

b yield as % of fraction applied to column.

{ label (or @symbol) needed for fn[@id='tfn3-mmr-07-05-1555'] } Rha, rhamnose; GalA, galacturonic acid; Gal, galactose; Ara, arabinose; Man, mannose; Glc, glucose; GlcA, glucoronic acid.

WRPP-1 consisted of Rha (3.0%), GalA (12.4%), Gal (24.7%) and Ara (53.9%), with minor amounts of Man, Glc and GlcA being detected (Table I). The amounts of Gal and Ara were >70% of total sugars. The ratio of Rha/GalA was 0.24, which was within the RG-I range of 0.05–1.0, defined by Schols and Voragen (20) and Yu et al(19). These results indicated small amounts of RG-I and HG domains, while a large proportion of AG and arabinan fragments was evident.

WRPP-2 consisted of Rha (7.8%), GalA (23.0%), Gal (15.0%) and Ara (48.7%), with minor amounts of Glc, GlcA and Man being detected. The ratio of Rha/GalA was ~0.34, suggesting the presence of RG-I, HG and AG.

WRPP-1 and WRPP-2 were mainly composed of Gal, Ara, Rha and GalA. Both had a large proportion of Gal and Ara, suggesting the existence of AG fragments. The amounts of GalA in WRPP-1 and WRPP-2 were 12.4 and 23%, respectively; one explanatory reason could be the elution order from the DEAE-Cellulose column. WRPP-2 contained more RG-I and HG domains compared to WRPP-1, while WRPP-1 contained more AG fragments compared to WRPP-2.

Antitumor effects in vitro

Human colon cancer HT-29 and HCT116 cells were treated with varying concentrations of various bee pollen polysaccharides from Rosa rugosa for 72 h. Cell proliferation was assessed using MTT assay. Following a 72-h treatment with total polysaccharides WRPP, cell proliferation was significantly inhibited in a dose-dependent manner. All the sub-fractions of WRPP also showed a concentration-dependent proliferation-inhibitory effect on HT-29 and HCT116 cells (Fig. 4). Notably, all the sub-fractions were less effective compared to the total fraction WRPP, suggesting that sub-fractions have a synergistic effect, which accounted for the anti-proliferative activity of bee pollen polysaccharides from Rosa rugosa on HT-29 and HCT116 cells in vitro.

Figure 4 Anti-proliferative effects of bee pollen polysaccharides from Rosa rugosa on HT-29 and HCT116 cells. HT-29 and HCT116 cells were treated with various polysaccharides at varying concentrations for 72 h, as indicated. Cell proliferation was determined using MTT assay. Columns, means from three independent experiments; bars, SD. *P<0.05, **P<0.01.

Discussion

In the present study, bee pollen polysaccharides from Rosa rugosa were extracted and fractionated. Total WRPP were purified to neutral (WRPP-N) and acidic polysaccharides (WRPP-1, WRPP-2). Acidic fractions contained different amounts of RG-I, HG and AG fragments. All the fractions inhibited the proliferation of HT-29 and HCT116 cells in a dose-dependent manner in vitro, indicating a potential antitumor activity. Sub-fractions showed a significant synergistic effect. To the best of our knowledge, this is the first study investigating the fractionation and antitumor activity of bee pollen polysaccharides from Rosa rugosa.

Acknowledgements

This study was supported by the Fundamental Research Funds for the Central Universities (no. 09SSXT125).

References