| Plantaginaceae has three genera. There are18species in China, including Plantago asiatica L. and Plantag depressa Willd.. Plantago was widely used in traditional Chinese medicine. Viscous polysaccharide from the seeds coat of Plantago was confirmed to have catharisis and preventing harmful substances from diffusion activities. And it could also regulate blood glucose and lipid level since it could extend remaining time of food in stomach. The seeds of Plantago asiatica L. from Ji’an in Jiangxi Province were subjected for study in this research. The viscous polysaccharide from the seeds of Plantago asiatica L.(Plantago asiatica L. crude polysaccharide, PLCP) was carried out for laxative study. PLCP was further purified by modern chromatographic technique. The purified fractions were subjected for primary structure determination. To explore the mechanism of high viscosity of the polysaccharide, that the effects of Ca2+on polysaccharide’s physico-chemical, structural and conformational properties were studied. The last part of this research was about the polysaccharide’s separation and purification method exploration. During the research, it was found that polysaccharide’s separation and purification process was difficult and time consuming. Therefore, polysaccharides from Plantago asiatica L, Ganoderma sinense, Radix Codonopsis, Radix Ginseng and Radix Astragali were carried for determination of partial coefficient (k value) of polysaccharides in aqueous two phase system (ATPS). Only the polysaccharide of Radix Astragali which had higher partial coefficient value was carried out for establishing a new method for polysaccharide purification by high speed counter current chromatography (HSCCC). Main conclusions are summarized as follows:1. The relaxation promotion effects of polysaccharide from the seeds of Plantago asiatica L. on constipated mice were studied. The crude polysaccharide, PLCP, was prepared by water extraction and ethanol precipitation. PLCP could promote mice intestine peristalsis in vivo. And it could shorten black stool exhausting time of Zao-jie or Shi-re constipatd mice, increase defecation grain number of Pi-wei-xu-han constipated mice. For Han-jie constipated mice, PLCP could shorten black stool exhausting time, increase defecation grain number and defecation grain weight. Therefore, the polysaccharide from the seeds of Plantago asiatica L. could improve defecation of constipation mice.2. PLCP was separated and purified into PLP-1, PLP-2and PLP-3by gel column chromatography. The three fractions’physico-chemical properties were determined. The weight average molecular weight of PLP-1, PLP-2and PLP-3were3.29×106,1.73×106and1.29×106Da, respectively. Their sugar contents were79.18%,87.32%and82.08%. Uronic acid contents were17.48%,14.66%and20.13%and protein contents were0.03%,1.16%and0.04%. Ultraviolet visible spectroscopy showed there was little absorption at260or280nm for the three fractions, which confirmed there was little protein in the three fractions. Characteristic absorption of carboxyl group and β-pyranose ring was found in FT-IR.3. Primary structure of PLP-1was determined. PLP-1was composed of Rha (2.46%), Ara (33.98%), Xyl (58.23%), Man (0.83%), Glc (2.23%) and Gal (2.36%). Its mainly uronic acid was GlcA. The majority residues were α-T-linked Araf (9.58%), β-T-linked Xylp (8.71%),α-1,3-linked Araf(18.22%), β-1,3-linked Xylp (8.05%), β-1,4-linked Xylp (5.85%), β-1,2,4-linked Xylp (16.98%) and β-1,3,4-linked Xylp (27.52%). GlcA was existed as α-T-linked GlcAp. And there were other residues, including1,2-linked Rhap, T-linked Glcp,1,6-linked Glcp, T-linked Galp,1,3-linked Galp and1,3,6-linked Galp, etc. Other methods, such as partial acid hydrolysis,1D and2D NMR, were further used to determine PLP-1’s primary structure.4. PLP-2’s primary structure and its morphology were determined. PLP-2was composed of Rha (1.58%), Ara (31.65%), Xyl (60.13%), Man (1.58%), Glc (1.90%) and Gal (3.16%). Its mainly uronic acid was GlcA. The majority residues were α-T-linked Araf(7.37%), β-T-linked Xylp (13.45%),α-1,3-linked Araf(11.23%), β-1,3-linked Xylp (4.09%), p-1,4-linked Xylp (4.33%), β-1,2,4-linked Xylp (12.28%) and β-1,3,4-linked Xylp (29.12%). GlcA was existed as a-T-linked GlcAp. And there were other residues, including1,4-linked Glcp,1,6-linked Glcp,1,3,4-linked Galp, 1,3,6-linked Glcp, T-linked Rhap,1,3-linked Rhap and1,4-linked Galp, etc. Other methods, such as partial acid hydrolysis,1D and2D NMR, were further used to determine PLP-2’s primary structure. SEM observation indicated PLP-2was in linear aggregation.5. Primary structure of PLP-3was determined. PLP-3was composed of Rha (1.85%), Ara (39.65%), Xyl (55.93%), Glc (0.83%) and Gal (1.83%). Its mainly uronic acid was GlcA. The majority residues were a-T-linked Araf(8.13%), β-T-linked Xylp (15.58%), α-1,3-linked Araf(16.82%), β-1,3-linked Xylp (7.47%), β-1,4-linked Xylp (5.24%), β-1,2,4-linked Xylp (17.87%) and β-1,3,4-linked Xylp (24.24%). GlcA was existed as a-T-linked GlcAp. And there were other residues, including T-linked Glcp,1,2-linked Glcp and T-linked Galp, etc. Other methods, such as partial acid hydrolysis,1D and2D NMR, were further used to determine PLP-3’s primary structure.6. Rheological properties of PLCP were determined. PLCP was high in apparent viscosity which increased with increase of polysaccharide’s concentration. It was pseudoplastic fluids.1.0%PLCP exhibited weak gel property. The polysaccharide’s apparent viscosity decreased significantly when temperature increased. NaCl and CaCl2could increase PLCP’s apparent viscosity, especially CaCl2.CaCl2could improve PLCP’s gel property and thermal stability, even at its low concentration. But NaCl only improve PLCP’s gel property at much higher concentration.7. The effect of Ca2+on physico-chemical properties of PLCP was studied. The optical conditions of removing Ca from PLCP were obtained by single factor experiment. The Ca removed polysaccharide was named as PLCP-E. There was little difference in structure between PLCP and PLCP-E, but the apparent viscosity of PLCP-E decreased significantly. Ca+could improve PLCP-E’s apparent viscosity significantly. The polysaccharide apparent viscosity was equivalent to that of PLCP at the same concentration, when re-added Ca2+was removed from PLCP-E by dialysis. Ferulic acid content of PLCP was0.61mg/g. Apparent viscosity of ferulic acid removed polysaccharide (PLCP-FAR) decreased significantly. When Ca2+was added into PLCP-FAR, the apparent viscosity increased greatly. But it was much lower than PLCP at the same concentration, when the added Ca2+removed again by dialysis. The probable interactions between Ca and the polysaccharide were concluded, further supported by SEM observation, thermal stability analysis and previous reports. Ca in polysaccharide supposed to be existed in divalent ion, Ca2+. Ca2+may have interactions with ferulic acid and GlcA, both of which were linked to the polysaccharide’s chain, but mainly with ferulic acid. Polysaccharide’s chain was linked each other to form crosslinking. The interactions contributed to the polysaccharide’s apparent viscosity, thermal stability and linear morphology.8. Comparing with PLCP and PLCP-E, the effects of Ca2+on the polysaccharide’s conformation were studied. In the same solvent, including water,0.1M NaCl and0.5M NaOH, the intrinsic viscosity of PLCP-E was always lower than that of PLCP. Light scattering analysis results showed that0.5M NaOH was good solvent for PLCP and PLCP-E. Weight average molecular weight (Mw) was determined to be1.60×106and1.17×106Da for PLCP and PLCP-E, respectively. Character ratio p (1.49for PLCP,1.89for PLCP-E) indicated it was more rigid for PLCP-E. HPSEC analysis showed that the polydispersity index(Mw/Mn) of PLCP-E increased while the Mw was lower than PLCP. That suggested EDTA chelation reduced the polysaccharide molecular weight and improved its molecular weight distribution. Mark-Houwink index value (a) was0.51and0.55for PLCP and PLCP-E, respectively. The persistence length Lp was1.1and7.8nm for PLCP and PLCP-E. The above results indicated that PLCP-E was more rigid than PLCP.9. HSCCC combined with ATPS was used to separate and purify the polysaccharide of Radix Astragali. Single factor experiments combined with orthogonal design were used to optimize the k value of the polysaccharide from Radix Astragali in ATPS of PEG-MgSOr-Water. The k value was the highest when the ATPS was consisted with16.0%MgSO4,12.0%PEG1000. The optimized ATPS combined with HSCCC was used to separate and purify the water extract of Radix Astragali (2.2g) directly. Two fractions were obtained, RAWE-H-A (43mg, yield of1.95%) and RAWE-H-B (50mg, yield of2.27%). RAWE-H-A was high in purity, while it was low for RAWE-H-B. Mw of RAWE-H-A was1.09×106Da. It was between1.33-2.32×106Da for RAWE-H-B. Structure characters analysis results showed that RAWE-H-A was similar to the polysaccharide of RAP which was purified by traditional method of gel column chromatogram from Radix Astragali. But their structure was still some difference. |
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