The Extraction Of Polysaccharides And Usnic Acid From Ramalina Sinensis And Antibacterial, Antioxidant Activity

There are abundant of lichen resourses in China, especially Ramalina. And all species in this genera are wild. The officinal value of Ramalina is very high and spread all over grass.Ramalina sinensis Jatta belongs to Ascolichens,Lecanorales,Ramalinaceae, Ramalina Ach. and comprises 200 species in the world ,32 species in china and 8 species in Xinjiang . Ramalina sinensis Jatta is one of the 15 edible lichen resources in china and one of the tobacco flavors of lichens widely used in overseas.The experiment material of this thesis is Ramalina sinensis Jatta that was collected from Nanshan August First tree farm . This thesis will optimize the extraction technology of polysaccharides and usnic acid from Ramalina sinensis Jatta and carry through qualitative analysis and quantitative analysis. Besides, this thesis also include that the primary study of antioxidant activity of polysaccharides and usnic acid ,antibacterial activity of usnic acid. The primary results were as follows:1. On the basis of single factor test and orthogonal test ,the optimum condition of the extraction of polysaccharide was carried out:temperature 80℃for 2h and two times,the ratio of solid-liquid was 1:25.It was knowable from results of variance analysis ,in four extraction factors ,only the ratio of solid–liquid have distinct influence to the extraction of polysaccharide .2. The polysaccharide that extracted from Ramalina sinensis was determinated by DNS method and carried through accurity test and stability test , The results showed that ,the consistency of sample was 1.624mg/ml and the polysaccharide content of Ramalina sinesis was 4.06%.The RSD of accuracy test was 1.62%,the absorbency was stable in an hour ,the RSD was 1.35%.3. The reduction and the anti-oxidant effects of the polysaccaride was determined by spectrophotometry method .The results showed that the IC50 of polysaccharide to·OH and O2-·were 6.45mg/mL and 10.63mg/mL,the polysccharide extrated from Ramalina sinensis have higher reduction energy.4. Analysis monosaccharide components of the polysaccharide in Ramalina sinensis with HPLC was established. .The chromatographic conditions were as follows :the wavelength of 250nm;the solvent flow lml/min; the solventA: 15%(V) acetonitrile+50mmol/L PhosPhate buffer (KH2PO4一NaOH,pH 6.9); the solvcntB: 40%(V) aetonitrile+50mmol/L Phosphate buffer(KH2PO4一NaOH,pH 6.9) ;time gradient:0,10,55min; corresponding solventB concentration gradient: 0% ,10% ,30%.The results showed that the polysaccharide from Ramalina sinensis was composed by four different monosaccharides,they were Glucose,Mannose,Rhamnose,Glactose,their molar ratio was 5.05:3.89:0.14:0.09.5. The usnic acid that was extracted from Ramalina sinensis was tested by Color test (CT) ,Microcrystal test(MCT), Thin layer chromatography(TLC).On the basis of orthogonal test ,the optimum condition of the extraction of usnic acid was carried out:temperature 70℃for 3h and three times,the ratio of solid-liquid was 1:20.6. The content of usnic acid was determinated by HPLC method, The chromatographic conditions were as follows: Kromasil C18(200 mm×4.6nm,5um) column was used with mobile phase as methyl alcohol–water-peracetic acid (81:14:5).The flow rate was 1 mL/min;temperature was 30℃; detection wavelength was 254 nm;The result showed that, the usnic acid content that extracted from Ramalina sinesis is 18.32% .7. The reduction and the anti-oxidant effects of the usnic acid was determined by spectrophotometry method .The results showed that the usnic acid have higher reduction energy and the IC50 of usnic acid to·OH was 54.56 ug/mL .8. The antibacterial activity of usnic acid extracted from Ramalina sinensis to four different bacterias and four different fungus with Filter Paper method , The minimal inhibitory concentrations(MIC) and the minimum bactericidal concentration were also determined. .The results showed that ,usnic acid had higher antibacterial activity .The minimal inhibitory concentrations (MIC)of Usnic acid from Ramalina sinensis were 29.2ug/ml (Escherichia coli) ,23.26ug/ml(Staphylococcus aureus), 40.88ug/ml(Bacillus subtilis), 35.04 ug/ml(Bacillus thuringiensis).The minimum bactericidal concentration , 29.2ug/ml (Escherichia coli), 29.2ug/ml(Staphylococcus aureus), 40.88ug/ml(Bacillus subtilis), 35.04 ug/ml(Bacillus thuringiensis).Usnic acid from Ramalina sinensis have antibacterial activity to four different fungus ,the effects to Aspergillus niger and Saccharomyces cerevisiae more apparent than Rhizopus sp. and Pencillium sp.