Study On Structures Of The Neutral Carbohydrates From Fifty Medicinal Plants

Medicinal plants are rich in carbohydrates,which act as major functional components and have diverse structures and biological activities.The medicinal plants carbohydrates could be classified into neutral and acidic carbohydrates according to the existence of uronic acid or not.They also could be classified according to the degree of polymerization(Dp)into oligosaccharides(Dp 2～19)and polysaccharides(Dp>19).Acidic carbohydrates in medicinal plants are pectin and pectin-derived oligosaccharides,which have been studied widely for years.However,there were few thorough studies about the structures and functions of neutral carbohydrates in medicinal plants,which blocked the comprehensive understanding of carbohydrates in medicinal plants.In this thesis,fifty kinds of medicinal plants were selected to prepare their neural carbohydrates,then the structures of representative neutral carbohydrates were analyzed by a combination of chemical,biological and instrumental methods.The specific research results are summarized as:The total carbohydrates were extracted from fifty kinds of medicinal plants by using hot-water extraction and ethanol precipitation,then were purified by a combination of ion-exchange chromatography and membrane ultrafiltration to obtain neutral carbohydrates.Among the fifty medicinal plants,twelve medicinal plants mainly contained neutral oligosaccharides,and the yields of neutral oligosaccharides in relation to raw materials ranged from 1.1%to 50.6%.Medicinal plants neutral oligosaccharides were classified into raffinose family oligosaccharide,fructo-oligosaccharides,gluco-oligosaccharides and hetero-oligosaccharides according to the monosaccharide compositions.Both raffinose family oligosaccharide and fructo-oligosaccharides are sucrose-based oligosaccharides.Thirteen raffinose family oligosaccharides were purified from neutral oligosaccharides of Salvia miltiorrhiza Bge,Stellaria dichotoma L.var.lanceolata Bge and Pseudostellaria heterophylla(Miq.)Pax by size exclusion and HILIC chromatographies.Comparative structural analyses were conducted on fructo-oligosaccharides from six kinds of medicinal plants including Cimicifuga foetida L,etc.The structures of gluco-oligosaccharides and hetero-oligosaccharides were not further analyzed because of difficult purification and their complex compositions.Other thirty-eight medicinal plants mainly contained neural polysaccharides,the yields of neural polysaccharides in relation to raw materials ranged from 0.2%to 10.3%.Medicinal plants neutral polysaccharides were classified into glucan,mannan,galactan and hetero-polysaccharides according to the monosaccharide compositions.Eight representative polysaccharides were chosen for further structural analysis.HPAEC-PAD,MALDI-TOF-MS,1D/2D NMR spectra and methylation analyses were combined to characterize the structural features of representative medicinal plants sucrose-based oligosaccharides.The thirteen raffinose family oligosaccharides contained four saccharides(manninotriose,stachynose,lychnose and stellariose)whose structures had been reported.The other nine saccharides were new natural compounds which were first reported in this study.Their structures were identified as:Galα1,6Galα1,6Glcα1,2Fruβ1,1αGal,G alα1,6Glcα1,2Fruβ1,1Galα6,1αGal,Glcα1,2Fruβ(1,1Galα)3,1Galα6,1αGal,Galα1,6(Galα1,4)Glcα1,2Fruβ1,1Galα6,1αGal,Galα1,6Galα1,4(Galα1,6)Glcα1,2Fruβ1,1αGal,Galα1,6Glcα1,2Fruβ(1,1Galα)3,1Galα6,1αGal,Galα1,6Glcα1,2Fruβ(1,1Galα4,1Galα)3,1Galα6,1αGal,Galα1,6(Galα1,4)Glcα1,2Fruβ(1,1Galα)3,1Galα6,1αGal and Galα1,4Galα1,6(Galα1,4)Glcα1,2Fruβ(1,1Galα)3,1Galα6,1αGalAll the nine saccharides were alternative raffinose family oligosaccharides and belonged to lychnose-based oligosaccharides.The fructo-oligosaccharides from six medicinal plants including Cimicifuga foetida L contained inulin type,levan type and mixed type fructo-oligosaccharides with different average Dps from 3 to 14.The structures of representative medicinal plants neutral polysaccharides were studied using methylation analysis,1D/2D NMR spectra,enzymolysis,HPAEC-PAD,MALDI-TOF-MS,HILIC-ESI-MS~2analyses.(1)Glucans from Adenophora tetraphylla(Thunb.)Fisch and Sophora flavescens Ait mainly contained linear or less branched(1→4)-α-D-glucan(>90%).Linear(1→6)-α-D-glucan and branched(1→5)-α-D-arabinan were purified from the degradation products of Adenophora tetraphylla(Thunb.)Fisch glucan after enzymolysis by amylase and glucoamylase.Significantly branched(1→4)-α-D-glucan were purified from the degradation products of Sophora flavescens Ait glucan after enzymolysis by amylase and glucoamylase.The results indicated the existence of small amounts of above polysaccharides in Adenophora tetraphylla(Thunb.)Fisch and Sophora flavescens Ait.(2)Mannans from Cassia obtusifolia L.and Anemarrhena asphodeloides Bunge were composed ofβ-1,4-D-Manp repeating units as backbone.Mannan of Cassia obtusifolia L.was branched galactomannan with single unitα-T-Galp branches attached to O-6 of mannose in backbone,and the average degree of substitution was21%.Mannan from Anemarrhena asphodeloides Bunge was galactoglucomannan containedβ-1,4-D-Glcp units(9%)in backbone,and the backbone was decorated with minor galactose and certain acetyl moieties.The average degree of acetyl moieties substitution was 0.47.(3)Galactans from Panax ginseng C.A.Meyer flowers and Polygonatum sibiricum Redoute mainly contained less branched(1→4)-β-D-galactan(>50%),and were good substances for preparing galacto-oligosaccharides withβ-(1→4)glycosidic bonds.Galactan from Panax ginseng C.A.Meyer flowers also contained significantly branched(1→6)-β-D-galactan,and the side chain of(1→6)-β-D-galactan was branched with Araf residues.Minor mannan also existed in Polygonatum sibiricum Redoute galactan,and could be separated from galactan by using Fehling reagent precipitation method.(4)Hetero-polysaccharides from Lonicera japonica Thunb.and Polygala tenuifolia Willd were composed of Glcp,Galp,Araf and Manp,which assembled as(1→4)-α-D-glucan、(1→4)-β-D-galactan、(1→5)-α-D-arabinan and(1→4)-β-D-mannan.The results indicated that hetero-polysaccharides were combinations of several neutral polysaccharide domains(Glucan,Galactan and Mannan).Above all,this thesis focused on the types and structural features of neutral carbohydrates in fifty kinds of medicinal plants.Nine alternative raffinose family oligosaccharides were first found and their structures were first reported.In addition,the structures of eight representative neutral polysaccharides were studied in detail,and the structural features of four types neutral carbohydrates were concluded.These results enriched the structural information of neutral carbohydrates in medicinal plants,and provided information for development of natural carbohydrates agents.