Preparation, Properties, Structure And Bioactivity Of Polysaccharides From Allium Macrostemon Bunge

Polysaccharide has become an important part of the research and development of natural medicines and health products. Most of the foreign pharmaceutical companies engaged in the carbohydrate drug research. As naturally occurring biological constituents, these high molecular weight polymers are highly appreciated for their multipurpose therapeutic properties, such as antioxidant, antitumor, immune-modulating and anti-inflammatory activities. A. macrostemon Bunge, a perennial herb that propagates vegetative and grows wild on the plains of China, Korea and Japan, belongs to the botanic family of Alliaceae. The young leaves and bulbs of this plant are used as a vegetable. Furthermore, the dried bulbs of A. macrostemon Bunge are well known as the traditional Chinese medicine "Xiebai".Up to now, research about A. macrostemon Bunge is mainly concentrated in the volatile oil and monomer compounds at home and abroad. The research in polysaccharide of A. macrostemon Bunge is far from perfect, which lack of an in-depth and a systematic study. In view of this, to establish the technological foundation for the development of modern multi-carbohydrate health food or sugar substances, to provide a theoretical basis for the further application of the A. macrostemon Bunge and its prescription, while to provide more references about research and development of polysaccharides in theory, academic and production practices were necessary. In this paper, we conducted a more systematic and in-depth research of polysaccharide from A. macrostemon Bunge about the optimization of the extraction process, the pure preparation methods, structural analysis and identification, activity screening evaluation and the structure and function relationship. Interpretation of these questions not only help to fully develop the use of our resources of Allium species, and to broaden its scope of application, but also to reveal its mechanism of action of traditional medicines will have a certain scientific value.A four-factor and three-level Box-Behnken design was used to optimise the extraction parameters for polysaccharides from A. macrostemon Bunge. Then, a graded ethanol precipitation was used to fractionate the water-extractable polysaccharides, resulting in three polysaccharides fractions. And the crude polysaccharides were purified by anion-exchange and gel filtration chromatography; The purified fractions were characterized by using the phenol-sulfuric method, periodic acid oxidation, Smith degradation, methylation combined with high-performance liquid chromatography (HPLC), gas chromatography (GC), Fourier-infrared spectroscopy (FT-IR), gas chromatography-mass spectrometry (GC-MS), etc. Finally, we investigated the antioxidant activities, the antitumor activities, antibacterial activity, neurotrophic activity, as well as hepatoprotective effects of polysaccharides from A. macrostemon Bunge, and described the initial relationship of the activity and structure of the polysaccharide. The main findings are as follows.1. Extraction optimisation of polysaccharides from Allium macrostemon BungeResponse surface methodology (RSM), based on a Box-Behnken design (BBD), was used to optimize the extraction conditions of polysaccharides (AMP). Four independent variables such as extraction temperature (℃), ratio of water to raw material (mL/g), extraction time (min) and extraction time were investigated. As a result, the optimal conditions for AMP extraction were the following:extraction temperature,87℃; ratio of water to raw material,12mL/g; extraction time,100min; and extraction times,3. Under these conditions, the experimental value was25.49±0.11%, which is well matched with value predicted by the model. The optimal conditions provide the basis of experimental data and theoretical analysis for the further development and utilization polysaccharides from A. macrostemon Bunge.2. Isolation, purification, physico-chemical characterization and structural elucidationThe dried pretreated bulbs of A. macrostemon Bunge was extracted with water by use of the optimal extraction conditions determined as mentioned above. The extract was then centrifuged, and the supernatant was mixed with absolute ethanol to a final ethanol concentration of40%,60%,80%. The resulting precipitate was obtained from centrifugation and dried, affording AMP40, AMP60and AMP80. The recovery rates were3.2%,11.8%and9.83%, respectively. The crude AMP were purified by DEAE-Cellulose52chromatography and Sephadex G-100chromatography to afford4fractions, AMP40-1, AMP40-2, AMP60-1and AMP80-1. Based on the characterization results of AMP by the phenol-sulfuric acid method, the Bradford assay method, the hydroxydiphenyl assay, we found that AMP40-2was quite different from the others, especially AMP40-2had relative higher content of uronic acid (30.22%). The molecular weights of AMP were determined by HPLC with size exclusion column. All of them showed a single and symmetrically sharp peak, indicated that they were homogeneous polysaccharide. According to the calibration curve of the elution times of standards, the molecular weights of AMP40-1, AMP40-2, AMP60-1and AMP80-1were estimated to be18.2kDa、105.0kDa、11.2kDa and8.1kDa, respectively.Monosaccharide composition analysis was acted by using the GC method. GC analysis showed that AMP40-1was composed of arabinose and glucose in a molar ratio of1.00:5.03. AMP40-2was composed of rhamnose, arabinose, glucose and galactose in a molar ratio of1.41:2.34:1.62:1.00. AMP60-1was composed of arabinose, glucose and galactose in a molar ratio of9.73:1.10:1.00. AMP80-1was composed of arabinose and glucose in a molar ratio of1.00:2.41. Results indicated that monosaccharide composition of AMP40-2was more complicated than the others.Infrared spectroscopy analysis indicated that the four samples showed typical peaks of polysaccharide. The characteristic absorption bands at833cm-1and894cm-1indicated that AMP40-2contained both a-glycosidic and β-glycosidic linkages, the band near1741cm-1was in coincidence with the fact that AMP40-2had relative higher uronic acid contents. UV analysis showed that the polysaccharides in the260-280nm no obvious absorption peak, indicate that it does not contain nucleic acids and proteins.The structural characterizations of purified polysaccharides were studied by using periodic acid oxidation, Smith degradation, methylation combined with HPLC, GC, GC-MS, FTIR and NMR spectra. Results showed that sugar rings of AMP40-1, AMP40-2, AMP60-1and AMP80-1were pyranose rings. AMP40-1were linked mainly by β-configuration glycosidic bond, and contained backbone chains of (1,2)-linked D-glucosyl and (1,2)-linked arabinosyl residues. The branch chains with non-reducing terminal glucose residues were attached to backbone chain by1�'6glycosidic bonds; AMP40-2were connected by a-configuration and β-configuration glycosidic bonds. Backbone chains of AMP40-2were linked by (1,6)-linked D-glucosyl and (1,6)-linked galactosyl residues. The branch chains with non-reducing terminal glucose, rhamnose and arabinose residues were attached to backbone chain by (1,2)-linked rhamnosyl glycosidic bonds; It was concluded that AMP60-1contained a backbone of (1,6)-linked D-glucosyl and (1,5)-arabinosyl residues with small amounts of the non-reducing terminal arabinose residues, also contained a minor (1,2,6)-D-glucose residue; AMP80-1were linked mainly by β-configuration glycosidic bond, and contained backbone chains of (1,2)-linked D-glucosyl residues. The branch chains with non-reducing terminal glucose and arabinose residues were attached to backbone chain by1�'6glycosidic bonds. Congo red dye experiments results indicated that AMP40-2possessed the triple helical structure.3. Antioxidant activities in vitro and hepatoprotective effects of polysaccharides from Allium macrostemon BungeThe antioxidant activities in vitro of polysaccharides from A. macrostemon Bunge were evaluated by measuring2,2-diphenyl-1-picrylhydrazyl (DPPH·) radical and hydroxyl radical (OH·) scavenging activity, superoxide radical scavenging activity (O2·), metal ion chelating activity and reducing power. During in vitro antioxidant assay, AMP40-2exhibited relative stronger scavenging activities than the others in most cases. The differences in antioxidant activities in vitro might be due to their differences in molecular weight, monosaccharide composition and uronic acid content.The antioxidant activities in vivo of crude AMP were investigated of against CCl4-induced acute liver damage in Kuming mice. The results showed that pretreatments with AMP40could improve markedly the activities of those antioxidant enzymes and the levels of CCl4-treated mice. The antioxidative system tended to be normalized by the protective action of AMP40. Results showed that molecular weight, monosaccharide composition and the uronic acid content of polysaccharide affects its antioxidant property directly.4. Antibacterial activity, the antitumor activity and neurotrophic activity of polysaccharides from Allium macrostemon BungeThe antimicrobial effects of extracts (AMW, AME and AMP) were evaluated by agar diffusion method. The results showed that:A. macrostemon Bunge has some anti-bacterial activity, its antibacterial ingredients present in the ethanol extract, may be a volatile oil or other small molecules, while the anti-bacterial activity of polysaccharides (water-soluble macromolecules) was weak.MTT assay was used to investigate the anti-proliferation activities against human gastric cancer cells (BGC-823) and human lung adenocarcinoma epithelial cells (A549) in vitro of AMP40-1、AMP40-2、AMP60-1and AMP80-1. The results showed that AMP40-1, AMP60-1and AMP80-1did not inhibit the growth of human lung cancer cell line A549, while AMP40-2inhibited proliferation of A549in a dose-dependent manner. For human gastric carcinoma cell BGC-823, AMP40-1、AMP40-2、AMP60-1and AMP80-1significantly inhibited proliferation in dose-dependent and time-dependent manner. The results demonstrate that the molecular weight, chemical property, monosaccharide composition and linkage type of polysaccharide play important roles in the antitumor activity of polysaccharide.The neurotrophic activity of polysaccharides from A. macrostemon Bunge were investingated by PC12cell assay. The screening results showed that AMP40-2induced the differentiation of PC12cell and has obvious neurotrophic activity.