Isolation, Purification, Structure And Bioactivity Of Polysaccharides From Mulberry (Morus Alba L.) Leaves

Mulberry (Morus alba L.) that belongs to the genus Morus in the family of Moraceae,is widely distributed in tropical, subtropical and temperate areas and is grown widely in different regions of north and south China. So, resources of this plant are abundant. The mulberry leaves have been used as a traditional Chinese herbal medicine for a long time.The main effects include dispelling wind and heat from the body, clearing lung-heat,moistening dryness, clearing liver, improving vision and so on. Early researches regarding mulberry leaves were mainly focused on alkaloid, flavonoid, polyphenol, etc. In recent years, mulberry leaves polysaccharides (MLP), the main active components of mulberry leaves, have attracted increasing attention. However, current research regarding MLP is still limited. In this dissertation, MLP was studied systemically, including optimization of extraction condition, determination of static rheological properties, isolation and purification, physicochemical and structure characterization, simulative digestive in vitro,antioxidation activity and immunomodulation activity to lay a theoretic foundation and to provide a technical support for the development and utilization of health foods and drugs regarding MLP. Main results are listed as follows:1. Research on the extraction condition and rheological property of MLPEffects of extraction parameters including extraction temperature, extraction time,ratio of water to raw and extraction cycles on the yields of MLP were investigated by single-factor tests. Accordingly, the factors (i.e. extraction temperature, extraction time and ratio of water to raw material) which markedly affected the MLP yield and their proper ranges were screened out based on the preliminary experimental results. A three-level,three-variable Box-Behnken design (BBD) was applied to determine the optimal levels of extraction variables for the extraction of MLP. By using the software of Design Expert version 8.0.6, the optimum extraction parameters were obtained as follows: extraction temperature 92 ?, extraction time 3.5 h, ratio of water to raw material 34 mL/g and extraction times 2. Under these conditions, the experimental value was 10.0 ± 0.50%.Effects of concentration, temperature, sucrose, pH and NaCl on the static rheological property of MLP were investigated. Result showed that the apparent viscosity of MLP increased with the increase in the MLP concentration, indicating that the MLP solution was a non-Newtonian pseudoplastic fluid. The MLP's apparent viscosity increased significantly when the temperature decreased. Acid treatment led to a sharp drop in the apparent viscosity of MLP. However,the alkali-treated groups had a slight effect on the apparent viscosity. The addition of sucrose made an increase of the viscosity. The apparent viscosity of MLP did not obviously changed with addition of low concentration of NaCl, and gradually increased with further increase of the NaCl concentration.2. Isolation, purification, physicochemical characterization and structure analysis of MLPThe crude MLP was separated by a DEAE-52 cellulose chromatography column,affording three polysaccharide fractions of MLP-1,MLP-2 and MLP-3. The main fraction MLP-3 was further purified with a Sephadex G-100 gel permeation chromatography column, resulting in two fractions of MLP-3a and MLP-3b. The homogeneity and molecular weights of MLP-3a and MLP-3b were analyzed by HPGPC. Results showed that both MLP-3a and MLP-3b were homogenerous polysaccharide and their average molecular weights were 80.99 kDa and 3.64 kDa,respectively.The content of carbohydrate, protein, uronic acid and sulfuric radical and total polyphenols in crude MLP, MLP-3a and MLP-3b were determined according to the reported methods of sulfuric acid-phenol coloration, coomassie brilliant blue coloration,meta-hydroxydiphenyl method, barium chloride-gelatin method and Folin-Ciocalteu assay,respectively. The carbohydrate contents were 52.09%, 89.74% and 37.20%, respectively.The protein contents were 2.16%,0.83% and 0.22%, respectively. The uronic acid contents were 32.45%, 6.53% and 65.29%, respectively. The sulfuric radical contents were 1.73%,1.40% and 1.22%, respectively. The total polyphenols contents were 1.93, 0.17 and 0.16(mg GAE/100 mg), respectively.Structures of crude MLP,MLP-3a and MLP-3b were determined by high-performance liquid chromatography (HPLC), infrared spectroscopy (IR), ultraviolet spectrum,methylation analysis and nuclear magnetic resonance spectroscopy (NMR). Results showed that the monosaccharide composition and molar ratios of crude MLP, MLP-3a and MLP-3b were determined as (Man: Rha: GlcA: GalA: Glc: Gal: Ara = 0.51: 5.13: 2.23: 3.02: 2.13:2.95: 2.55), (Man: Rha: GlcA: GalA: Glc: Gal: Ara = 0.77: 4.53: 0.81: 1.21: 3.47: 12.55:11.14), (Rha: GlcA: GalA: Gal: Ara = 1.57: 0.20: 6.10: 1.27: 0.89), respectively. The backbone chain of MLP-3a was linked by ?-1,3-Ara and ?-1,4-D-Gal residues. The branch chains contained 1,3,5-Ara, 1,3,6-Glc, 1,2-Rha and 1,4-GalA. The GalA residues in MLP-3a were partially acetylated. The backbone of MLP-3b could be expressed by[-1,4-?-GalA-1,4-?-GalA-]n-[-1,2-a-Rha-1,4-a-GalA-]m and some GalA residues were methyl esterified and acetylated. There were side chains at the C4 of Rha in MLP-3b, which were composed of ?-1,4-Gal, ?-1,5-Ara and ?-1,3-Ara.3. In vitro digestion of MLP by saliva, simulated gastric juice and gastrointestinal juiceThe saliva, simulated gastric juice and gastrointestinal juice digestion of crude MLP and its purified fractions (MLP-3a and MLP-3b) were investigated in vitro. It was found that after digestion in saliva, the molecular weight of MLP remained unchanged and no reducing sugar and monosaccharides were detected, indicating that MLP were not digested in saliva. After digestion in gastric juice and gastrointestinal juice, the molecular weight of MLP remained unchanged. Reducing sugar contents of digested production at different times did not change significantly. In addition, there was no monosaccharide released during the gastric juice and gastrointestinal juice digestion. Therefore, MLP were resistant to the gastric juice and gastrointestinal juice.4. Antioxidant activity of MLPAntioxidant activities in vitro of MLP were measured by using the chemical methods including scavenging activities of DPPH radical, hydroxyl radical, superoxide radical,ABTS radical, chelating activity of metal ions, determination of reducing power, activity of anti-lipid peroxidation, inhibition of (3-carotene-linoleate autoxidation and evaluation by rat pheochromocytoma PC 12 cell model. The results indicated that crude MLP and its purified fractions (MLP-3a and MLP-3b) had weak reducing power, moderate DPPH radical scavenging activity, strong scavenging activities of hydroxyl radical, superoxide radical and ABTS radical, and strong ferrous ion chelating activity. They also showed strong inhibition effects on liver lipid peroxidation and ?-carotene-linoleate oxidation. Crude MLP and MLP-3b can protect PC 12 cell against H2O2-induced oxidative lesion.5. Immunomodulatory activity of MLP-3aBy using the RAW264.7 model, the in vitro immunomodulatory activity of MLP-3a was evaluated. It was found that MLP-3a could significantly stimulate the proliferation of macrophages, enhance the capability of macrophage phagocytosis, increase the activity of acid phosphatase, and promote the production of nitric oxide, mouse tumor necrosis factor(TNF)-a, mouse interferon (IFN)-?,and mouse interleukin (IL)-1?. The results preliminarily suggested that MLP-3a possessed potent immunomodulating effect. The immunomodulatory activity of MLP-3a was further evaluated by using Cyclophosphamide(Cy)-induced immunosuppression mice models. The results showed that MLP-3a can increase the thymus index, spleen index, hemolysin level, lysozyme content and the activities of CAT, T-AOC, T-SOD, GSH-Px, and reduce the MDA level.