Structural features of pectic polysaccharide from Angelica sinensis (Oliv.) Diels
Structural features of pectic polysaccharide from Angelica sinensis (Oliv.)Diels
Yuanlin Sun a,*,Steve W.Cui b ,Jian Tang c ,Xiaohong Gu c
a
Department of Life Sciences,Yuncheng University,Yuncheng 044000,China
b
Food Research Program,Agriculture and Agri-Food Canada,Ont.,Canada N1G 5C9c
State Key Laboratory of Food Science and Technology,School of Food Science and Technology,Jiangnan University,Wuxi 214122,China
a r t i c l e i n f o Article history:
Received 2September 2009
Received in revised form 13December 2009Accepted 15December 2009
Available online 23December 2009Keywords:
Angelica sinensis Structure
Pectic polysaccharide Methylation analysis Partial acid hydrolysis Enzymic digestion
a b s t r a c t
The structure of the pectic polysaccharide (ASP3)isolated from roots of Angelica sinensis (Oliv.)Diels was investigated using partial acid hydrolysis,enzymic digestion combined with methylation analysis,and further supported by 1H and 13C NMR spectroscopy techniques.The results indicated that ASP3contained a backbone of linear homogalacturonan fragments as ‘‘smooth regions”and rhamnogalacturonan frag-ments as ‘‘hairy regions”with repeating unit of [?4)-a -D -Gal p A-(1?2)-a -L -Rha p -(1?].A total of 58.8%rhamnopyranose residues in the backbone were substituted at O -4position by the side chains.The side chains contained mainly b -1,6-and b -1,4-galactopyranan bearing 3,6-and 4,6-substituted b -D -galactopyranose residues as branched points and short a -1,5-arabinofuranan possessing 3,5-substi-tuted a -L -arabinofuranose residues as branching points.In addition,b -1,6-galactopyranan side chains were highly branched with a -1,5-arabinofuranan carrying 3-O -substituents (1,3,6-Gal)and terminated by the a -arabinofuranose residues which form arabinogalactan.
ó2009Elsevier Ltd.All rights reserved.
1.Introduction
Pectins are a family of complex heterogeneous polysaccharides that constitute a large proportion of the cell wall of many higher plants where greatly in?uence growth,development and senes-cence (O’Neill,1990;Ridley,O’Neil,&Mohnen,2001).Pectins are also traditional gelling and thickening agents for the production of jams and jellies,and the area of the use extends to the produc-tion of fruit,dairy,and dessert products and pharmaceuticals (Thakur,Singh,&Handa,1997;Voragen,Pilnik,Thibault,Axelos,&Renard,1995).
In recent years,pectin is increasingly recognized as an impor-tant precursor of substrates improving gastrointestinal functions.It plays an important role in the regulation of some physiological processes and therefore in the prevention of hyperlipidemia,as well as bowel cancer (Lim,Yamada,&Nonaka,1998;Willats,McCartney,Mackie,&Knox,2001).Earlier,we isolated a pectic polysaccharide named ASP3from roots of Angelica sinensis (Sun,Tang,Gu,&Li,2005)which is a well-known oriental herb (Zhang &Cheng,1989).The sugar chain of ASP3was found to contain res-idues of galacturonic acid,arabinose,galactose,and rhamnose as the main constituents.Our previous study has shown that ASP3can protect leucocytes and lymphocytes of mice against radia-tion-induced damage,which has potential radioprotective effect on acute radiation injured mice.
According to reports,many of the bioactivities of pectins from various sources have been shown to have a relationship with com-plex branched structures (Wang,Dong,Zuo,&Fang,2003;Yamada,1994;Yu,Kiyohara,&Matsumoto,2001),so elucidation of molec-ular ?ne structural features of the pectic substances is necessary for understanding the mechanism of physiological activity and clarifying the structure–activity relationships.
The primary structure of pectin obtained from various sources has been studied extensively by methods of partial chemical or enzymatic degradation (Bushneva,Ovodova,Shashkov,Chizhov,&Ovodov,2003;Dong &Fang,2001;Habibi,Mahrouz,&Vignon,2005;Polle,Ovodova,Chizhov,Shashkov,&Ovodov,2002a;Polle,Ovodova,Shashkov,&Ovodov,2002b;Singthong &Cui,2004).However,the precise chemical structure of pectin remains under debate,although the structural elements of pectin are rather well described (Coenen,Bakx,Verhoef,Schols,&Voragen,2007).The present work is devoted to further elucidation of the detailed struc-tural features of the pectic polysaccharide ASP3from A.sinensis using mild acid hydrolysis and enzymic digestion followed by NMR spectroscopy and methylation analysis of fragments obtained.2.Experimental
2.1.Plant materials and preparation of ASP3
The roots of A.sinensis (Oliv.)Diels,cultivated in Minxian County,Gansu Province,China,were provided by Shanhe
0144-8617/$-see front matter ó2009Elsevier Ltd.All rights reserved.doi:10.1016/j.carbpol.2009.12.030
*Corresponding author.Tel.:+863592090036;fax:+863592097068.E-mail address:sylwts@https://www.sodocs.net/doc/3f9740319.html, (Y.Sun).Carbohydrate Polymers 80(2010)
544–550
Contents lists available at ScienceDirect
Carbohydrate Polymers
j o u r n a l ho m e p a g e :w w w.e l s e v i er.c om/loc
ate/carbpol
Pharmaceutical Co.Ltd.(Wuxi,China).Isolation followed by puri?-cation of the pectic polysaccharide ASP3from roots of A.sinensis was performed as described earlier(Sun et al.,2005).
2.2.General methods
Uronic acid content was determined by photometry with m-hydroxybiphenyl at520nm(Blumenkrantz&Asboe-Hansen, 1973),using D-galacturonic acid as standard.Total neutral sugar content was determined by the reaction with phenol in the pres-ence of sulfuric acid using galactose and arabinose as standards.
A correction was made for the response of galacturonic acid in the neutral sugar test.Neutral sugar composition was analyzed by GC after conversion of the hydrolysate into alditol acetates,as described earlier(Sun et al.,2005).The percentage of monosaccha-rides in the sample was calculated from the peak areas using re-sponse factors.The speci?c optical rotation was determined in H2O at25°C using a WZZ-2A polarimeter.
2.3.Partial acid hydrolysis
Fragment ASP3(150mg)was hydrolyzed with0.2M tri?uoro-acetic acid(TFA)for1h at121°C.After cooling,TFA was evapo-rated under a stream of N2.The hydrolysate was dissolved in distilled water and dialyzed against distilled water(M w cut-off 3500Da).The retentate and dialysate were concentrated and puri-?ed respectively on size exclusion chromatography(SEC)of Se-pharose CL-6B column(D1.0?120cm,Amersham Bioscience)at room temperature and eluted with degassed distilled water (12mL/h).A puri?ed high-molecular weight fraction(ASP3-PH) and a low-molecular weight fraction(ASP3-PL)were collected, concentrated and then lyophilized.
2.4.Enzymic hydrolysis
Fragment ASP3(100mg)was dissolved in24mL of water,and 1M NaOH(6mL)was added.The solution was kept for2h at 25°C.Excess alkali was neutralized with acetic acid to pH5.5.Mould endo-a-(1,4)-polygalacturonase(EndoPG,EC3.2.1.15,Fluka–467 U/g)was added and the mixture was incubated at30°C for72h. The enzyme was inactivated by heating at100°C for10min and the denatured protein was removed by centrifugation.The digestion product obtained was concentrated and subjected to Sepharose CL-6B column(D1.0?120cm,Amersham Bioscience)to give an en-zyme-resistant fraction(ASP3-EH)and an enzyme-sensitive fraction (ASP3-EL).Fractions were collected,concentrated and then lyophilized.
2.5.Determination of the glycosidic linkage composition
The glycosidic linkage analysis was determined by methylation and gas chromatography–mass spectroscopy(GC–MS).Prior to methylation,the sample containing uronic acid was reduced to the corresponding neutral sugar by using1-cyclohexyl-3-(2-mor-pholinoethyl)-carbodiimide methyl-p-toluenesulfonate(CMC,Flu-ka)and sodium borodeuteride(NaBD4,Acpos),following a procedure described by Taylor and Conrad(1972)and York,Darvill, McNeil,Stevenson,and Albersheim(1986)with slight modi?cation (Cui,2005).After this reduction,methylation analysis was carried out according to the method of Ciucanu and Kerek(1984)with slight modi?cation to give a fully methylated product(Cui, 2005).The methylated product was then converted into partially methylated alditol acetates(PMAA)by hydrolysis,reduction with NaBH4,and acetylation followed by linkage analysis using GC–MS(OV1701capillary column,0.25mm?30m,0.25mm?lm thickness coupled to a Trace Mass Spectrometer,Finnigan).The carrier gas was helium;3°C/min gradient from150to250°C. The temperature of the interface was250°C;Energy of ionizing electrons was70eV.Peak identi?cation was based on retention times using partially methylated alditol acetates as standards. The percentage of the methylated sugars was estimated as ratios of the peak areas(total ion current).
2.6.1H and13C NMR spectroscopy
The polysaccharide samples were exchanged3times in D2O(at concentrations of approximately40mg/mL),with intermediate freeze-drying.Finally,samples were dissolved in D2O.1H and 13C NMR spectroscopy were performed on a Brucker AMX500 NMR spectrometer(Germany)using standard pulse sequences with5and10mm tubes at65°C.Internal1,4-dioxane was used as an internal chemical-shift reference for spectra.Two-dimen-sional spectra(COSY-45,TOCSY,and HMQC)were recorded using the standard Bruker procedures(Cui,Eskin,Biliaderis,&Marat, 1996).
3.Results and discussion
3.1.Preparation of ASP3
The pectic polysaccharide named ASP3has been isolated and puri?ed from roots of A.sinensis as described earlier(Sun et al., 2005).The sugar composition of ASP3was listed in Table1.
3.2.Partial acid hydrolysis of ASP3
Partial degradation of polysaccharides by acid hydrolysis is based on the fact that some glycosidic linkages are more labile to acid than others.For example,linkages between neutral sugars are the most susceptible to acid hydrolysis;hence controlled acid hydrolysis is frequently used to remove neutral sugars(Coenen et al.,2007).To study the linkages between backbone and side chains of polysaccharide,the native polysaccharide ASP3was par-tially hydrolyzed with0.2M TFA.Partial acid hydrolysis resulted in two subfractions of ASP3:high-molecular(ASP3-PH)and low-molecular(ASP3-PL)ones.The sugar composition of fractions was given in Table1.It showed that for the polymer fraction ASP3-PH,the amount of arabinose,galactose,mannose and glucose decreased considerably compared with ASP3,whereas the amount of rhamnose and galacturonic acid increased,suggesting that link-ages between two GalA sugars are more stable then aldobiuronic linkages(GalA-Rha)or pseudo-aldobiuronic(Rha-GalA)sugars and linkages between neutral sugars are most susceptible to acid hydrolysis.ASP3-PH,yield70%of the parent ASP3,composed of galacturonic acid(76.5%),rhamnose(2.7%),galactose(19.2%)and small amount of arabinose(0.2%),suggesting the presence of a typ-ical homogalacturonan and rhamnogalacturonan substituted by the side chains of mainly galactosyl and arabinosyl residues.
For the oligomer fraction ASP3-PL,the sugar composition pre-sented in Table1showed that the neutral sugars(arabinose,gal-actose,mannose and glucose)were the main constituent.These results con?rmed that galacturonic acid and rhamnose present in the backbone which were not capable of mild hydrolysis,whereas the neutral sugars were attached to the side chains and easy to be hydrolyzed.
The subfraction ASP3-PH possessed higher positive rotation ?a 25D+194°than that of the raw fraction ASP3(+131°)measured at the same conditions(c0.1;H2O;25°C)due to the prevalence of D-Gal p A,the main constituent of ASP3-PH.
Y.Sun et al./Carbohydrate Polymers80(2010)544–550545
3.3.Enzymic digestion of ASP3
Fraction ASP3,which previously shown a symmetrical SEC peak on Sepharose CL-6B(Fig.1A),was de-esteri?ed with0.5M NaOH, digested with EndoPG,and then subsequently fractionated and puri?ed on the same chromatography.
The SEC elution pro?le of modi?ed ASP3is shown in Fig.1B.A small proportion of enzyme-resistant fraction with high-molecular weight(ASP3-EH)and a large proportion of enzyme-sensitive frac-tion with low-molecular weight(ASP3-EL)were obtained.ASP3-EH,yield29.71%of the parent ASP3,?a 25D+16°(c0.1;H2O),was polymeric and eluted in the void volume.It contained residues of galactose(46.0%),arabinose(9.4%),galacturonic acid(35.6%),and rhamnose(4.6%).ASP3-EL,yield62.3%of ASP3,contained large amount of free galacturonic acids or oligomers(83.0%)which were determined by HPAEC-PAD(results not shown).The results con-?rmed that the linear homogalacturonan was present in the parent ASP3(Yu et al.,2001).
Sugar composition of the fractions was given in Table1.It showed that the Gal p A content of ASP3-EH decreased(35.6%)com-pared with the parent ASP3(58.3%),indicating a signi?cant degra-dation of the polygalacturonan backbone.However,the presence of Gal p A residue also indicated the occurrence of methyl esters, acetyl groups or neutral side chains attached to the backbone of Gal p A units,thus limited the action of EndoPG by steric hindrance (Bonnin,Dolo,&Goff,2002).Fragment ASP3-EH was differed in content of neutral sugar residues,especially rhamnose(4.6%),in comparison with fragment ASP3(1.9%).With an increase of rham-nose residues and other neutral sugars in ASP3-EH,we presumed that some of Gal p A residues were connected with rhamnose resi-dues in repeating units to give a typical rhamnogalacturonan back-bone,and the neutral sugars(arabinose,galactose,mannose and glucose)were attached to the backbone as side chains,thus resis-tant to the digestion of EndoPG.
3.4.Determination of the glycosidic linkage composition of ASP3and ASP3-PH
To differentiate galactose arising from the reduction of galact-uronic acids residues and galactose existed in the side chains,the carboxyl groups of ASP3were reduced with NaBD4into the corre-sponding6,60-d2-D-galactosyl residues before methylation.
The results of methylation analysis of carboxyl reduced ASP3 and ASP3-PH were given in Table2.It showed that the puri?ed ASP3was composed mainly of1,4-D-linked galacturonic acid, 1,2-and1,2,4-linked rhamnose,terminal,1,5-,1,3,5-linked arabi-nose,terminal,1,6-,1,3,6-,1,4-and1,4,6-linked galactose,as was commonly reported in pectic polysaccharide(Habibi,Heyraud, Mahrouz,&Vignon,2004;Polle et al.,2002b).Methylation analysis revealed that2,3,6-tri-O-methyl galactitol(6,60-d2)arising from the reduced1,4-D-galacturonic acid was the main sugar compo-nent.The low amount(1.8%)of3-O-methyl rhamnitol and3,4-di-O-methyl rhamnitol suggested that ASP3contained the main part of homogalacturonan fragments as‘‘smooth regions”(a linear chain of1,4-D-Gal p A units)and certain amount of rhamnogalactu-ronan segments as‘‘hairy regions”.Regarding the rhamnosyl resi-dues,The ratio41.2:58.8of1,2-to1,2,4-linked rhamnose indicated a total of58.8%rhamnose residues in the backbone were substituted at O-4position by side chains.
The proportion of terminal,1,6-,1,3,6-,1,4-,and1,4,6-linked galactose was,respectively,in the ratio of14.9:30.7:34.9: 12.3:7.2.These results suggested that the galactan side chains con-tained a central core of1,6-linked galactose residues,as65.6%of the units were1,6-linked,more than half of which(53.2%)were substituted at O-3position(1,3,6-linked galactose).In addition, there was a core of1,4-linked galactose residues(19.5%),in which
Table1
Sugar composition of fractions from ASP3by partial acid hydrolysis and degradation of EndoPG.
Fragments Uronic acid(wt.%)Content of the sugar residues(wt.%)
Rha Ara Man Glc Gal
ASP358.3 1.910.50.40.924.9 ASP3-PH76.5 2.70.2Trace0.419.2 ASP3-PL 6.3Trace31.9 1.9 3.462.8 ASP3-EH35.6 4.69.4 2.0 1.346.0 ASP3-EL83.0Trace 1.0Trace 3.7 1.8
Table2
Glycosidic linkage composition of methylated ASP3and ASP3-PH.
Residues Linkage PMAA Mol.%Mol.%
ASP3ASP3-PH ASP3ASP3-PH
D-Gal p A1,4-2,3,6-Me3-Gal p100.0100.052.176.9
L-Rha p1,2-3,4-Me2-Rha p41.273.8 1.8 2.4
1,2,4-3-Me-Rha p58.826.2
L-Ara f T-2,3,5-Me3-Ara f62.453.213.60.3
1,5-2,3-Me2-Ara f19.346.8
1,3,5-2-Me-Ara f18.3—
D-Gal p T-2,3,4,6-Me4-Gal p14.942.427.918.9
1,6-2,3,4-Me3-Gal p30.723.2
1,3,6-2,4-Me2-Gal p34.912.2
1,4-2,3,6-Me3-Gal p12.312.0
1,4,6-2,6-Me2-Gal p7.210.2
D-Glc p A T-2,3,4,6-Me4-Gal p100.0100.0 3.9 1.3
D-Glc p1,4-2,3,6-Me3-Glc p100.0100.00.70.2
546Y.Sun et al./Carbohydrate Polymers80(2010)544–550
36.9%of the units(1,3,6-linked galactose)were substituted at O-6 position.The low proportion of terminal galactosyl residues (14.9%)indicated that a part of the galactopyranan side chains were terminated by the a-arabinofuranose residues which form arabinogalactan.
The proportion of terminal,1,5-,1,3,5-linked arabinose was, respectively,in the ratio of62.4:19.3:18.3.These results suggested that the arabinan side chains contained a central core of1,5-linked arabinosyl residues,as37.6%of the units were1,5-linked.The de-gree of branching(around33.3%)was relatively high,for almost half of which(48.7%)were branched at O-3position.62.4%of ara-binosyl residues were at the nonreducing ends.The high propor-tion of terminal arabinose residues suggested that not all of the terminal arabinose units present in the arabinan side chains,but also attached to the highly branched galactan side chains or con-nected to the backbone directly(Ros,Schols,&Voragen,1996).
Compared with the parent ASP3,carboxyl reduced ASP3-PH was relatively enriched in galacturonic acid and rhamnose(76.9%and 2.4%,respectively)whereas the amount of arabinose,galactose, mannose and glucose decreased considerably.The results con-?rmed that galacturonic acid and rhamnose were present in the backbone,whereas other neutral sugars were attached to the side chains which were easy to be hydrolyzed under weak acid.From the GalA:Rha ratio32:1it can be concluded that homogalacturo-nan(HG)and rhamnogalacturonan I(RG I)fragments remain pres-ent in the sample after TFA hydrolysis(Coenen et al.,2007).
Methylation analysis(Table2)also showed that the content of 1,2,4-linked rhamnose(26.2%)of the total rhamnose in the back-bone of ASP3-PH decreased compared with that of ASP3(58.8%), whereas the amount of1,2-linked rhamnose increased,indicating the branching points of the backbone with neutral side chains ap-peared to be at O-4position of1,2,4-linked rhamnose residues. Further information was then deduced that1,2-and1,2,4-linked rhamnose residues were substituted in O-2position by1,4-D-linked galacturonic acid.
After partial acid hydrolysis,most arabinose residues(97.5%. mainly T-and1,3,5-Ara f)were removed and partial O-6substi-tuted galactosyl residues(32.4%.mainly1,6-Gal p and1,3,6-Gal p) were hydrolyzed,while the proportion of terminal galactose resi-dues(42.4%)increased considerably,compared with ASP3 (14.9%).All the results above indicated that most of arabinose res-idues in side chains were located at the end of the side chains.The high molar ratio of terminal arabinose(62.4%)in the original poly-saccharide ASP3suggested that in addition to connecting directly with O-4position of1,2-linked rhamnosyl in the backbone or attached to O-5position of1,5-L-Ara f residues into short arabinan chains,most of T-L-Ara f should be attached to O-3of1,6-linked D-galactosyl residues which forms arabinogalactan.During the course of partial acid hydrolysis,these furanosyl rings are usually considered as weak glycosidic linkages that are more labile to acid than pyranosyl rings(Cui,2005;Habibi et al.,2005),which cause an increase of terminal galactose.There was a minor change in the amount of1,4-and1,4,6-linked galactose residues in ASP3-PH after partial acid hydrolysis,which suggested that the side chains consisted by these two kinds of galactose residues were sta-ble in weak acid condition.The detection of2,3,4,6-Me3-Gal p(6,60-d2)indicated that Gal p A were1,4-linked.
Based on the results of partial acid hydrolysis and enzymic digestion,we supposed that the backbone of the pectic polysaccha-ride ASP3consisted of galacturonic acid and rhamnose residues. The neutral side chains attached to O-4position of rhamnose res-idues.In addition to the a-1,5-linked arabinofuranan side chains substituted at O-3position(1,3,5-Ara),there were dominantly two other types of neutral side chains existed in ASP3:one was arabinogalactan in which1,6-D-linked galactopyranan was highly branched with a main core of1,6-D-linked galactose carrying3-O-substituents(1,3,6-Gal)and terminated by a-arabinofuranose residues.The other was1,4-D-linked galactopyranan branched with a main core of1,4-D-linked galactose carrying6-O-substitu-ents(1,4,6-Gal).
3.5.1H and13C NMR spectroscopy analysis
The1D and2D NMR spectra of the three fragments were ana-lyzed,respectively.According to the characteristic signals,the1H and13C spectra of the polysaccharide fraction ASP3were com-pletely assigned by two-dimensional COSY-45,TOCSY and HMQC NMR experiments,and the corresponding chemical shifts of signals were summarized in Table3.
The1H and13C NMR spectra of ASP3were given in Fig.2.In the proton spectrum(Fig.2A),signals at5.0–5.4and4.4–5.0ppm were corresponded to the anomeric protons of a-arabinofuranose and b-galactopyranose residues.H-1of a-rhamnopyranose and a-galact-uronic acid had signals at5.25and5.08/4.95ppm,respectively. Two weak resonance signals at1.28and1.38ppm were identi?ed to H-6from methyl group of the rhamnopyranose units.These methyl rhamnose signals appeared generally as two well-resolved doublets,due to the presence of two different rhamnose residues, which were,respectively,the rhamnosyl residues linked only at O-2and the rhamnosyl residues linked both at O-2and O-4(Cui et al., 1996;Habibi et al.,2005).This assignment was in agreement with the results of methylation analysis.
Table3
Chemical shifts for the resonances of glycosyl residues of ASP3in1H and13C NMR spectra.
Glycosyl residues Chemical shifts,d(ppm)
H1/C1H2/C2H3/C3H4/C4H5/C5H6/C6CH3O ?4)-a-Gal p A-(1?4 5.08/102.3 3.70/70.7 4.10/71.3 4.41/81.2n.d.a/73.2173.5 3.78/55.6 ?4)-a-Gal p A-(1?2 4.95/103.0 3.70/70.9 4.00/71.3n.d./81.0n.d./n.d.176.9—?2)-a-Rha p-(1? 5.25/n.d.a 4.10/78.5 3.86/72.2 3.41/76.0 3.70/70.2 1.25/19.5—?2,4)-a-Rha p-(1? 5.25/n.d. 4.15/79.0 3.90/72.2 3.82/84.7 3.47/72.5 1.38/19.7—b-Gal p-(1? 4.42/105.5 3.53/73.8 3.64/73.5 3.93/71.4 3.69/77.4 3.78/63.3—2-O-Me-b-Gal p-(1? 4.49/105.0 3.29/84.5 3.36/75.7 3.96/71.4 3.73/78.6 3.70/63.3 3.48/62.1 ?6)-b-Gal p-(1? 4.61/106.5 3.47/73.0 3.66/73.5 3.91/70.9 3.88/75.3 3.98/3.72/71.3—?3,6)-b-Gal p-(1? 4.68/105.5 3.79/72.9 3.83/84.6 4.21/69.9 3.76/74.2 3.72/71.3—?4,6)-b-Gal p-(1? 4.46/105.5 3.54/73.5 3.66/73.5 3.95/78.5 3.66/75.3 3.72/71.3—?4)-b-Gal p-(1? 4.69/105.5 3.69/73.0 3.94/73.0 4.14/78.5 3.71/n.d. 3.88/3.82/63.3—a-Ara f-(1?3 5.40/111.0 4.19/84.3 3.91/76.9 4.05/87.0 3.91/3.79/64.0——a-Ara f-(1?3 5.23/112.0 4.22/84.3 3.93/76.9 4.12/86.8 3.82/3.69/64.0——a-Ara f-(1?5 5.13/110.0 4.12/83.8 3.95/76.9 4.02/86.5 3.82/3.72/64.0——?5)-a-Ara f-(1? 5.07/108.0 4.12/83.8 4.02/77.0 4.21/86.8 3.80/3.89/71.6——?3,5)-a-Ara f-(1? 5.09/108.0 4.28/82.8 4.09/84.0 4.44/82.0 3.94/3.82/72.0——
a Not determined.
Y.Sun et al./Carbohydrate Polymers80(2010)544–550547
The proton signals near2.1ppm arise from the–CH3of the O-acetyl groups(Fig.2A).The two doublets suggested that ASP3con-tained two kinds O-acetyl groups at the different positions of the sugar residues(Bushneva,Ovodova,Shashkov,&Ovodov,2002). Meanwhile,the carbon signal at22.8ppm(Fig.2B)in13C NMR spectra supported the above deduction(Wang,Liu,&Fang,2005).
The1H/13C HMQC and TOCSY spectra of fragments demon-strated the presence of regions consisting of a-1,4-linked homoga-lacturonan.In the1H and13C NMR spectra of fraction ASP3-PH,a group of intensive signals were observed for the residues of a-1,4-linked galacturonic acid(Table3).According to the literature (Keenan,Belton,Matthew,&Howson,1985),these resonances were characteristic for a pure a-1,4-D-galactopyranosyluronan which occupied the main part of the fraction ASP3.
In the low-?eld region of13C NMR,typical signals were ob-served for the C-6carboxyl group of galacturonic acid units at 176.9and173.5ppm.The occurrence of two carboxyl signals con-?rmed the presence of free and esteri?ed carboxyl groups of a-D-Gal p A(Catoire,Goldberg,&Pierron,1998;Wang et al.,2005). The carbon signal at55.6ppm was assigned to methyl ester groups of the native polysaccharide.The presence of methyl esteri?ed Gal-p A residues was also con?rmed by the signal of55.6/3.78ppm in HMQC spectrum of fraction ASP3-PH(Bushneva et al.,2002).In addition,the COSY-45correlation peak at4.95/4.41ppm observed for fraction ASP3-PH indicates the H2/H4interaction of neighbor a-(1,4)-linked Gal p A residues of linear homogalacturonan regions (Bushneva et al.,2002).
The anomeric carbon signal of rhamnopyranose was not observed in13C NMR spectrum for its low content.However,anal-ysis of the homonuclear COSY-45,TOCSY and HMQC spectra of the digested fraction ASP3-EH revealed the presence of a-1,2-linked rhamnopyranose residues.In the HMQC spectrum of fraction ASP3-EH(Fig.3),the minor response at19.5ppm was easily iden-ti?ed to C-6from methyl group of rhamnopyranose units,and the COSY-45and TOCSY spectra showed the corresponding proton sig-nals associated with a-1,2-linked rhamnopyranose residues.In particular,some of residues were observed to be substituted at O-4position from the correlation peak of C4/H4-atoms at84.7/ 3.82ppm in the HMQC spectrum(Fig.3),which agree well with the results of the methylation analysis.Other characteristic signals were assigned and listed(Table3)according to the2D NMR spectra and reference(Cui et al.,1996).Finally,a complete assignment of the signals originating from a-1,2-and a-1,2,4-rhamnopyranose residues was obtained by comparison of the observed chemical shifts with corresponding values in literature(Habibi et al.,2005; Keenan et al.,1985
). 548Y.Sun et al./Carbohydrate Polymers80(2010)544–550
In the HMQC spectrum of ASP3-PH(Figure not shown),the signals of a-arabinofuranose and partial b-galactopyranose al-most disappeared and became https://www.sodocs.net/doc/3f9740319.html,pared with these changes,a-galacturonic acid and a-rhamnopyranose residues were largely retained,and the signal of1,2-linked rhamnopyra-nose at1.25ppm became stronger compared with the signal of 1,2,4-linked rhamnopyranose at 1.29ppm.These results con-?rmed that a-arabinofuranose and b-galactopyranose residues were side chains linked to O-4of1,2,4-linked rhamnopyranose.
Galacturonic acid,rhamnose,arabinose and galactose were de-tected as the main sugars in fragment ASP3-EH,suggesting the presence of a rhamnogalacturonan.The signals in the heteronu-clear1H/13C HMQC spectrum of fragment ASP3-EH(Fig.3)also indicated the occurrence of linear a-1,2-L-rhamno-a-1,4-D-galac-turonan region.
In the anomeric region,the signals at105–107ppm corre-sponded to the anomeric carbons of terminal or branched galacto-pyranose residues,and signals at108–110ppm were assigned to the C-1of terminal or branched arabinofuranosyl units(Cui, 2005).The signals at102.3and103.0ppm arose from C1of a-Rha p and a-Gal p A,respectively(Cui et al.,1996)(Fig.2B).Resonance sig-nals in the region of75–85.1ppm were corresponded to secondary hydroxyl groups(C-2,C-3)of arabinofuranosyl units,while the sig-nals of C-2–C-4of b-D-Gal p appeared in the region of65–85ppm (Cui,2005).Resonance signals in the region of60–64ppm arose from C-5of terminal arabinofuranosyl units(64.0ppm)and C-6 of terminal galactopyranose residues(63.3ppm).
According to the literature data published for pectic substances, 1H and13C NMR spectroscopy allowed detection of the terminal,6-, 3,6-,4-and4,6-substituted b-galactopyranose residues in the polysaccharide fragment ASP3.In addition,the residues of a-arab-inofuranose substituted in5-,and3,5-positions as well as the ter-
minal a-arabinofuranose residues were identi?ed(Table3).The connectivity from H-1to H-5was clearly established from COSY-
45spectra.These data were con?rmed by two-dimensional homo-
nuclear COSY-45,TOCSY,and the heteronuclear HMQC spectrum.
In addition,the residues of2-O-methyl-b-D-galactopyranose
were detected.The resonance of C-2at84.5ppm in the1H/13C
HMQC spectrum of the fragment ASP3-EH(Fig.3)indicated that
the O-methyl group(62.1/3.48ppm)was linked at this position,
which otherwise would be in the range of72–74ppm(Polle
et al.,2002a,2002b).A correlation peak of the O-methyl group
with H-2of the terminal b-galactopyranose residue(OMe/H2
3.48/3.29ppm)in the COSY-45spectrum of ASP3-EH con?rmed
this interpretation.Other signals for these substituted residues
were fully assigned and listed(Table3)from homonuclear COSY-
45,TOCSY spectra and heteronuclear HMQC,compared with liter-
ature data(Bushneva et al.,2002;Catoire et al.,1998;Habibi et al.,
2005;Polle et al.,2002a,2002b).
4.Conclusion
The structural features of ASP3were studied using partial acidic
hydrolysis and enzymic digestion,combined with methylation
analysis and NMR spectral data.The results indicated that a-1,4-D-galactopyranosyluronan fragments occupied the main part of the pectic polysaccharide ASP3as‘‘smooth regions”.Some residues
of galacturonic acid in the linear region were methoxylated and
contained O-acetyl groups on C-2and/or C-3.The rami?ed region
appear to be rhamnogalacturonan blocks with repeating unit
of
[?4)-a-D-Gal p A-(1?2)-a-L-Rha p-(1?].A total of58.8%rhamno-syl residues in the backbone were substituted at O-4position by the side chains.The side chains,which contained residues of termi-nal,b-1,6-,b-1,3,6-,b-1,4-and b-1,4,6-linked galactopyranose and terminal,a-1,5-,a-1,3,5-linked arabinofuranose,were linked as blocks of galactan,arabinan,and arabinogalactan,which were the mainly constitution of the branch,attached to O-4of the back-bone rhamnose residues.A part of terminal Gal p were substituted at O-2position by methyl into2-O-Me-b-D-Gal p. Acknowledgements
The authors thank Mr.Liping Wang(Testing&Analysis Center, Jiangnan University)for the help in GC–MS experiments of the samples.This work was supported by a grant from Natural Science Foundation of Shanxi(2007021042).
References
Blumenkrantz,N.,&Asboe-Hansen,G.(1973).New method for quantitative determination of uronic acid.Analytical Biochemistry,54,484–489.
Bonnin,E.,Dolo,E.,&Goff,A.L.(2002).Characterisation of pectin subunits released by an optimised combination of enzymes.Carbohydrate Research,337, 1687–1696.
Bushneva,O.A.,Ovodova,R.G.,Shashkov,A.S.,Chizhov,A.O.,&Ovodov,Y.S.
(2003).Structure of Silenan,a pectic polysaccharide from Campion Silene vulgaris(Moench)Garcke.Biochemistry(Moscow),68,1360–1368. Bushneva,O.A.,Ovodova,R.G.,Shashkov,A.S.,&Ovodov,Y.S.(2002).Structural studies on hairy region of pectic polysaccharide from campion Silene vulgaris (Oberna behen).Carbohydrate Polymers,49,471–478.
Catoire,L.,Goldberg,R.,&Pierron,M.(1998).An ef?cient procedure for studying pectin structure which combines limited depolymerization and13C NMR.
European Biophysical Journal,27,127–136.
Ciucanu,I.,&Kerek,F.(1984).A simple and rapid method for the permethylation of carbohydrates.Carbohydrate Research,131,209–217.
Coenen,G.J.,Bakx,E.J.,Verhoef,R.P.,Schols,H.A.,&Voragen,A.G.J.(2007).
Identi?cation of the connecting linkage between homo-or xylogalacturonan and rhamnogalacturonan type I.Carbohydrate Polymers,70,224–235.
Cui,S.W.(2005).Food carbohydrates:Chemistry,physical properties,and applications (pp.60–63).Boca Raton:CRC Press.
Cui,W.W.,Eskin,M.N.A.,Biliaderis,C.G.,&Marat,K.(1996).NMR characterization of a4-O-methyl-b-D-glucuronic acid-containing rhamnogalacturonan from yellow mustard(Sinapis alba L.)mucilage.Carbohydrate Research,292,173–183. Dong,Q.,&Fang,J.N.(2001).Structural elucidation of a new arabinogalactan from the leaves of Nerium indicum.Carbohydrate Research,332,109–114.
Habibi,Y.,Heyraud,A.,Mahrouz,M.,&Vignon,M.R.(2004).Structural features of pectic polysaccharides from the skin of Opuntia?cus-indica prickly pear fruits.
Carbohydrate Research,339,1119–1127.Habibi,Y.,Mahrouz,M.,&Vignon,M.R.(2005).Isolation and structural characterization of protopectin from the skin of Opuntia?cus-indica prickly pear fruits.Carbohydrate Polymers,60,205–213.
Keenan,M.H.J.,Belton,P.S.,Matthew,J.A.,&Howson,S.J.(1985).13C-NMR study of sugar-beet pectin.Carbohydrate Research,138,168–170.
Lim,B.O.,Yamada,K.,&Nonaka,M.(1998).Dietary?bers modulate indices of intestinal immune function in rats.Journal of Nutrition,127,663–667.
O’Neill,M. A.(1990).The pectic polysaccharides of primary cell walls.In D.
Harborne(Ed.),Methods in plant biochemistry(pp.415–444).London:Academic Press.
Polle,A.Y.,Ovodova,R.G.,Chizhov,A.O.,Shashkov,A.S.,&Ovodov,Y.S.(2002a).
Structure of Tanacetan,a pectic polysaccharide from Tansy,Tanacetum vulgare L..Biochemistry(Moscow),67,1371–1376.
Polle,A.Y.,Ovodova,R.G.,Shashkov,A.S.,&Ovodov,Y.S.(2002b).Some structural features of pectic polysaccharide from tansy,Tanacetum vulgare L..Carbohydrate Polymers,49,337–344.
Ridley,B.L.,O’Neil,M.A.,&Mohnen,D.(2001).Pectins:Structure,biosynthesis,and oligogalacturonide-related signaling.Phytochemistry,57,929–967.
Ros,J.M.,Schols,H.A.,&Voragen,A.G.J.(1996).Extraction,characterisation,and enzymatic degradation of lemon peel pectins.Carbohydrate Research,282, 271–284.
Singthong,J.,&Cui,S.W.(2004).Structural characterization,degree of esteri?cation and some gelling properties of Krueo Ma Noy(Cissampelos pareira)pectin.
Carbohydrate Polymers,58,391–400.
Sun,Y.L.,Tang,J.,Gu,X.H.,&Li,D.Y.(2005).Water-soluble polysaccharides from Angelica sinensis(Oliv.)Diels:Preparation,characterisation and bioactivity.
International Journal of Biological Macromolecules,36,283–289.
Taylor,R.L.,&Conrad,H. E.(1972).Stoichiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-activated carboxyl groups.Biochemistry,11, 1383–1388.
Thakur,B.R.,Singh,R.K.,&Handa,A.K.(1997).Chemistry and uses of pectin.
Critical Reviews in Food Science and Nutrition,37,37–47.
Voragen,A.G.J.,Pilnik,W.,Thibault,J.F.,Axelos,M.A.V.,&Renard,C.M.G.C.
(1995).Pectins.In A.M.Stephen(Ed.),Food polysaccharides and their applications (pp.287–340).New York:Marcel Dekker.
Wang,X.S.,Dong,Q.,Zuo,J.P.,&Fang,J.N.(2003).Structure and potential immunological activity of a pectin from Centella asiatica(L.)Urban.
Carbohydrate Research,338,2393–2402.
Wang,X.S.,Liu,L.,&Fang,J.N.(2005).Immunological activities and structure of pectin from Centella asiatica.Carbohydrate Polymers,60,95–101.
Willats,W.G.,McCartney,L.,Mackie,W.,&Knox,J.P.(2001).Pectin:Cell biology and prospects for functional analysis.Plant Molecular Biology,47,9–27. Yamada,H.(1994).Pectic polysaccharides from Chinese herbs:Structure and biological activity.Carbohydrate Polymers,25,269–276.
York,W.S.,Darvill,A.G.,McNeil,M.,Stevenson,T.T.,&Albersheim,P.(1986).
Isolation and characterization of plant cell walls and cell wall components methods in enzymology(p.118).New York:Academic Press.
Yu,K.W.,Kiyohara,H.,&Matsumoto,T.(2001).Characterization of pectic polysaccharides having intestinal immune system modulating activity from rhizomes of Atractylodes lancea DC.Carbohydrate Polymers,46,125–134. Zhang,S.Y.,&Cheng,K.C.(1989).Angelica sinensis(Oliv.)Diels:In vitro culture regeneration,and the production of medicinal compounds.In Y.P.S.Bajaj(Ed.), Biotechnology in agriculture and forestry(pp.1–10).Heidelberg,New York: Springer.
550Y.Sun et al./Carbohydrate Polymers80(2010)544–550