Study On Extraction, Purification, Structure And Antioxidative Activities Of Scutellaria Barbata Polysaccharide

Scutellaria barbata D.Don,a herb belonging to the Lamiaceae family,is widely distributed in china.This herb is known in traditional Chinesse Medicine Ban-Zhi-Lian and has been used as an antitumor,anti-inflammatory,antifebrile agent.The main chemical compositions are flavonoids,alkaloid,essential oils and polysaccharide.Polysaccharide which is the one of major component of Scutellaria barbata D.Don have many kinds of bioactivities.But the study were seldom carried out on isolation,purification,structure and property of polysaccharides from Scutellaria barbata D.Don.This paper studied the extraction,separation,purification and sulfation of Scutellaria barbata D.Don polysaccharide,the antioxidant activity of SBP,SBP₁,SBP₂,SBPâ… ,SBP-L,SBP-L₁ and SBP-L₂ and their chemical structure,which gave a foundation for the further research..The main results and innovations were as follows:1.Based on the single factor experiment,designing orthogonal experiments to optimize the extraction process of crude Scutellaria barbata polysaccharide,the optimal parameters of extracting Scutellaria barbata polysaccharide by hot water were:the extraction temperature was 60℃, solid-liquid ratio was 1:20,extraction time was 2 h and extraction times were 4,and the rate of polysaccharides extraction was 2.82%.The amount of carbohydrates was measured with phenol-sulfuric acid method.2.This paper studied on de-coloration of Scutellaria barbata polysaccharide.which compared the de-coloration capacity of five different kinds of de-coloration agents:active carbon,neutral alumina,macroporous adsorption resins AB-8,LSA-21 and NKA-9.The better de-coloration agents was AB-8.Decoloring experiments were carried out by single factor experiment.The best conditions for de-coloration were determined as follows:the pH is 7,the temperature is 40℃,the content of polysaccharides is 1046.5 mg/L.Adopt the best condition,holding rate of polysaccharides is 88.46%,de-coloration rate is 90.65%.3.Polysaccharides(SBP) were treated after de-coloring by quaternary ammonium salt precipitation method.SBP₁ and SBP₂ were obtained.SBP was isolated using DE-52 cellulose chromatography in series processes.SBPâ… and SBPâ…¡was obtained respectively eluting the DE-52 cellulose chromatography with distilled water and the solution of 0.1 moL/L NaCI.SBPâ… was identified as homogeneous polysaccharide component by SephadexG-200 gel chromatography,HPLC,UV spectrum and freeze-thawing. 4.Chemical modification of sulfation reaction about SBP,Scutellaria barbata polysaccharide Sulfate(SBP-L) was obtained and its water-sohibility was increased.By scanning it with UV spectrum,the results showed that it had absorption peaks at 262 nm.The content of sulphate groups was 5.73 mg/mL,the proportion of sulfate substitution was 2.89.IR showed that it has a-O-SO^3- absorption peaks at 1258 cm^-1.5.The monosaccharide components of Scutellaria barbata polysaccharide were measured by GC.Three methods of TMS,acety.lated aldononitriles and sugar alcohol acetates were compared to confirm acetylated aldononitriles which is one of the best methods.Hydrolyzing experiment were carried out by single factor experiment.The best conditions were determined as follows:the concentration of sulphuric acid was 0.5mol/L,the volume was 2 mL,the time was 12 h and the temperature was 100℃.GC result showed that SBP was mainly composed of rhamnose,arabinose, xylose,mannose,glucose and galactose,and the mole proportion of monosaccharide was 3.26:0.37:4.85:5.16:4.10:9.47.SBP-L was mainly composed of arabinose,mannose,glucose and galactose,and the mole proportion of monosaccharide was 0.84:6.67:3.62:5.53.SBP₁,SBP₂,SBP-L₁, SBP-L₂ and SBP I were mainly composed of rhamnosexylose,mannose,glucose,galactose and the mole proportion of monosaccharide respectively were 4.51:5.50:4.86:4.90:8.22,3.25:6.20:5.85: 4.05:11.30,3.86:4.39:4.96:4.95:7.73,3.04:6.87:5.51:4.37:10.90,3.53:4.62:7.22:4.08:8.37.6.The average molecular weights of SBP₁,SBP₂,SBPâ… ,SBP-L₁ and SBP-L₂ were approximately 290000,340000,300000,360000 and 390000Da.,determined respectively by HPLC. Partial hydrolysis with sulphuric acid,periodate oxidation,Smith degradation,GC and IR was used to determine their structure.The results showed that:The main chain of SBP₁ and SBP₂ was made up ofβ—(1→2)—pyranose glucosan and galactose,and the side chain of SBP₁ was composed of(1→3, 4,6)—rhamnose,arabinose,xylose,mannose and glucose.The mole proportion of bond(1→6): (1→3):(1→2 or 1→4)was 9%:22%:69%.The side chain of SBP₂ was composed of(1→3, 4,6)—rhamnose,arabinose,mannose and glucose.The mole proportion of bond(1→6):(1→3): (1→2 or 1→4) was 12%:26%:62%.The main chain of SBPâ… was made up ofβ—(1→2,3)—pyranose glucosan and galactose,and the side chain of SBPâ… was composed of(1→4,6)—rhamnose,arabinose,mannose and glucose.The mole proportion of bond(1→6):(1→3):(1→2 or 1→4)was 10.6%:47%:42.4%.The main chain of SBP-L₁ was made up ofβ—(1→2)—pyranose glucosan and galactose,and the side chain of SBP-L₁ was composed of(1→3,4,6)—rhamnose, arabinose,xylose,mannose and glucose and had a fewα—pyranose glucesidal bond.The mole proportion of bond(1→6):(1→3):(1→2 or 1→4) was 9.6%:38.6%:51.8%.The main chain of SBP-L₂ was made up ofβ—(1→2)—pyranose glucosan,galactose and arabinose,and the side chain of SBP-L₂ was composed of(1→3,4,6)—rhamnose,mannose and glucose.The mole proportion of bond(1→6):(1→3):(1→2 or 1→4) was10.6%:24.2%:65.2%.7.Antioxidative activities of seven kinds of Scutellaria barbata polysaccharides(SBP,SBP₁, SBP₂,SBPâ… ,SBP-L,SBP-L₁ and SBP-L₂) in vitro were studied:the capability of the polysaccharides to eliminate O^2- was determined through pyrogallol auto oxidation;the ability of the polysaccharides to scavenge hydroxyl radicals was determined by the system of ferrosin-iron-H₂O₂ and the ability of the polysaccharides to scavenge NO₂^- in the condition of pH for simulation gastric juice in vitro.The result showed that the seven kinds of Scutellaria barbata polysaccharides had different antioxidation activities in vitro,and their activities depended on their amounts.The antioxidation activities of SBP was better than separate polysaccharides SBP₁ and SBP₂.The antioxidant activities of sulfation of Scutellaria barbata polysaccharide SBP-L and SBP-L₂ were increased than SBP and SBP₂,through comparing with SBP₁ and SBP-L₁ the ability of the SBP₁ to scavenge hydroxyl radicals and NO₂^- is better,but the capability of the polysaccharides to eliminate O^2- was reverse.This paper studied that seven kinds of Scutellaria barbata polysaccharides had different antioxidation activities in vitro because all kinds of them had different percent ofβ-(1→3) glucesidal bond.