wente

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the pectin molecule as a whole, i.e., its backbone andside chains. An AFM image gives immediately a pic-ture of all molecules [13]. Using AFM images, it is pos-sible to characterize mixtures of pectin molecules andindividual molecules inside a particular mixture on thebasis of their dimensions and shape. It was proposedusing this method that pectin from the cell walls of agreen tomato has a branched galacturonan backbone

[14]. Later, this suggestion was confirmed experimen-tally for comaruman, a pectin from the marsh cinque-foil Comarum

palustre

L. [17].

By comparing the AFM images of two pectin frac-tions extracted from a tomato by different methods, acorrelation between the length of side chains and thecontent of neutral monosaccharides was established. Inpectin fractions, mixtures of separate polymer mole-cules and their aggregates were found [14]. Aggregatesare observed even after strong dilution, when only a fewmacromolecules are discernible. This indicates thatpectin substances exist as a multipolymer complex inwhich individual components are coupled by intermo-lecular interactions. An aggregate of separate polymersreflects the heterogeneity of a pectin polysaccharidesample. Linear polymers with a chain length from tensto hundreds of nanometers are clearly seen, togetherwith branched polymers carrying the side chains.

The methylation analysis provides informationabout the middle member of a mixture of macromol-ecules. It should be born in mind that some branches arepresent as short chains which are difficult to detect byAFM. In this case, the results obtained by the methyla-tion studies are also of great importance [13, 14].

A study of the physical structure of pectin from afresh sugar beet by AFM showed for the first time thatthe pectin extract contains a mixture of linear pectin, abranched pectin polysaccharide, and a complex of pec-tin with a protein linked to one end of the pectin chain[24]. Previously [25], it has been proposed that there isa covalent link between pectin and the protein. The dataobtained by AFM confirm this proposal but are notdirect evidence for the presence of this link: the possi-bility of the physical association between pectin and theprotein is not ruled out. Further studies are needed toidentify the type of the proteinpectin bond in com-plexes and determine the role of pectin and the proteinin the complex and the plant cell wall [24].

Finally, by using AFM it is possible to obtain struc-tural information regarding supramolecular nano-sized

particles of a native pectin polysaccharide that are eas-ily observable by this method [26, 27].

Most recently, AFM has been used to study thestructure of pectin (from the albedo of an orange peel)in aqueous solutions (water and citrate buffer) and ofgel networks formed by pectin [28]. Varying the con-centration of pectin in solutions has resulted in greatlydifferent AFM images. The results obtained have led tosome important conclusions. Thus, pectin in pure waterat concentrations above 10

g/ml forms aggregates of

network structures; at lower concentrations, molecularnetwork structures dissociate to their constituent com-ponents. These components tend to expand due to inter-charge repulsion along the carbohydrate chain, a phe-nomenon known as the polyelectrolyte effect. At aconcentration of 6.5

g/ml, small bars, segment rods,rings, screwed bars, branched molecules, and densering-shaped bodies are observed. The same structures

are also distinguishable at a concentration of about13.1

g/ml, at which pectin macromolecules formmolecular networks in both pure water and a citratebuffer. At a concentration of 10

g/ml, pectin in the gelcan be fixed in an unstable state due to insufficient sal-vation of the pectin network in the presence of a highconcentration of sucrose in acidic citrate buffer [28].

A very important area of the application of AFM isa study of pectin substances as food fibers for the foodindustry, which involves qualitative and quantitativeassays of the structures of food fibers and depicts theproperties of food fibers and their changes during pro-cessing and storage [26].

A General Scheme of Pectin Structure

A general structure al pattern of pectin polysaccha-rides is given in many papers, e.g., in [29, 30].

The linear region

of homogalacturonan consists of1,4-linked

-

D

-galactopyranosyluronic acid residues.These regions are joined by one or two

-

L

-rhamnopy-ranose residues which are involved in the linear chainby a 1,2-linkage. The backbone of many pectins hasthis very structure. They differ only by the length of thechain [2931].

The ramified region

consists of three subunits: RG-I,arabinogalactan, and xylogalacturonan, which can bepresent in different ratios, as it is shown for apple pectin[32]. The data obtained for pectins from a peach, carrot,onion, leek, and potato are fully consistent with thisstructure [31]. In some cases (zosteran, a pectin fromsea phanerogram, and lemnan, a pectin from duck-weed), the polymer contains an apiogalacturonan frag-ment [1, 6].

The RG-I fragment considerably varies in differentpectins. Its backbone consists of alternating residues of1,4-linked galacturonic acid residues and 1,2-linkedrhamnose residues partially substituted for by singlegalactose residues linked to rhamnose residues by 1,4-linkages. Also, the RG-I subunit can have long arabinan

and galactan side chains. Arabinose residues can be ter-minal 1,3- and 1,3,5- linked residues. RG-I can alsocontain either a xylogalacturonan unit in which singlexylopyranose residues are 1,3-linked to the backbone,as it is in apple pectin [31], or an apiogalacturonan frag-ment in which single or 1,3-linked D

-apiose residuesare linked to theD

-galacturonic acid of the core by 1,2-and/or 1,3-linkages [1, 6].

A general model for apple, citrus, and beet pectinshas been suggested which has an alternating linear 1,4-

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CURRENT VIEWS ON PECTIN SUBSTANCES 271

Linear region Linear region Branched regionBranched region

:

arabinan, galactan,

Smooth regions Ramified (hairy) regionsHomogalacturonan

Weakly ramified region

GalA

RG-I

Rha

GalAra

Rhamnogalacturonan

Branched (hairy) regions

RG-I

Xyl

Fer

of homo-galacturonan

of rhamno-galacturonan

of rhamnogalacturonan

I (RG-I)

and arabinogalactan.Weakly branched region:

xylogalacturonan

Fer

Xyl XylGal

Ara

Ara

Ara

Ara

Gal Gal GalAra

Schematic structure of pectin polysaccharides. Fer, ferulie acid residue.

linked

-

D

-galacturonan chain and a branched regioncontaining most of the neutral monosaccharides [33].Original pectins from these sources differ by molecularmass (citrus > apple > beet) and content of rhamnose(beet > apple > citrus). From apple, beet, and citrus pec-tins, homogalacturonan with a polymerization degreeof 72120, 91108, and 114138 kDa, respectively,were isolated. Sugar beet pectin was found to containferulic acid (Fer) residues which are linked to the neu-tral monosaccharides of side chains (mainly to L

-ara-binofuranose residues) by an ester bond [34].

The structure of a molecule of apple and beet pec-tins, as well as related pectins from other vegetables

and fruits, can be represented by a scheme from [31].

RG-II

, a minor component of primary cell walls, isdistinguished by a very complex structure [1, 30, 3537]. RG-II is resistant to

-1,4

-

endo

-polygalacturonaseand is isolated after the preliminary treatment of pectinswith this enzyme. Among the constituent sugar residuesof RG-II, the widely occurring monosaccharides suchas D

-galacturonic acid, L

-rhamnose, D

-galactose, L

-arabinose,D

-xylose,D

-glucose,L

-fucose,D

-mannose,and D

-glucuronic acid have been identified [37,38].

However, along with these residues, rather unusualmonosaccharides have been found as follows: D

-apiose, 2-

-methyl-

L

-fucose, 2-

O

-methyl-

D

-xylose,AceA, DHA, and KDO. AceA was first found in pectinsubstances as an unidentified sugar residue of RG-IIfrom cell walls of a suspension of a sycamore culture in1978 [39] and identified in 1983 as an acidic branchedmonosaccharide [40].

H

H

OHOH

CH2OH

O

OH

H3C

OH

COOH HO

O

H, OH

OOH OH

CH2OH

OH

COOH

HO

OOH

OH

COOH

HO

HOOC

AceA

KDO DHA

D-Api

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DHA was identified by combined gas-liquid

chromatography-mass spectrometry and proton reso-

nance spectroscopy in 1988 [41]. The information

about the presence of KDO in pectins was first reported

in 1978 [42] and later entirely confirmed [41]. This was

the first evidence of the presence of KDO in pectin sub-

stances of plants, in particular in RG-II of primary cell

walls. Earlier, KDO had been detected only in bacteriallipopolysaccharides. It was proposed that the three

above mentioned acidic monosaccharides (AceA,

DHA, and KDO) are the necessary components of RG-

II and should be present in any plant.

RG-II was first isolated from the cell walls of the

sycamore Acer

pseudoplatanus

[39]. Also, RG-II was

found in cell walls of rice, Douglas spruce, onion, kiwi,

radish, roots of the falcate hares ear Bupleurum

falca-

tum

, and leaves ofArabidopsis

thaliana

(see [1]). It was

also isolated from the pulp of a sugar beet, the commer-

cial enzyme preparation Pectinol AC, the products ofprocessing of a grape vine as the major component of

red wine polysaccharides, as well as from the juices of

apple (

Malus

domestica

), carrot (

Daucus

carota

), and

tomato (

Solanum

licopersicum

) treated with enzyme

preparations. RG-II is present as a dimer (see below) in

pectin polysaccharides of black currant and blueberry

(in cell walls, juice, and fruit press residues). In RG-II

located near the plasma membrane, the galacturonic

acid residues of the main chain are not esterified by

methanol, whereas RG-II located in primary cell walls

contains a great number of methoxyl groups, as was

shown by immunochemical methods. Interestingly,after the softening of fruits and vegetables during ripen-

ing, RG-II becomes a dominant polysaccharide in

apple, tomato, and carrot juices. Thus, RG-II that accu-

mulates in juices during the processing of fruits and

vegetables is an important pectin polysaccharide in the

industrial production of fruit juices [43].

RG-II is a comparatively small-sized polysaccha-

ride of extremely complex structure which has not yet

been completely established, although some variants

have been suggested [35].

It was found that RH-II contains boron, which formsboratediol esters that cross-link RH-II molecules to

form a dimer [38, 43]. It was shown that borate esters

cross-linking two RG-II molecules are localized on

-1,3'

-linked apiose residues, which consequently play

an important role in the growth of plant tissues. The

dimer has acid-labile borate ester bonds which are

hydrolyzed as pH in the cell wall and decrease during

the auxin-induced cell growth [38].

It is known that RG-II is the only polysaccharide inprimary cell walls that has borate bridges which form adimer from RG-II [38, 4446]. This borate complex isa part of a macromolecular pectin complex consistingof homogalacturonan, RG-I, and RG-II; borate esters ofRG-II form molecules of cross-linked macromolecularpectin [46]. However, it is not ruled out that homogalac-turonan, RG-I, and RG-II are covalently linked to each

other without the participation of borate [29, 47], sincepolysaccharides are not separated from each other bygel-permeation chromatography.

Although RG-II is contained in the cell wall inminor amounts, it plays a key role in its architectonics[48]. The structure of RG-II is rather conservative fordifferent plant species [41], indicating the importanceof its biological functions. RG-II not only influencesplant growth, but is also involved in the interactions ofplants with phytopathogens by affecting the specifictransport systems during the passage of amino acidsthrough membranes. Of importance for the realizationof the biological functions of RG-II are rather long

galacturonan regions in its macromolecule (of sevenand more galacturonic acid residues) and, in the opin-ion of the authors of [49], the residues of such unusualmonosaccharides as KDO and apiose.

Commercial Pectins

Commercial pectins find use in the food industryowing to their gelling capacity. Pectins obtained by acidextraction from vegetables and fruits (sugar beets,apples, citrus fruits) are predominantly homogalactur-onans and contain minor amounts of neutral sidechains.

It is just these pectins that form a group of com-mercial pectins [50, 51]. According to Europeanrules, commercial pectins intended for food productsmust contain more than 65% of GalA per dry weightof a sample, and according to American pharmaco-poeia, more than 74%. Homogalacturonans differfrom one another by substituents: GalA residues cancontain free carboxyls or methanol-esterified car-boxyls. Also, at C2 and C4 positions, GalA residuescan be acetylated, as is the case in pectins from sugarbeet and potato tubers. The gelation properties of

O O

CH2

OO

O

O

H2C

OO

O

OB

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pectins with a low DM (LM pectins) can be enhancedby chemical amidation in methanol, which leads to

the occurrence of amide groups at position C6 ofGalA residues (LMA pectins).

The physical properties of commercial pectinsdepend on their molecular masses, and primarily onDM. DM is determined by the number of moles ofmethanol per 100 moles of galacturonic acid [50]. Themodern methods of the direct quantitative analysis ofmethanol involve gasliquid chromatography (GLC) orHPLC. HPLC requires much less time compared withthe earlier methods, in particular ion-exchange chroma-

tography on DEAE cellulose [52]. A method for deter-mining DM by Fourier transform IR spectroscopy hasbeen elaborated: the value of the absorption band of thecarboxyl at 1756 cm

1

correlates well with the meanvalue of DM [53]. The method is rapid but requires themaintenance of a strictly specified pH value and precisecalibration using the characterized pectins. Recently, amethod of quantitative analysis of methoxyl groups inpectins by GLC on columns with a special attachmenthas been developed [54]. The results of this assay fullyagree with the data of HPLC.

In addition, a method for the simultaneous determi-nation of the content of methoxyl and acetyl groups in

pectin polysaccharides has been devised. The methodconsists in that methanol and acetic acid that split outafter alkaline hydrolysis are separated by GLC in aChrompak PoraPlot Q capillary column and aredetected by mass spectrometry. By using this proce-dure, methanol and acetic acid are readily determinedquantitatively in 1 mg of pectin [55].

A well-suited method for determining DM and thehomogeneity of pectin fractions is capillary electro-phoresis [56]. It was used to determine the mean valueof DM of a pectin sample since there is a direct relation-ship between the electrophoretic mobility of the mole-cule and the mean value of the charge per one monosac-charide residue [57]. Subsequently, this method wasused to study the distribution of galacturonic acidmethyl esters in pectins [5880]. Samples were frac-tionated by gel chromatography according to molecularmasses and then by electrophoresis according tocharge. The results confirmed the assumption that elec-trophoretic mobility depends on molecular mass andDM and showed that capillary electrophoresis providesreal information regarding the homogeneity and chargedensity distribution in the pectin chain [56].

Pectins with a high DM (HM pectins) contain 50%or more esterified GalA residues. LM pectins areobtained by deesterification of HM pectins under par-ticular controlled conditions (pH, temperature, andtime). Both groups of pectin polysaccharides form gels,but under different conditions: LM pectins at low pHvalues or in the presence of calcium cation, and HMpectins form gels by hydrophobic interactions, espe-

cially in the presence of sucrose [51].As a rule, commercial pectins are obtained using cit-

rus fruit peels and apple wastes, byproducts of the man-ufacture of juices, and sucrose from sugar beet, and, toa lesser degree, byproducts of the manufacture of starchfrom potatoes, sunflower heads in oil production, andonions.

The pectin extraction procedure should be rapid toprevent the degradation of polysaccharides by enzymesin the starting materials. The degradation of pectins byenzymes during the storage of the initial materials canlead to samples with quite different gel forming proper-ties. The starting material should be dried immediately

after processing to avoid degradation [51].Pectins from the byproducts of sugar beet process-ing have a low gelation capacity owing to their lowmolecular masses and a high content of acetyl groups.Treatment with acidic methanol removes acetyl groupsand increases DM but substantially lowers the molecu-lar mass [61]. Nevertheless, acetylated pectins findapplication owing to their emulsifying properties. Thegelation of acetylated pectins can be enhanced by treat-ment with pectinesterase and pectinacetylesterase [62].

As mentioned above, sugar beet pectin contains fer-ulic acid residues [63] linked by ester bonds primarilyto O2 residues of arabinose and O6 residues of galac-tose of neutral carbohydrate chains. The side chains ofarabinan and galactan consist of 1,5

-linked

-

L

-arabi-nose and 1,4

-linked

-

D

-galactose residues, respec-tively. When treated with a mixture of H2O/peroxidaseor ammonium persulfate, these molecules are easilyjoined to form dimers, thereby increasing the viscosityand gelling of sugar beet pectin. About 20% of the totalnumber of ferulic acid residues in pectin are present asdimers. The structural identification of dimeric oli-gosaccharides from sugar beet pectin, linked by ferulicacid residues, showed the presence of covalent (intra-

O

OH

OH

COOCH3

O O

O

OH

OH

COOH

O

O

OH

OH

CONH2

O

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and intermolecular) cross-links of pectin arabinans andgalactans through diferuloyl bridges in sugar beet cellwalls [63].

Owing to valuable physical properties, commercialpectins are widely used as thickening agents, gels,adhesives, emulsifiers, and stabilizers of solutions.

Sources of Pectins Investigated in the Last Century(19982008)

Pectin polysaccharides of the European North ofRussia, which were studied in the Department ofMolecular Immunology and Biotechnology of the Insti-tute of Physiology (Komi Science Centre, The UralsBranch, Russian Academy of Sciences), such assilenan, a pectin from the catchfly Silene vulgaris(Moench) Garke (ObernabehenL.), tanacetan, a pectinfrom the tansy TanacetumvulgareL., lemnan, a pectinfrom the lesser duckweedLemnaminorL., comarumanfrom the marsh cinquefoil C.palustreL., and some oth-ers have been described in great detail in review [6]. We

will not dwell on these compounds.Plants of the genusLupinuscan serve as sources ofvaluable pectins. It has been established that they con-tain from 1.5 to 7% of pectic substances depending onthe variety, year, and vegetation stage. Varieties con-taining about 7% of pectins are promising for industrialproduction of pectins. Lupin pectins have perfect swell-ing and water-absorbing abilities and form a stable gelat a 2% concentration [64]. The molecular mass of pec-tins varies between 100 and 400 kDa, and DM is 6585%. The basis of a pectin macromolecule is D-galac-turonic acid; of other monosaccharides, galactose andarabinose have been identified as the main components.Pectin has a complex branched structure and represents

a mixture of linear galacturonans and highly-branchedpectin polysaccharides [65].

It was proposed to use mandarin orange peels toexpand the spectrum of pectin sources. The composi-tion and properties of pectins from mandarin orangepeels depend on the conditions of growth and the vari-ety of the fruits [66].

It was also proposed to use burdock (genusArc-tium)as plant row material for obtaining pectin. Bur-dock is characterized by a high content of pectic sub-stances [67]. The highest content of pectin is in leaf cut-tings (1.5%). In dry press residues of burdock, 21% ofpectic substances accumulate, whereas in dry apple

marcs, only 11%. Consequently, burdock leaf cuttingscan compete with traditional row material for obtainingpectin. Unfortunately, burdock pectin gelatinizespoorly, which is related to a low DM.

Pumpkin pectin was isolated by extraction with0.1 M HCl after preliminary digestion using threeenzyme preparations: cellulase from Trichoderma vir-ide, hemicellulase from Aspergillus niger, and a gly-cosidase complex fromXanthomonas campestris[68].The method was optimized as follows: pectin was

obtained from the pumpkin pulp preliminarily treatedwith an enzyme preparation from Asp. awamori[69].The main function of the enzymes is to digest celluloseand other insoluble components of plant tissue. Thetreatment with purified enzymes of a known specificityenables one not only to isolate pectin but also to obtainadditional information about its structure and links withother cell wall components.

It should be noted that pumpkin is a nontraditionalbut a very interesting and promising source of pectin.Pumpkin pectin forms gels at concentrations muchlower than the commercial citrus pectin. The use ofenzymes substantially increases the yields of pectinsubstances and leads to the complete solubilization ofplant material.

Pectins of amaranthwere isolated from differentspecies of this widely distributed plant [70]. On the ter-ritory of Russia, 17 amaranth species exist.AmaranthusretroflexusL. or prostrate amaranth, known as a plant,occurs most widely. Many amaranth species find appli-cation in nontraditional medicine. Thus, an aqueous

infusion of leaves of this plant is used in the treatmentof diseases of the GIT and as a hemostatic. A decoctionof the apices ofAm. cruentosis used for the treatmentof rheumatism, womens diseases, and as an antitussic.Amaranth decoctions are used to remove radionuclides.Several methods of isolating pectin from amaranth havebeen developed. Good results with a high efficiency ofisolation have been obtained by the method using arotor-pulsation device. The raw material is thoroughlydivided by the device and is subjected to cavitation andultrasound treatment, which results in the disintegra-tion of the raw material and the highly effective extrac-tion of pectin [71].

Amaranth pectin is characterized by a high contentofD-galacturonic acid residues (more than 80%) and ahigh DM (75%). Structurally, this is a typical represen-tative of this class of polymers: it has a linear region ofgalacturonan (rhamnogalacturonan) and a ramifiedregion of RG-I in which the side chains are composedprimarily of arabinose and galactose residues [70].

Amaranth pectin is used in medicine as a hypoten-sive drug, in nutrition, and as a regulator of growth anddevelopment of plants [70].

A new pectin has been isolated from leaves of theThailand ligneous vine Cissampelos pareira (familyMenispermaceae) which widely occurs in warmregions of Asia, East Africa, and South America [72].The plant is considered to be a medicinal plant and isused in the treatment of asthma, dysentery, urologicaldiseases, and post-traumatic pains. Aqueous extracts ofleaves form gels which are used as a dessert in Thai-land. These gels can serve as a source for obtaining nat-ural polysaccharides, in particular pectin. Pectin con-sists predominantly of galacturonic acid residues (7075%) with a low DM and minor amounts of neutralmonosaccharides. Pectin forms a good gel whose sta-

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linked xylopyranose and -1,3-and -1,6-linked galac-topyranose residues (galactan). In addition, the sidechains contain arabinofuranose residues of xylose resi-dues linked at position 2 by a -1,4-linkage and of galac-tose residues linked at position 3 by a -1,6-linkage.

Pectin stimulates the proliferation of B lymphocytesinduced by a lipopolysaccharide but does not stimulatethe proliferation of T lymphocytes induced by con-

canavalin A. It was shown that arabinose residues in theside chains are not necessary for immune activity. Ofprimary importance is the backbone of galacturonan.

Centellaasiaticais used in China and India as a sed-ative and an antiulcerous preparation, as well as in thetreatment of lepsory. Pectin isolated from this plantcontains Rha, Ara, Gal, Glc, and GalA in the molarratio of 1.0 : 0.6 : 1.5 : 0.2 : 1.1 [85]. It was found thatthe backbone of this pectin consists of the repeating dis-accharide unit 4)--D-GalpA(1 2)--L-Rhap-1 and hence belongs to the RG-I group. The sidechains are predominantly linked to the rhamnopyranoseresidues of the core by a 1,4-linkage. These chains con-

sist of residues of arabinose and galactose, or arabinoseresidues in combination with galactose residues; also,short glucan chains are present. About 45% of Rhapres-idues in the core are substituted for by side chains. Ara-binose residues form for the most part oligoarabinosylside chains and are occasionally attached to 1,6-linkedgalactose residues.

Pectin possesses no immune activity. However,some of its derivatives, e.g., those devoid of acetylgroups and containing a lesser amount of arabinose res-idues, show immunostimulating activity. Treatmentwith periodate and the removal of 1,6-linked Gal resi-dues also lead to a derivative with immunostimulatingactivity. Derivatives containing the core with a minor

amount of side chains have the highest activity.Some species of cactus (family Cactaceae) find

practical application [8689]. Thus, the opuntia Opun-tia ficus indica Mill is cultivated in Chile as a fruitplant, and young shoots of this cactus are also used insome countries by humans as a food product. The pec-tin polysaccharide obtained from the fruit peel has themain homogalacturonan chain of -1,4-linked galactu-ronic acid residues; some regions of homogalacturonanare joined by 1,2-linked -L-rhamnose. The side chainsare composed of 1,5--L-arabinan and 1,4--D-galac-tan. Also, the endosperm of opuntia seeds was found tocontain pectins rich in arabinan.

From the ginsengPanax ginsengC.A. Mey (familyAraliaceae), a series of pectin polysaccharides havebeen isolated and thoroughly studied by Japaneseinvestigators [90]. The structures and physiologicalactivity of some of these polysaccharides have beenconsidered in detail in a recently published review [91].

Most recently [92], one more acidic pectin-likepolysaccharide has been isolated from ginseng. It has ahigh antiadhesive activity against pathogenic bacteria,in particular against the adhesion by epithelial cells of

the GIT of the bacteriumHelicobacterpylori, one of themain ethiological agents of chronic gastritis, ulcers ofstomach and duodenum, stomach carcinoma, and lym-phoid tumors. Pectin polysaccharide has a molecularmass of 12 kDa and consists of residues of galacturonicand glucuronic acids, rhamnose, arabinose, and galac-tose. A partial hydrolysis of the polysaccharide by pec-tinase furnishes an oligosaccharide fraction whose

activity is comparable with the activity of the originalbiopolymer.

Hares earBupleurum falcatum (family Umbel-liferae)is a medicinal plant widely distributed in Japan,China, and Primorye Territory. Its roots are tradition-ally used in the treatment of chronic hepatitis, nephro-logical syndrome, and various autoimmune diseases.The physiologically active pectin polysaccharide fromthis plant, named bupleuran 2IIc, has been studied indetail by a group of Japanese investigators headed byProf. Yamada [90, 93]. They have established the struc-tures of bupleuran 2IIc and its antigenic epitope. Pectincontains approximately 70% of 1,4-linked-D-galacto-pyranosyluronic acid, of which 30% is esterified bymethanol. Some galacturonic acid residues are thepoints of branching of the carbohydrate chain of thepectin polysaccharide. Also, bupleuran 2IIc has a typi-cal ramified region of RG-I. The polysaccharide alsocontains a minor region of RG-II which contains thesame rarely-occurring monosaccharide residues:apiose, AceA, KDO, and DHA. It was shown thatbupleuran 2IIc has high antiulcerous and anticomple-mentary activities. Intravenous injection of pectinresults in the formation of antibodies, and the polysac-charide is found in the liver. After peroral administra-tion, the polysaccharide is also detected in the liver andPeyers patches. It was found that the antigenic epitope

of antiulcerous bupleuran 2IIc is located in the externalmoiety of the ramified region and represents two differ-ent oligosaccharides with the terminal side residues ofglucuronic or 4-O-methylglucuronic acids, which arelinked to the galactan chain by a 1,6-linkage [94]:

Also, bupleuran 2IIc has a mitogenic action: it pro-liferates B cells in the absence of macrophages, andactivated B cells are transformed to antibody-formingcells in the presence of interleukin IL-6. Pectin alsoenhances the secretion of macroglobulin IgM from nor-mal murine B cells. As a result, long-term cell prolifer-ation is induced. When B cells are stimulated bybupleuran 2IIc, the protein of retinoblastoma is phos-phorylated and the expression of cyclins regulating thecell cycle is enhanced, which correlates with cell prolif-eration. The ramified region of pectin enhances thesecreting activity of IL-6, thereby causing an increasein macroglobulin secretion [95].

3Galp1 3Galp1 3Galp1

6GlcpA1

or4MeGlcpA1

Structure of the epitopeof bupleuran 2IIc

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From leaves of the plantain Plantago major L.(family Plantaginaceae), two polysaccharides show-ing a high activity toward the complement have beenisolated: arabinogalactan RMI and pectin polysaccha-ride PMII [96]. The first polysaccharide belongs to ara-binogalactans of AG-II type. It consists of the 1,3-linked galactan core with branching 1,6-linked galactanside chains coupled to the backbone by a 1,6-linkage.

Terminal and 1,5-linked arabinofuranose residues arelinked to these side chains via a 1,3-linkage. Also, PMIcontains a protein component characteristic for AG-II.The pectin polysaccharide PMII has a typical structureof RG-I [91].

A study of the interaction between the human com-plement and PMII has shown that PMII is a potent acti-vator of both the classical and alternative pathway ofcomplement activation. It induces the secretion ofhuman immunoglobulin IgG, which is directly relatedto the well-known wound-healing effect of leaves of thegreater plantain P. major[91, 97].

It has been shown on mice that regular prophylactic

administration of PMII protects well against pneumo-coccal infection, and this protective effect is due to thestimulation of inborn rather than adoptive immunity[91, 98100].

Argane tree (Argania spinosaL.), family Sapota-ceae, is an endemic of south-west Morocco [101, 102],has a hard wood pulp, and gives fruits similar to olives.It protects soil against water and wind erosion. Differ-ent parts of the plant find wide and various applications:seeds are used to obtain valuable oil for nutrition, cos-metic, and medicinal purposes (they lower the choles-terol level and improve blood flow); substances beingpart of cell wall polymers are used in food and cosmetic

industries and as drugs.Along with cellulose, cell walls contain pectins, an

arabinogalactanprotein complex, and xyloglucan.

In the fraction of pectin substances from fruit pulp,homogalacturonan, RG-I, and RG-II have been isolatedand fractionated. RG-I is the main constituent of pectinand contains, along with galacturonic acid, a greatamount of arabinose residues and a lesser amount ofgalactose residues as branched side chains. The resi-dues of other monosaccharides: glucose, xylose,apiose, and glucuronic acid are the minor componentsof RG-I. Arabinans and/or arabinogalactans containedin the pectin fraction are present either in a free state oras part of RG-I side chains. The presence of galactanside chains is also not excluded. RG-II is present in thepectin fraction as a minor polysaccharide in the form ofa dimer. Arabinose residues amount to 34% of allmonosaccharides of this polysaccharide, which consid-erably exceeds the content of arabinose in RG-II of themajority of other plants [102]. For comparison, the con-tent of arabinose in RG-II of Arabidopsis thaliana is14% [103], and in RG-II of pumpkin, as low as 9%[104].

Peach (Persica vulgaris L.), family Rosaceae, isgrown as a fruit culture in countries of moderatelywarm and subtropical zones. In China, six species ofthis plant grow. Many varieties are cultivated in MiddleAsia, in the Caucasus, in southern Ukraine, and inMoldova. Despite the wide distribution of the peach,pectin substances from its fruits have been little inves-tigated. There are only a few articles on the extraction

of pectin from peach fruits [105108]. Temperature,pH, and the time of acid treatment affect the yield ofpectin from fresh and stored peach grist. The yield ofpectin varies from 2 to 10% depending on pH and tem-perature. The yield is maximal at a higher temperatureand low pH values. The DM value in all cases remainshigh and ranges from 72.1 to 95.4%. It was also foundthat the yield of pectin from stored press residues issubstantially higher than that from fresh husks; how-ever, the DM and specific viscosity of solutions in thiscase decrease.

The yield of pectin substances also depends on the

ratio of ethanol and extract during precipitation of pec-tin. As this ratio increases, a higher yield and greaterDM values are observed. A maximal yield (9.94 2%)was obtained at the ethanolextract ratio of 1.5 : 1.0 andwith the time of acid treatment of about 120 min [108].

Pectin from spinach (Spinaca oleraceaeL.), fam-ily Chenopodiaceae, has been studied by treating thecell walls of a suspension culture with the enzymedriselase [109] which easily hydrolyzes glycosyl bondsof -D-GalpA and -L-Rhapin homogalacturonan andRG-I after the preliminary removal of methyl estergroups by cold diluted alkali. Driselase is also capable

of partially and completely hydrolyzing methylesteri-fied oligogalacturonides to form free GalA and metha-nol. However, other esters (acetates and feruloyl estergroups) are resistant to driselase; as a result, oligosac-charides carrying -acetyl or -feruloyl ester groupscan result during fermentolysis. In addition, the pres-ence of -acetyl groups protects one or several methylester groups from hydrolysis by driselase. This makesit possible to map methyl ester and acetyl ester groupsinside the pectin core. By enzymatic hydrolysis of spin-ach pectin with driselase, oligogalacturonides andrhamnogalacturonides were isolated. Five out of six oli-gosaccharides from homogalacturonan contained 3--

acetyl-GalpA residues, whereas methylesterified GalAresidues were located near both 2--acetyl-GalA and3--acetyl-GalA. The oligosaccharide GalA-Rha-GalA-Rha-GalA obtained from RG-I also contained3--acetyl-GalAresidues.

Thus, the distribution of methyl ester and acetylgroups in galacturonan and RG-I of spinach pectin isirregular and is still far from being completely under-stood [105].

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Physical Properties of Pectins

The gelling properties of pectin polysaccharidesdepend mainly on DM [33, 110, 111].

Pectins are widely used as gelling agents, stabilizersof solutions, thickening agents, adhesives, and emulsi-fiers in many food products. It has been shown that animportant role in the formation of pectin gels is played

by sucrose, which can stabilize the structure of junctionzones of gel formation [1, 112].

The interaction of proteins and peptides with pectinsresults in the formation of soluble and insoluble com-plexes whose structures depend on the ratio of biopoly-mers and pH [113115]. In this process, covalent bondsbetween the tyrosine residues of the protein and the car-boxyl group of uronic acid of pectin are formed, whichcan lead to the formation of a pectin network. There isan alternative hypothesis, according to which proteinsform ionic complexes with pectins [114, 116].

Globular proteins, such as bovine serum albumin(BSA) and -lactoglobulin, form gels, and the mixing

of these gels with LM pectin results in the formation ofmixed gels. During the formation of gels, a kineticcompetition between gel formation and the phase sepa-ration process takes place. The control of relative ratesof these processes by external factors, such as environ-mental conditions, leads to a wide spectrum of micro-structures [116, 117]. The formation of gels during theinteraction of pectin polysaccharides and globular pro-teins has been extensively studied. A variety of struc-tures and the rheological properties of resulting gelshave been described which differ depending on thenature of biopolymers and the conditions of gel forma-tion (see, e.g., [117125]). It has been found that thepresence of Na+and Ca2+cations and their ratio play a

decisive role in the gel formation of mixtures of globu-lar proteins with pectins [117]. Increasing the amountof pectin and calcium concentration makes the mixedgel more rigid [119].

During the treatment and storage of gels, reactionsbetween pectin and a protein or a peptide can takeplace: hydrolysis of ester groups in pectin, decarboxy-lation, -elimination, or the hydrolysis of glycosidebonds due to the presence of pectinase in the mixture.These processes should be taken into considerationduring the production of pectinprotein gels [126].

The high bioadhesive capacity of pectins was usedto obtain a medicinal preparation based on lactoferrin,a milk glycoprotein producing the bactericide action inthe treatment of chronic inflammation in stomatitis[127]. It was shown that HM pectins are best suited forpreparing bioadhesive tablets since, along with a highbioadhesive capacity, they have the property of releas-ing the active medicinal substance. Moreover, this pro-cess can be regulated by the concentration of calciumions, which cross-react with the galacturonan fragmentof the pectin macromolecule and facilitate the releaseof lactoferrin.

It is well known that the gel-forming capacity ofpectins depends on many factors, including DM, thelocalization of methyl ester groups, pH, the concentra-tion of biopolymer and calcium ions, the ionic strengthof solution, temperature, and others [72, 110, 128130].

LM pectins form gels in the presence of calciumions. Commercial LMA pectins require a lesser amount

of calcium for gelation and are less sensitive to synere-sis induced by a high concentration of calcium ions; inthis case, the gel formation is thermally reversible[131].

The range of calcium ion concentrations at whichrigid gels are formed is relatively narrow in the casethat gel formation occurs in the absence of other salts orwith the use of NaCl alone. This range is extendedtowards higher Ca2+concentrations in the presence ofsodium citrate and potassium and sodium tartrates, as ithas been shown in a study of the effect of this mixtureof salts on the gelation of LMA pectin [128]. Thesesalts make gels more elastic and thermally reversible,

even during mechanical destruction. The treatment ofHM pectins with pectinmethylesterase leads to the for-mation of LM pectins whose behavior in solutionsgreatly differs from the behavior of HM pectins andstrongly depends on the concentration of monovalentcations [129, 132]. When solved in the presence ofmonovalent ions of Li+, Na+,and K+at concentrationsfrom 0.005 to 0.05 M, the resulting LM pectin exhibitsa higher specific viscosity than HM pectin, whereasHM pectin has a higher specific viscosity in the pres-ence of 0.2 M monovalent salts. Interestingly,Mwsub-stantially increases during the transformation of HMpectin to LM pectin, which is probably related to theaggregation of molecules of LM pectin being formed

[129, 132].Mixtures of HM and LM pectins are often used in

the food industry for the manufacture of products withparticular properties. By varying the conditions of gelformation, it is possible to substantially change therheological behavior and the microstructure of mixedgels of HM and LM pectins [130, 133, 134]. A strongsynergetic effect in the rheological behavior of mixedgels was noted in the presence of Ca2+and 60% sucroseat pH 3, i.e., under conditions promoting the gelation ofboth HM and LM pectins.

Also, during the production of gels with a lowsucrose content, a strong synergetic effect is observed

in the rheological behavior of mixed gels in which theconcentration of HM pectin is three times as high as theconcentration of LM pectin. In this case, a mixed gelwith a lower sucrose concentration has the same rheo-logical parameters as HM pectin in the presence ofsucrose of a considerably higher concentration [130].

Interesting results have been obtained in studies ofthe gelling properties of natural citrus pectin (DM64%). Pectin was preliminarily either esterified bymethanol to pectin with a higher DM or saponified to

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obtain pectin with a low DM value, and the resultingsamples were then reduced by sodium borohydride[110]. As a result, two thirds of galacturonan carboxylgroups were transformed to primarily alcohol groups,and only one third remained in the form of free car-boxyl groups. Eventually, the resulting samples gavesolutions with a substantially higher dynamic viscosityand better rheological parameters. It has been shown

earlier that the gelation and subsequent stability of gelsare determined by hydrogen bonds and hydrophobicinteractions, in particular between the hydroxyl groupsof galacturonan and sucrose and between the methylester groups, respectively [135]. In the absence ofsucrose, these bonds are weak and gelation does notoccur. In the case of citrus pectin modified by saponifi-cation, the number of hydroxyl groups increases, whichleads to the formation of a considerably greater amountof hydrogen bonds. Therefore, the stabilization of theremaining hydrophobic interactions and the formationof gels require a lesser amount of sucrose than in thecase of the original citrus pectin [110].

Gel matrices based on biopolymers, among thempectin substances, are widely used in food, agricultural,and pharmaceutical industries as systems for the encap-sulation and subsequent release of active substances.One of the trends in the food industry is the release ofaromatic compounds from colloid solutions and gels[136138]. It has been shown that the intensity of theodor emitted by colloid solutions and gels weakenswith increasing the concentration and rigidity of the gelowing to the direct binding of aromatic substances withpolymeric chains [136, 138140]. Gels of citrus HMpectin at different concentrations for the limonene odorconservation have been studied, and a correlationbetween a decrease in the intensity of the emitted odor

and an increase in gel rigidity has been established[138].

At present, in the field of biopolymer research it isproposed to use biopolyelecrtolyte multilayers for thecreation of encapsulation systems and the preparationof coatings which enable one to control cell adhesion[141, 142]. Thus, the formation of multilayers duringthe interaction of citrus pectin (DM 36.6%) with theweak oppositely-charged polyelectrolytes poly-L-lysine and chitosan has been studied [143145]. Poly-L-lysine can cross-link molecular networks of pectinand bind with a high affinity to the surface of a pectinmacromolecule. Pectin and chitosan in the ratio 1.2 : 1at pH 5.6 form on solid surface alternating layers ofpectin and chitosan; during this process, the binding ofthe polymers to the surface takes place. The multilayerformation is observed at pH values at which both poly-mers carry a charge on macromolecules. The thicknessof an individual layer depends on the concentration ofbiopolymer. Changes in pH that suppress the charge ofone of the polyelectrolytes lead to the destruction of themultilayer composition, indicating the importance ofelectrostatic interactions for the formation and stabilityof multilayers [145].

Effect of Pectins on the Growth and Developmentof Plants

Pectins fulfill important biological functions inplants [1, 29]. They regulate ion and water exchange,participate in the structure formation of plant cell walls,facilitate seed germination and growth of plantlets, pro-vide plant turgor, etc. Changes in the composition andstructure of pectin substances during the growth and

development of plants have been studied by an exampleof pectins from stalks and leaves of the common flaxLinumusitatissimumL. [146]. The content of pectins instalks of the plant decreases from 7% in the initial stageof development to 2% in the stage of wilting, whereastheir content in leaves during the development remainsunchanged (~4%). The content of galacturonic acid inpectin polysaccharides of the stalk increases from 53 to66% in the wilting phase, whereas in pectins of leavesonly insignificant changes during plant growth areobserved. The DM of flax pectin polysaccharides ishigh, 80100%, and varies slightly during the develop-ment of the plant. The degree of acetylation plays an

important role in the development of the plant and isequal to 2340%; it only slightly changes during plantgrowth and development. Pectin substances have highmolecular masses and are synthesized at the initialstage of plant development [146]. The content of solu-ble -galactans localized in flax stalks falls during thedevelopment of the plant: they are either destroyed orare fixed inside cell walls [147].

It has been shown that during ripening of fruits, thepolygalacturonase-induced solubilization of pectinpolysaccharides and the release of ethylene take place,which enhances fruit ripening. These processes proceeddifferently in different fruits [148153]. Thus, the rateof ethylene release by the fruits of the Peruvian cherry

(ground cherry PhysalisperuvianaL.) increases duringripening and is maximal on day 50 after flowering,which is accompanied by the synchronous release ofCO2, and the high level of the polygalacturonase activ-ity correlates well with an increase in the amount ofreleased ethylene [152]. It should be noted that fruitsoftening is always associated with a decrease in theamount of the insoluble form of pectic substances,which is controlled by specific enzymes, primarily bypolygalacturonase [152, 153].

Physiological Activity of Pectins

Pectins have a wide spectrum of physiological activ-ity [133, 154], including the immunomodulating action[1, 155]. The gastroprotective action of pectins is alsowell known [1, 156, 157].

Pectins have been an integral part of food at allstages of the evolutionary development of humans,which has governed the practically ideal adaptation ofthe human organisms to them [154].

A property of great importance is the anticarcino-genic and/or antimetastatic effects of pectins, in partic-

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ular apple pectin, which have been demonstrated by theexample of apple pectin in experimental models ofintestinal carcinogenesis and liver metastases. Theapplication of apple pectin in the treatment of intestinalcancer has been patented in Japan.

It has been shown that pectins with different esteri-fication degrees, as well as polysaccharides from thesepectins, induce the apoptosis of adenocarcinoma HT29

of the human large intestine cancer in vitro. The great-est effect has been observed with oligosaccharide prep-arations from LM pectins [158].

It is assumed that on the surface of cancer cells, pec-tin polysaccharides bind proteins responsible for theadhesion of tumor cells by healthy tissues. A commer-cial preparation which represents modified citrus pectinsuppresses the growth of cancer cells and metastasizing[159, 160]. It has been shown that it also increases theprostate-specific antigen doubling time, the majoroncomarker of prostate adenocarcinoma, in 70% ofmen suffering from prostate cancer [161].

Also, modified citrus pectin facilitates the removal

of toxic elements out of the organism with urea [160].The use of pectin as a carrier matrix of biologically

active components or drugs merits attention [162, 163].Among these are the products of interaction of citruspectin with helminthicides. Studying the immobiliza-tion of the antituberculous drug isoniasid on pectin sub-stances has shown that the preparation obtained has ahigher tuberculostatic activity than pure isoniasid. Theapplication of chemically modified pectins as carriersof drugs also is promising for the treatment of the intes-tine. Cross-linked pectin, which is less soluble and lesssubject to degradation in the organism, as well as cal-cium pectinate gel [162, 163] containing NaHCO3and

3are effective for these purposes. These gels arecharacterized by a high porosity and proper density.The acidification of the gelling medium increases thepore sizes, which provides a good and quick release ofthe medicinal preparation in the proper place. On theother hand, the method makes it possible to regulate therealization of the drug and provide its prolonged release[163].

Pectins attract increasing interest in gastroenterol-ogy as peroral preparations and therapeutical means inthe treatment of diseases associated with mucosalinflammation. Also, pectins are used as a physical bar-rier to protect epithelium against opportunistic micro-organisms during stress [164167]. Among differentaspects of the action of pectins on the GIT, its interac-tion with mucus is the subject of most intensive study;thus, it has been shown that pectins bind to mucin, themain component of the mucus of the GIT, to form a gelnetwork [167]. In this manner, pectin polysaccharidesenhance the protective barrier properties of mucus andhence can be applied to treat lesions of the GIT andinfectious diseases. By varying the molecular charac-teristics of pectin, it is possible to change the rigidity ofthe resulting gels and the pattern of pectin distribution

in the pectinmucin complex. This enables one to reg-ulate the interaction of pectin with mucin to meet thedemands for drug delivery and clinical therapy [168].

Conclusions

Pectic substances represent a manifold class of com-plex plant polysaccharides which constitute a function-

ally important moiety of primary cell walls. At present,great advances of fundamental importance have beenmade, especially in the elucidation of the main featuresof the chemical structure, physicochemical properties,and biological activity of pectins. Nevertheless, manyproblems are yet to be studied to use this big class ofnatural compounds purposefully and with awarenessfor treating various diseases and increasing the durationof active human life.

ACKNOWLEDGMENTS

This work was supported by the program: LeadingScientific Schools (NSh-2383.2008.4), the RussianFoundation for Basic Research (project no. 06-04-48079), the integration project of basic research of theUrals and the Siberian Divisions of the Russian Acad-emy of Sciences, the Programs of the Presidium of theRussian Academy of Sciences: Fundamental Sciencesto Medicine and Molecular and Cellular Biology,and the Foundation for the Promotion of National Sci-ence.

The author is grateful to the senior engineer of theInstitute of Physiology (Komi Science Centre, TheUrals Branch, Russian Academy of Sciences)Yu.A. Ovchinnikova for help in the preparation of thereview.

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