| Sugarcane Leaves are leaves of the plants named Saccharumsinensis Roxb. and Saccharum officinarum Linn. which belongs to thegenus of Saccharum in the family of Poaceae(Gramineae), and sugarcaneis widely distributed in tropic and subtropic area including more than100countries in the worls. Global total planting areas reached19millionhectares. Sugarcane also widely cultivated in China, leading to the outputof15million tons per year.Yield of sugarcane in Guangxi ZhuangAutonomous Region, one of the major sugarcane producing area,accounts for60percent of the nation’s harvest. Sugarcane Leaf is one ofthe agricultural residues which come from the process of sugarcaneplanting, showing a big amount with15percent of the sugarcaneyield.Apart from some animal feed, this rich resource is abandoned orburned that create very big resource waste and environmentalpollution.On the other hand, shortage of the resources of Chinesemedicine is a serious sustainable development problem in China.Variousfactors, such as deterioration of the natural environment, the changes of the plant species and disorder of the collection of herbs, aggravated theunfavourable situation. It is significant for protection and development ofChinese medicine resources to study on this crop with medicinal value，discover its new use.The thesis consists of two parts of studies on the chemicalconstituents and bioactivity from Sugarcane Leaves.20Compounds wereobtained and identified from Sugarcane Leaves for the first time.Polysaccharides were isolated, purified and identified,then they wereevaluated for hypoglycemic effects and machanism, neuroprotectiveeffects, anti-neuroinflammatory activities using in vitro and in vivoexperiments.Moreover, inflammatory effects of total flavonoids fromSugarcane Leaves, acute toxicity of Sugarcane Leaves were observed.Theresearches provide experimental foundation and scientific basis for thefurther medicinal study and development of Sugarcane Leaves.Chapter1: Chemical Constituents of Sugarcane LeavesExperiment1: Preliminary Tests for Chemical Components ofSugarcane Leaves in two Different Cultivated SpeciesObjective: To identify the chemical components of thetwo different cultivated species of Sugarcane Leaves usingcomprehensive preliminary tests． Methods: Aqueous extract,95%ethanol extract, and petrol ether extract were tested by test-tubeexperiment method to preliminarily demonstrate the chemicalcomponents of two different cultivated species of SugacaneLeave．Results: The two different cultivated species of Sugarcane Leavesmight contain amino acids, sugar， polysaccharides, organic acids,flavonoids, phenols, coumarins, lactones, sterols, triterpenes. Conclusion: Sugarcane Leaves are rich in effective components and have certainproperties can be used for some medical purpose．Experiment2: Chemical Constituents of Dichloromethane Fractionfrom Sugarcane LeavesObjective： To study the chemical constituents ofdichloromethane fraction from Sugarcane Leaves. Methods：Compoundswere isolated and purified by silica gel column chromatography, gelchromatography, polyamide column chromatography, medium-pressureliquid chromatography(MPLC) and high performance liquidchromatography. Their structures were established by physical andchemical identification methods and spectroscopic methods. Results：12compounds were isolated, then the structures of10compounds wererespectively established as: methyl4-hydroxy benzoate(14),(E)-methyl3-(4-hydroxyphenyl)acrylate(15), dehydrovomifoliol(16),vomifoliol(18),4-hydroxy-3,5-dimethoxybenzoic acid(20),7-(((2S,3R,4R)-3,4-dihydroxy-4-(hydroxylmethyl)tetrahydro-furan-2-yl)oxy)-2-(3,4-dihydroxyphenyl)-4H-chromen-4-one(22), loliolide(27)3-hydroxy(3-hydroxy-4-methoxyphenyl)-5-(4-hydroxy-3-methoxyphenyl)-4-(hydroxymethyl)dihydrofuran-2(3H)-one(28),3a,7-dihydroxy-4-(4-hydroxy-3-methoxyphenyl)-6-methoxy-3a,4,9,9a-tetrahydronaphtho[2,3-c]furan-1(3H)-one(29),1-(4-hydroxy-3-methoxy-phenyl)heptan-1-one(32). Conclusion：The10Compounds were obtained from Sugarcane Leaves for the first time.Experiment3: Isolation, Purification, Characterization ofPolysaccharides from Sugarcane Leaves Objective： Isolation, purification, identification,component analysis and structural elucidation were performed on thepolysaccharides from Sugarcane Leaves. Methods：The polysacchareideswere isolated by water-extraction and alcohol-precipitation method,further purified by DEAE column chromatography, Sephadex columnchromatography, Q-Sepharose fast flow ion exchange chromatography,Sepharcryl S-300gel chromatography; molecular weight was measuredby HPGPC; monosaccharide composition of the polysaccharide wasanalyzed by precolumn derivatization HPLC; structure of thepolysaccharide was analyzed by IR,1D-NMR,2D-NMR, GC-MS.Results：The powder of Sugarcane leaves were extracted3times byboiling water for2h per time. The aqueous extracts were then fractionatedby adding3times95%EtOH, resulting in one water-soluble crudepolysaccharide fraction. Crude polysaccharide was applied toDEAE-Cellulose, gel filtration chromatography, Q-Sepharose fast flowion exchange chromatography, Sepharcryl S-300gel chromatography,leading to isolation of8water-soluble polysaccharide fraction in whichtwo polysaccharide SLP0A and SLP3A were further evaluated. The purityof SLP0A is95.1%, total sugar content is82.7%, protein contentcontent is7.8%, glucuronic acid content is3.2%. The relative molecularweight of SLP0A is10.7kDa，as well as it is mainly composed of Glc,Gal, Ara, Man, Xyl, Rha, GlcA in the mole ratio of33.3:29.1:14.5:9.4:4.0:1.2:1.0; The purity of SLP3A is88.7%, total sugarcontent is70.8%, protein content content is14.7%, glucuronic acidcontent is4.6%. The relative molecular weight of SLP3A is59.8kDa, it iscomposed of Gal, Ara, Glc, as well as a small concentration of Xyl, Man, Rha, GlcA, in the mole ratio ofGlc:Gal:Ara:Man:Xyl:Rha:GlcA=7.3:15.5:9.4:2.4:3.6:2.2:1.0. Differentend-groups of sugar ring of SLP0A can be α-configuration orβ-configuration according the different spectrum：End-group A is α-Glc;end-groups B and D are'3)-L-Ara-(α-1'and'2,4)-L-Ara-(α-1'respectively; end-groups C and E are α-Xyl and α-Rha respectively;end-group F is β-Man; end groups G and H are β-Gal in various linkages.Conclusion： SLP0A and SLP3A, two heteropolysaccharides withdifferent MW and structures, were obtained from Sugarcane Leaves forthe first time.Experiment4: Chemical Constituents of the Supernatant Obtainedby the Water Extraction and Alcohol Precipitate Method fromSugarcane LeavesObjective：To study the chemical constituents of theSupernatant obtained by the water extraction and alcohol precipitationmethod from Sugarcane Leaves. Methods：Compounds were isolated andpurified by D101macroporous resin column chromatography, gelchromatography, medium-pressure liquid chromatography(MPLC) andhigh performance liquid chromatography. Their structures wereestablished by physical and chemical identification methods andspectroscopic methods. Results：8compounds were isolated, then theirstructures were respectively established as:(2R,3R,4S,5S,6R)-2-(2-(4-hydrooxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-(methoxy-2,3-dihydrobenzofuran-5-yl)ethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-trio(1),2,3,4,5-tetrahydroxy-5-(4-hydroxy-3,5-dimethoxyphenyl) pentanoic acid(2),1-(4-hydroxy-3,5-dimethoxyphenyl)butane-1,2,3,4-tetraol(3)、2-(2-(2,3-dihydroxy-3-(3,4,5-trimethoxyphenyl)propoxy)-4,5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-3-yloxy)-6-(hydroxymethyl)-tetrahydro-2H-pyran-3,4,5-triol(4),1-(3,5-dimethoxy-4-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxylmethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)butane-1,2,3,4-tetraol(8),5-3,5-dimethoxy-4-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-2,3,4,5-tetrahydroxypentanoicacid(9),4-hydroxy-3-methoxybenzoic acid(11),1-(4-hydroxy-3-methoxyphenyl)ethanone(12). Conclusion： The8Compounds were obtained from Sugarcane Leaves for the first time.Chapter2: Studies on Pharmacodynamics of Sugarcane LeavesExperiment1: Acute Toxicity Study of Sugarcane Leaves in MiceAbstract Objective: The acute toxicity test of Sugarcane Leaves inmice was carried out to esgablish base for further research. Methods: Thetoxic effect was observed, MTD and MD were test, LD50were calculatedby Kaber method and Sequence Method。Results: Aqueous extraction,30%ethanol extraction and50%ethanol extraction of Sugarcane Leavesshowed toxicity except SLP. Conclusion: The results suggested thatSugarcane Leaves are of toxicity, but its leading cause of death needmore research to confirm.Experiment2: Protective Effects of Sugarcane Leaf Polysaccharideson PC12CellsAbstract Objective: To study the protective effects of SugarcaneLeaf polysaccharides against serum-deprived injury and rotenone-inducedinjury respectively in PC12cells. Methods: All experiments were carry out with PC12in vitro, which were induced by serum deprivation androtenone respectively. Cell viabilities were measured by MTT assay.Results: The results show that comparing with the model group, SLPgroups could increase the survival rate of serum-deprived PC12cells（P<0.05）, whereas aggravated rotenone-induced injuries of PC12cellson a small scale that was not statistically significant. Conclusion: SLPhas neuroprotective effects but its mechanism requires further research toclarify.Experiment3: Anti-neuroinflammatory Effects of Sugarcane LeafPolysaccharidesAbstract Objective: To observe the anti-neuroinflammatory effectsof polysaccharides from Sugarcane Leaves. Methods: The model ofneuroinflammation was established by stimulating microglia BV2withlipopolysaccharide(LPS) leading to NO production, and treated withRSLP1and RSLP1-2respectively, the cells viability was determined byMTT assay．Results: SLP(10-5mol/L) and curcumin(10-7mol/L) are equalin the inhibition ratio on inflammation of BV2cells. Conclusion: SLPhas a certain anti-neuroinflammatory effects．Experiment4: Anti-inflammatory Effects of Total Flavonoids fromSugarcane LeavesAbstract Objective: To study the anti-inflammatory effects of totalflavonoids from Sugarcane Leaves. Methods: The mice models of acuteauricle swelling induced by dimethyl benzene，capillary permeabilityinduced by acetic acid and granuloma induced by tampon wereestablished to observe the anti-inflammatory effects of SLTF via oraladministration. Results: Compared with the model group, the ear edema resulted from dimethyl benzene were inhibited in the medium-dose andlow-dose groups(P<0.05), as well as significantly inhibited in thehigh-dose group(P<0.01); the increase of vascular permeability caused byacetic acid were inhibited markedly at each dose(P<0.05). Moreover, thehigh-dose and medium-dose of SLTF can inhibit the granulomahyperplasia in mice(P<0.05), the low-dose showed slight inhibition thatwas not statistically significant.Conclusion: The total flavonoids ofSugarcane Leaves have anti-inflammatory effects．Experiment5: Screening of Hypoglycemic Activity of DifferentExtracts from Sugarcane LeavesAbstract Objective: To evaluate the hypoglycemic activity of thedifferent extracts of sugarcane leaves. Methods: Adrenalin, alloxan andstreptozocin were used to induce hyperglycemia in three mice models,and then treated them with the different extracts of sugarcane leaves toobserve the impact of blood glucose via oral administration. Results: Inthe adrenalin-induced mice, aqueous,50%alcohol extract, petroleumether extract and n-butanol extract of sugarcane leaves can inhibit theblood glucose level in varying degrees and in normal mice they have littleeffect on the blood glucose level(P<0.05). In the alloxan-inducedmice,each extract of sugarcane leaves can inhibit the blood glucose levelin varying degrees(P<0.05). In the streptozocin-induced mice,aqueous,30%and50%alcohol and ethyl actate extract of sugarcaneleaves can decrease the blood glucose level in varying degrees(P<0.05).Conclusion: aqueous,30%and50%alcohol extract of sugarcane leaveswere the active fractions of hypoglycemic effects because of their betteractivities than other extracts. Experiment6: Hypoglycemic Activities of Polysaccharides fromSugarcane Leaves and their Mechanism in the Diabetic RatsAbstract Objective: To study hypoglycemic activities ofpolysaccharides from sugarcane leaves,and investigate the mechanism.Methods:Rats with streptozotocin-induced diabetes were used as model,hypoglycemic activities of polysaccharides from sugarcane leaves wereobserved. Blood glucose was determined by blood glucosemornitoring.Insulin was detected by ELISA method.The pancreaticinflammatory cell infiltration, pathological indicators were observed byHE staining. Secreting levels of IL-2、TNF-α and IL-17in serum weredetected by ELISA method. The mechanisms of SLP on regulating bloodglucose in diabetic rats were discussed. Results: SLP can reduced theblood glucose and insulin in the diabetic rats. The pathologicalexperimental results showed that SLP has protective effects on damagedpancreatic islets more or less.In addition, SLP has the inhibitory effectson over-expression of IL-2, IL-17, TNF-α in serum of diabetic rats.Conclusion: SLP is the hypoglycamic active component of SugarcaneLeaves and its mechanism might be related to the protective effects onpancreatic islets and immunoregulation．... |
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