Aceric Acid Research Articles

Antitumor and antimetastatic activities of rhamnogalacturonan-II-type polysaccharide isolated from mature leaves of green tea via activation of macrophages and natural killer cells

To investigate the antitumor and antimetastatic polysaccharide from the mature leaves of green tea, GTE-II was purified using size exclusion chromatography. GTE-II consisted of 15 different sugars including rarely observed sugars such as 2-O-methyl-fucose, 2-O-methyl-xylose, apiose, aceric acid, 3-deoxy-d-manno-2-octulosonic acid, and 3-deoxy-d-lyxo-2-heptulosaric acid, which were characteristics of pectic polysaccharide rhamnogalacturonan-II. Treatment of peritoneal macrophages with GTE-II not only increased interleukin (IL)-6 and IL-12 production, but also had significantly increased tumoricidal activity against Yac-1 tumor cells than those obtained from untreated mice. In an assay of natural killer (NK) cell activity, intravenous administration of GTE-II significantly stimulated NK cytotoxicity against Yac-1 tumor cells. Furthermore, the depletion of NK cells by injection of rabbit anti-asialo GM1 serum eliminated the inhibitory effect of GTE-II on B16BL6 melanoma cells. These data suggest that GTE-II inhibits tumor metastasis, and its antitumor effect is associated with activation of macrophages and NK cells.

감잎 유래 다당체가 면역억제 마우스의 면역조절에 미치는 영향

감잎으로부터 새로운 생리활성 다당 소재를 개발하기 위하여 감잎의 열수추출물로부터 다당획분 PLW-0, pectinase 처리 추출물로부터 다당획분 PLE-0를 분리하여 정상 및 면역 억제 동물모델에서 면역자극활성을 측정하였다. 감잎 유래 다당체들은 정상모델에서 마우스의 면역기관인 비장과 흉선의 무게를 증가시켰으며, 비장세포 유래 림프구의 증식에 농도 의존적으로 증식활성을 보였는데, pectinase 처리 다당 획분 PLE-0의 활성이 열수추출 다당 획분 PLW-0에 비해 전체적으로 높은 활성을 보이는 것을 확인할 수 있었다. 또한 CY에 의해 면역이 억제된 동물모델에서도 CY처리에 의해 저하된 임파구 증식, NK 세포 활성 및 백혈구 수를 현저하게 증가시켰다. 이러한 면역억제 극복 활성 역시 유의적인 차이는 보이지 않았으나 PLE-0가 PLW-0에 비해 높음을 확인할 수 있었다. CY는 악성 종양의 치료 및 장기이식 후 나타나는 거부반응의 억제제로 사용되고 있는 대표적인 약물로 알려져 있다. 그러나 CY의 비선택적인 독성으로 인해 암세포뿐만 아니라 정상세포에도 독성을 나타내어 백혈구 수 감소, 혈소판 감소, 빈혈 등과 같은 부작용을 나타내고 있다. 따라서 최근에는 이와 같은 부작용이나 면역독성을 최소화 할 수 있는 물질을 발굴하고자 하는 연구가 이루어지고 있는데, 본 실험의 결과로부터 감잎 유래 다당체는 정상모델에서의 면역자극 및 CY에 의해 초래된 면역억제를 감소시킴으로써 면역조절제로서의 개발가능성을 제시해 줄 수 있다고 생각된다. 또한 pectinase 처리 다당 PLE-0가 열수추출 다당 PLW-0에 비해 비교적 높은 활성을 보인 것을 감안해 보았을 때 효소처리가 감잎 유래 다당의 활성증가에 도움을 준다는 것을 확인하였다. We prepared 2 different crude polysaccharides from persimmon leaves, by hot water (PLW-0) and pectinase digestion (PLE-0), respectively. PLW-0 and PLE-0 showed similar sugar compositions and contained 11 different sugars, including rarely observed sugars such as 2-methyl-fucose, 2-methyl-xylose, apiose, and aceric acid. However, the uronic acid content of PLE-0 was lower than that of PLW-0, because of pectinase treatment. In normal mice, administration of PLW-0 and PLE-0 increased the spleen index and splenocyte proliferation. The effect of PLE-0 on the spleen index and splenocyte proliferation was greater than that of PLW-0. We subsequently assessed the immunomodulatory activities of PLW-0 and PLE-0 on cyclophosphamide (CY)-induced immunosuppressed mice. We revealed that mice treated with PLW- 0 or PLE-0 showed increased splenocyte proliferation, NK cell activity, and white blood cell numbers. Taken together, our results indicate that PLW-0 and PLE-0 can enhance immune function in normal mice and modulate CY-induced immune suppression.

감잎에서 분리한 다당의 면역자극 활성

Abstract In order to develop new physiologically active polysaccharides from persimmon leaves, two different crude polysaccharides were prepared using hot water (PLW-0) and pectinase digestion (PLE-0) and their immuno-stimulating activities were estimated. PLW-0 and PLE-0 showed similar sugar compositions with 15 different sugars, including rarely observed sugars in general polysaccharides such as 2-O-methyl-fucose, 2-O-methyl-xylose, apiose, aceric acid, 3-deoxy-D-manno-2-octulosonic acid, and 3-deoxy-D-lyxo-2-heptulosaric acid, but the uronic acid content of PLE-0 was lower than that of PLW-0 caused by pectinase treatment. Both PLW-0 and PLE-0 showed potent anti-complementary activity in a dose-dependent manner which was similar to a known immuno-stimulating polysaccharide, PSK, from Coriolus versicolor. The activity of PLE-0 at a low concentration (100 ㎍/㎖) was higher than that of PLW-0. In an in vitro cytotoxicity analysis, PLW-0 and PLE-0 (up to 1,000 ㎍/㎖) did not affect the growth of peritoneal macrophages and Colon 26-M3.1 carcinoma cells. In contrast, they enhanced lymphocyte proliferation activity. Peritoneal macrophages stimulated with PLW-0 and PLE-0 produced various cytokines, such as IL-6 and IL-12. However, PLE-0 was more effective on the cytokine production. Intravenous administration of PLW-0 and PLE-0 significantly augmented natural killer (NK) cell cytotoxicity against Yac-1 tumor cells 3 days after the treatment of polysaccharide fractions. But NK cells obtained from the PLE-treated group showed higher tumoricidal activity even at a low dose of 40 ㎍/mouse. In experimental lung metastasis of Colon 26-M3.1 carcinoma cells, prophylactic administration of PLW-0 and PLE-0 significantly inhibited lung metastasis in a dose-dependent manner and PLE-0 was more effective on the inhibition of cancer metasasis. The results lead us to conclude that the pectinase-treated process is indispensable to preparing polysaccharides with higher immune-stimulating activity from persimmon leaves.Key words: persimmon leaves, polysaccharide, immune-stimulating activity, anti-metastatic activity

The Synthesis and Origin of the Pectic Polysaccharide Rhamnogalacturonan II – Insights from Nucleotide Sugar Formation and Diversity

There is compelling evidence showing that the structurally complex pectic polysaccharide rhamnogalacturonan II (RG-II) exists in the primary cell wall as a borate cross-linked dimer and that this dimer is required for the assembly of a functional wall and for normal plant growth and development. The results of several studies have also established that RG-II structure and cross-linking is conserved in vascular plants and that RG-II likely appeared early in the evolution of land plants. Two features that distinguish RG-II from other plant polysaccharides are that RG-II is composed of 13 different glycoses linked to each other by up to 22 different glycosidic linkages and that RG-II is the only polysaccharide known to contain both apiose and aceric acid. Thus, one key event in land plant evolution was the emergence of genes encoding nucleotide sugar biosynthetic enzymes that generate the activated forms of apiose and aceric acid required for RG-II synthesis. Many of the genes involved in the generation of the nucleotide sugars used for RG-II synthesis have been functionally characterized. By contrast, only one glycosyltransferase involved in the assembly of RG-II has been identified. Here we provide an overview of the formation of the activated sugars required for RG-II synthesis and point to the possible cellular and metabolic processes that could be involved in assembling and controlling the formation of a borate cross-linked RG-II molecule. We discuss how nucleotide sugar synthesis is compartmentalized and how this may control the flux of precursors to facilitate and regulate the formation of RG-II.

Isolation and characterization of immuno-stimulating polysaccharide in Korean black raspberry wine

To characterize polysaccharides existing as soluble form in Korean fruit wines, polysaccharides were isolated from Korean black raspberry wine, and their immuno-stimulating activities were examined. A complex pectic polysaccharide, RB-2 was purified from the crude polysaccharide (RB-0) in raspberry wine by subsequent size-exclusion chromatography. RB-2 had relatively potent macrophage activity and splenocyte mitogenic activity in vitro. The intravenous administration of RB-2 significantly augmented the cytotoxicity of natural killer cells against Yac-1 tumor cells. RB-2 consisted of 15 monosaccharides including rarely observed sugars in general polysaccharides, such as 2-O-methyl-fucose, 2-O-methyl-xylose, apiose, aceric acid, 3-deoxy-D-manno-2-octulosonic acid, and 3-deoxy-d-lyxo-2-heptulosaric acid. Methylation analysis indicated that RB-2 comprised at least 20 different glycosyl linkages such as 3,4-linked fucose, 2,3,4-linked rhamnose, 3′-linked apiose, and 2,3,3′-linked apiose, characteristic of rhamnogalacturonan II (RGII). RB-2 was eluted as two peaks having a high (9,000 Da) and a low molecular mass (5,000 Da) on high performance size exclusion chromatography, and the peak of high molecular mass was down shifted to the low molecular mass under the acidic pH conditions less than 2.0. Results indicated higher molecular weight polysaccharide in RB-2 was mainly presented as an RG-II dimer, and Korean black raspberry wine contains selected polysaccharides, which provide immunostimulating activities beneficial to human health.

Presence of rhamnogalacturonan II in the juices produced by enzymatic liquefaction of Agave pulquero stem ( Agave mapisaga)

Rhamnogalacturonan II (RG-II), a small complex pectic polysaccharide is released from Agave pulquero stems ( Agave mapisaga), after the production period of aguamiel. RG-II was obtained by treatment with two commercial liquefying enzyme preparations, it was isolated by size-exclusion chromatography and characterized. RG-IIs contains diagnostic sugars such as apiose, 2- O-methyl- l-fucose, 2- O-methyl- d-xylose, aceric acid, Kdo and Dha. Glycosyl-linkage compositions of the Agave pulquero RG-II like structures are similar to the theoretical model described through sycamore RG-II structure. The presence of 3′-linked apiose indicates that the obtained juice from Agave pulquero plant contains the free RG-II dimer. Thus, when pectinolytic enzyme preparations are used to process Agave pulquero ( A. mapisaga), RG-II is released as one of the main soluble polysaccharide fraction.

Indirect approach to C-3 branched 1,2- cis-glycofuranosides: synthesis of aceric acid glycoside analogues

Aceric acid (3- C-carboxy-5-deoxy-α- l-xylofuranose) residues are present in pectic polysaccharide rhamnogalacturonan II (RG II) in the form of synthetically challenging 1,2- cis-glycofuranosides. To access synthetic fragments of RG II incorporating aceric acid, a four-step procedure based on C-2 epimerisation of initially prepared 1,2- trans-glycofuranoside was developed. Readily available derivatives of branched-chain l-lyxofuranose bearing a 3- C-vinyl group as a masked 3- C-carboxyl group were investigated as potential precursors of aceric acid units. In the first step of the procedure, installation of a participating group at C-2 of the furanose ring ensured stereocontrol of the O-glycosylation, which was carried out with the thioglycoside of 2- O-acetyl-3,5-di- O-benzyl-3- C-vinyl- l-lyxofuranose. After the glycosylation step, the 2- O-acetyl group was removed, the free 2-OH group was oxidised and the resulting ketone was finally reduced to form the C-3-vinyl- l-xylofuranoside. The use of L-Selectride in the key reduction reaction was essential to achieve the required stereoselectivity to generate 1,2- cis-furanoside.

De Novo Synthesis of Aceric Acid and an Aceric Acid Building Block

The de novo synthesis of an aceric acid thioglycoside building block and the total synthesis of the plant carbohydrate aceric acid are described via a highly convergent strategy. Aldol reaction of acetaldehyde and a protected tartaric acid derivative provided the open chain carbohydrate. Subsequent acid treatment yielded the aceric acid thioglycoside in 35% total yield over five steps. Oxidative cleavage of the thioketal in the open chain carbohydrate and basic hydrolysis of the methyl ester furnished fully deprotected aceric acid in 31% yield over six steps.

Synthesis of the Branched-Chain Sugar Aceric Acid: A Unique Component of the Pectic Polysaccharide Rhamnogalacturonan-II

Described herein is the synthesis of 3-C-carboxy-5-deoxy-L-xylose (aceric acid), a rare branched-chain sugar found in the complex pectic polysaccharide rhamnogalacturonan-II. The key synthetic step in the construction of aceric acid was the stereoselective addition of 2-trimethylsilyl thiazole to 5-deoxy-1,2-O-isopropylidene-alpha-L-erythro-pentofuran-3-ulose (2), which was prepared from L-xylose. The thiazole group was efficiently converted into the required carboxyl group via conventional transformations. Aceric acid was also synthesized by dihydroxylation of a 3-C-methylene derivative of 2 followed by oxidation of the resulting hydroxylmethyl group. The C-2 epimer of aceric acid was also synthesized using thiazole addition chemistry, starting from L-arabinose.

Pectic substances from red beet ( Beta vulgaris L. var. conditiva). Part II. Structural characterisation of rhamnogalacturonan II

Cell wall material from ripe red beet ( Beta vulgaris L. var. conditiva) was isolated as alcohol-insoluble residue (AIR). The cyclohexane- trans-1,2-diaminotetraacetate (CDTA) soluble extract (CDTA-soluble pectin) was fractionated by anion exchange and gel filtration chromatography. The main fraction was degraded with an endo-α-(1→4)-polygalacturonase and further fractionated by gel filtration chromatography. Methylation analysis of the intermediate-molecular weight fractions was performed after carbodiimide-activated reduction of the pectic polysaccharides with NaBD 4. Besides galacturonic acid and rhamnose, the fraction contained rare sugar residues, such as apiose, 2- O-methyl-xylose, 2- O-methyl-fucose, 3,4-linked fucose, 2,3,4-linked rhamnose, 2-linked glucuronic acid, aceric acid, 3-deoxy- d-manno-2-octulosonic acid (Kdo), and 3-deoxy- d-lyxo-2-heptulosonic acid (Dha) which are characteristic for rhamnogalacturonan II (RG-II). The constituents were present in amounts which approximately corresponded to the structural model for RG-II. The molecular weight of RG-II was determined by MALDI-TOF-MS and showed to contain two populations with molecular weights of about 4100 and 4300, respectively. Arabinopyranose residues were shown to occur only terminally linked. This result suggests that the aceric acid containing side-chain of red beet RG-II is shorter than the one from other plants. 1,3,4-linked galactose and 1,2,3,4-linked galacturonic acid were detected as well, their positions within the RG-II molecule remain to be established.

Determination of the neutral and acidic glycosyl-residue compositions of plant polysaccharides by GC-EI-MS analysis of the trimethylsilyl methyl glycoside derivatives

A method, using GC-EI-MS analysis of the trimethylsilyl methyl glycoside derivatives, has been developed to identify the neutral and acidic glycosyl-residue compositions of plant polysaccharides. Monosaccharides (15) including four pentoses (arabinose, xylose, 2- O-methylxylose, and apiose), three hexoses (galactose, glucose, and mannose), three 6-deoxy hexoses (rhamnose, fucose, and 2- O-methylfucose), two hexuronic acids (galacturonic and glucuronic acids), two 2-keto-3-deoxy sugars (Kdo and Dha), and aceric acid were quantified. The monosaccharide derivatives were identified by their retention times and by their characteristic GC-MS fragmentation patterns. These monosaccharides are components of plant cell wall polysaccharides and red wine polysaccharides. Thus, GC-MS analysis of trimethylsilyl methyl glycoside derivatives is suitable for determining the neutral and acidic glycosyl-residue compositions of plant cell wall polysaccharides and of polysaccharides present in plant-derived foods and beverages.

Characterization of pectic polysaccharides having intestinal immune system modulating activity from rhizomes of Atractylodes lancea DC

Two acidic polysaccharides (ALR-a and ALR-b, former names ALR-5IIb-2-2 and 5IIc-3-1, respectively; although ALR-5IIb-2-2 and 5IIc-3-1 were used as their abbreviations in a previous paper (Planta Med., 64 (1998) 714), here the polysaccharides have been abbreviated to ALR-a and ALR-b, respectively, in order to avoid complexity) have been purified from rhizomes of Atractylodes lancea DC. as intestinal immune system modulating polysaccharides (Planta Med., 64 (1998) 714). Endo-α- d-(1→4)-polygalacturonase digestion of ALR-b gave large proportions of a fragment (PG-1) eluted in the void volume, and the lowest-molecular-weight fraction (PG-3) in addition to a small proportion of intermediate fraction (PG-2). Component sugar and methylation analyses using base-catalyzed β-elimination indicated that PG-1 consisted of a rhamnogalacturonan core with side chains rich in neutral sugars and that PG-3 mainly contained (1→4)-linked galacturono-oligosaccharides. PG-2 comprised characteristic component sugars such as 2-Me-Fuc, 2-Me-Xyl, apiose (Api) and aceric acid (AceA), but PG-2 lacked some glycosidic linkages compared with those of the typical rhamnogalacturonan II (RG-II). PG-2 showed potent intestinal immune system modulating activity, but PG-1 and galacturono-oligosaccharides in PG-3 had no activity. Further gel filtration and anion-exchange chromatography of ALR-a gave a potent intestinal immune system modulating polysaccharide (ALR-a-Bb). Component sugar and methylation analyses indicated that ALR-a-Bb also comprised unusual component sugars characteristic in RG-II as well as PG-2 derived from ALR-b. ALR-a-Bb or PG-2 from ALR-b little affected directly the proliferation of bone marrow cells. PG-2 from ALR-b expressed similar significant intestinal immune system modulating activity to RG-II (GL-RI) isolated from leaves of Panax ginseng C.A. Meyer, but RG-II obtained from a pharmacologically active pectin (bupleuran 2IIb) of Bupleurum falcatum L. had no activity.

An NMR solution study of the mega-oligosaccharide, rhamnogalacturonan II.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic mega-oligosaccharide that is released enzymatically from the primary cell wall of higher plants. It contains roughly 30 monosaccharide units (MW approximately 5 kDa) including very unusual residues such as Kdo, Dha, aceric acid and apiose. Previous studies have demonstrated that these monomers are arranged into four structurally well-defined oligosaccharide side chains (A-D), linked to a homogalacturonan mainchain, but the specific attachment sites of these branches on the pectic backbone have not yet been elucidated. In the present work, fairly complete assignments of the 750 MHz 1H NMR spectra and partial assignments of the 13C NMR spectra of the sodium-borohydride-reduced RG-II monomer were obtained for a 5 mM sample isolated from red wine. On the whole, these data corroborate the primary structures of the sidechains previously established by methylation analysis, partial hydrolysis and FAB-MS spectrometry but some heterogeneity has been demonstrated (partial substitution at B5, B6, and A5). The preferred orientations of the majority of the sidechain glycosidic linkages in the RG-II monomer have been determined from the sequential nOe data and the solution structure is generally in good agreement with the stable conformers previously obtained by molecular modeling (MM3) of the disaccharide and sidechain oligosaccharide building blocks. All of a two-residue, a three-residue, and a four-residue segment of the backbone have been tentatively identified from long range interactions between sidechain protons as well as in the mainchain. Taking into account the length of the 9-mer galacturonan mainchain described in prior work, these building blocks constitute almost the complete structure of RG-II (Scheme 2).

The preferred conformations of the four oligomeric fragments of Rhamnogalacturonan II

Rhamogalacturonan II (RG-II) is a structurally complex pectic mega-oligosaccharide that is released enzymatically from the primary cell wall of higher plants. RG-II contains 28 monosaccharide units (MW≅6KDa) which belong to 12 different families of glycosyl residues, including very unusual ones such as Kdo, Dha, aceric acid, and apiose. Eighteen different disaccharide segments can be identified, and so far the primary structure has not yet been determined. These monomeric units are arranged into four structurally well-defined oligosaccharide side chains, linked to a pectic backbone made up of 1,4-linked α- d-galactosyluronic acid residues. The specific attachment sites of these four side-chains on the pectic backbone remains to be elucidated. The present work presents a three-dimensional database of all the monosaccharide and disaccharide components of RG-II. The conformational behavior of d-Api f and l-AceA f monosaccharide has been assessed through computations performed with the molecular mechanics program MM3 using the flexible residue approach. For each furanosyl residue, energies of various envelope and twist conformers were systematically calculated as a function of the puckering parameters Q and φ. Energy minima are observed in both the Northern and Southern zones of the conformational wheel of each monosaccharide. As for the constituting segments, the conformational behaviour of 18 different disaccharides was evaluated using the flexible residue procedure of the MM3 molecular mechanics procedure. For each disaccharide, the adiabatic energy surface, along with the locations of the local energy minima and drawings of the conformations of each local minimum located in the energy maps have been established. The geometries of the minima and the potential energy surfaces of the different fragments were included in the database of the POLYS, a program for building oligo and polysaccharides. All these results were used for the generation, prior to a complete optimization, of the complete structure of each fragment of RG-II. It is shown that both A and B fragments are very flexible about the two sidechain glycosidic linkages which are closest to the backbone. The remaining part of the sidechain is rigid for the heavily branched A fragment, it is flexible for the more linear B fragment. The lowest energy conformer of each fragment results in good exposure of the hydroxyl groups of the apiosyl residues. Some possible implications of these features in boron complexation are presented.

Rhamnogalacturonan II from cell walls of Cryptomeria japonica

Rhamnogalacturonan II (RG-II) was isolated from cell walls of sugi ( Cryptomeria japonica). The oligosaccharides which could not be generated from sycamore ( Acer psuedoplatanus, angiosperm) RG-II were produced from sugi RG-II by lithium treatment. The structure of sugi RG-II is supposed to be partially different from that of sycamore RG-II. The pectic polysaccharides isolated from cell walls of xylem-differentiating zones of sugi (Cryptomeria japononica) were degraded with endo- α-(1 → 4)-polygalacturonase and the polysaccharides, composed mainly of rhamnogalacturonan II (RG-II), were obtained from the degradation products. These polysaccharides consisted of rhamnosyl, fucosyl, arabinosyl, xylosyl, galactosyl, glucosyl, galacturonic acid, glucuronic acid and the characteristic sugars of RG-II, namely, 2- O-methylfucose, 2- O-methylxylose, apiose, aceric acid and thiobarbituric acid assay-positive glycosyl {probably, 3-deoxy- d- manno-2-octulosonic acid (Kdo) and 3-deoxy- d- lyxo-heptulosaric acid (Dha)}. The polysaccharides contained the glycosyl residues of RG-II, besides small amounts of the glycosyl linkages of RG-I. The RG-II was structurally analysed by partial acid hydrolysis and lithium treatment in ethylenediamine. The glycosyl sequences of three compounds generated by partial hydrolysis were not identical to the partial structure of the sycamore ( Acer pseudoplatanus) RG-I and RG-II structures previously proposed by Albersheim et al. Furthermore, five novel glycosyl sequences were detected in the products from lithium treatment. The results suggest that the structure of sugi RG-II is somewhat different from that of sycamore RG-II.

Rhamnogalacturonan II dimers cross-linked by borate diesters from the leaves of Panax ginseng C.A. Meyer are responsible for expression of their IL-6 production enhancing activities

Leaves of Panax ginseng C.A. Meyer contain larger amounts of thiobarbituric acid (TBA)- positive material, suggested to be rhamnogalacturonan II (RG-II), than the roots. Starting from leaves, three different polysaccharides, GL-RI, GL-RII and GL-RIII, were isolated from polysaccharide subfraction GL-5 by subsequent anion-exchange and size-exclusion chromatography. Sugar and methylation analyses indicated that the three polysaccharides consisted of the same substitution patterns of 2- O-methyl-fucose, 2- O-methyl-xylose, apiose, 3- C-carboxy-5-deoxy- L-xylose (aceric acid, AceA), 3-deoxy- D- manno-2-octulosonic acid (Kdo), and 3-deoxy- D- lyxo-2-heptulosaric acid (Dha), being characteristic monosaccharides of RG-II, but no other pectic components. Another RG-II (GL-4IIb2) has also been isolated without endo-polygalacturonase digestion from leaves of P. ginseng, and turned out to be a macrophage Fc receptor expression enhancing polysaccharide [K.-S. Shin, H. Kiyohara, T. Matsumoto, and H. Yamada, Carbohydr. Res., 300 (1997) 239–249]. GL-4IIb2 and GL-RIII had relatively potent interleukin-6 (IL-6) production enhancing activity of macrophages, however, GL-RI and GL-RII had negligible and weak enhancing activities, respectively. Partial acid hydrolysis (0.1 M trifluoroacetic acid, 40°C, 24 h) of GL-RI, GL-RII and GL-RIII gave in each case a large-size fraction (AH-1), an intermediate-size fraction (AH-2) and a short oligosaccharide fraction (AH-3). EIMS and FABMS analyses indicated that AH-3 consisted of Rha-(1→5)-Kdo and Ara-(1→5)-Dha, and that AH-2 comprised non-, mono- and di-acetylated AceA-containing hexa- to nona-saccharides. Also for AH-1 no significant structural differences were observed among the polysaccharides, but the presence of microheterogeneity is suggested. The AH-1 fractions did not show any IL-6 production enhancing activity. Size-exclusion HPLC indicated that GL-RIII mainly comprised RG-II of higher molecular mass (12,000), and that GL-RI and GL-RII mainly contained RG-II of lower molecular mass (7000). Boron content and 11B NMR analyses indicated that the higher molecular weight polysaccharide in GL-RIII was mainly present as a RG-II dimer cross-linked by borate diesters. Dissociation of the RG-II dimer containing borate diester in GL-RIII to the monomer by mild acid treatment significantly decreased its IL-6 production enhancing activity whereas re-dimerization of dissociated GL-RIII recovered the enhancing activity. Artificial re-formation of the RG-II dimer containing borate diester in GL-RI also potentiated the IL-6 production enhancing activity. The present observation is a first report of biological activity related to the RG-II dimer.

Rhamnogalacturonan II from the leaves of Panax ginseng C.A. Meyer as a macrophage Fc receptor expression-enhancing polysaccharide

A complex pectic polysaccharide (GL-4IIb2) has been isolated from the leaves of Panax ginseng C.A. Meyer, and shown to be a macrophage Fc receptor expression-enhancing polysaccharide. The primary structure of GL-4IIb2 was elucidated by composition. 1H NMR, methylation, and oligosaccharide analyses. GL-4IIb2 consisted of 15 different monosaccharides which included rarely observed sugars, such as 2- O-methylfucose, 2- O-methylxylose, apiose, 3-C- carboxy-5-deoxy- l- xylose (aceric acid, AceA), 3-deoxy- d- manno-2-octulosonic acid (Kdo), and 3-deoxy- d- lyxo-2-heptulosaric acid (Dha). Methylation analysis indicated that GL-4IIb2 comprised 34 different glycosyl linkages, such as 3,4-linked Fuc, 3- and 2,3,4-linked Rha, and 2-linked GlcA, which are characteristic of rhamnogalacturonan II (RG-II). Sequential degradation using partial acid hydrolysis indicated that GL-4IIb2 contained α-Rha p-(1 → 5)-Kdo and Ara f-(1 → 5)-Dha structural elements, an AceA-containing oligosaccharide, and uronic acid-rich oligosaccharide chains in addition to an α-(1 → 4)-galacturono-oligosaccharide chain. FABMS and methylation analyses suggested that the AceA-containing oligosaccharide was a nonasaccharide in which terminal Rha was additionally attached to position 3 of 2-linked Ara p of the octasaccharide chain observed in sycamore RG-II. Component sugar and methylation analyses assumed that the uronic acid-rich oligosaccharides possessed a similar structural feature as those in sycamore RG-II. GL-4IIb2 had a larger molecular mass (11,000) than sycamore RG-II (∼5000). A structure of macrophage Fc receptor expression-enhancing polysaccharide from the leaves of Panax ginseng C.A. Meyer has been characterised to be a rhamnogalacturonan II type polysaccharide containing a characteristic nonasaccharide sequence.

Rhamnogalacturonan II, a dominant polysaccharide in juices produced by enzymic liquefaction of fruits and vegetables

Rhamnogalacturonan II (RG-II), a small complex pectic polysaccharide, is released from apple ( Malus domestica), carrot ( Daucus carota), and tomato ( Solanum lycopersicum) by treatment with two commercial liquefying enzyme preparations. RG-II was isolated by size-exclusion chromatography from apple, tomato, and carrot juices obtained by enzymic liquefaction. All the RG-IIs contained the diagnostic sugars, apiose, 2- O-methyl- l-fucose, 2- O-methyl- d-xylose, aceric acid, Kdo and Dha. Glycosyl-linkage compositions of the neutral and acidic sugars, including aceric acid, were consistent with the hypothetical model described for sycamore RG-II confirming the conservation of RG-II in plants. Thus, when pectinolytic enzyme preparations are used to process fruits and vegetables, RG-II is released as a main soluble polysaccharide fraction while other pectic polysaccharides are heavily degraded. © 1997 Elsevier Science Ltd.

Structural characterization of red wine rhamnogalacturonan II

The pectic polysaccharide rhamnogalacturonan II (RG-II), which accounts for ~ 20% of the ethanol-precipitable polysaccharides in red wine, has been isolated from wine polysaccharides by anion-exchange chromatography. Four fractions enriched with RG-II were obtained and the RG-II then purified to homogeneity by Concanavalin A affinity and size-exclusion chromatographies. The glycosyl-residue compositions of the four RG-IIs are similar; all the RG-IIs contain the monosaccharides (apiose, 2-O- methyl- l-fucose , 2-O- methyl- d-xylose , Kdo, Dha, and aceric acid) that are diagnostic of RG-II. The glycosyl-linkages of the neutral and acidic sugars, including aceric acid, were determined simultaneously by GC-EIMS analysis of the methylated alditol acetates generated from per- O-methylated and carboxyl-reduced RG-II. Two of the RG-IIs contain boron, most likely as a borate di-ester that cross-links two molecules of RG-II together to form a dimer. The dimer contains 3′- and 2,3,3′-linked apiosyl residues whereas the monomer contains only 3′-linked apiosyl residues which suggests that the borate di-ester is located on at least one of the apiosyl residues of RG-II. Although the wine RG-IIs all have similar structures they are not identical since they differ in the length and degree of methyl-esterification of the RG-II backbone and in the presence or absence of borate di-esters. Nevertheless, these studies show that the major structural features of wine and primary cell wall RG-II are conserved.

Pectic polysaccharides from xylem-differentiating zone of Cryptomeria japonica

Pectin was extracted from the cell walls of xylem-differentiating zones of sugi ( Cryptomeria japonica) with the chelating agent, trans-1,2-cyclohexanediamine- N, N, N′, N′-tetraacetic acid at 20° and Na 2CO 3 solution at 1° and 20°. The extracted pectin fractions were purified by anion-exchange chromatography and digested with pure endo-polygalacturonase from Aspergillus niger. The hydrolysates were then fractionated by gel permeation chromatography into three or four fractions. Sugar composition and glycosyl linkage composition analyses of each fraction showed that the void volume fractions contained 2- and 2,4-linked rhamnosyl, 5-linked arabinosyl, terminal galactosyl and 4-linked galacturonic acid residues, indicating that they were rhamnogalacturonan I. The fractions obtained from the included volume had rare sugar residues, such as apiosyl, 2-O- methyl- l-fucosyl , traces of 3-O- methyl- l-fucosyl , 2-O- methyl- d-xylosyl residues aceric acid and thiobarbituric acid assay-positive sugar residues, showing that these fractions contained rhamnogalacturonan II-like polysaccharides. Several linear oligosaccharides composed of (1 → 4)-linked α- d-galacturonic acid residues were purified from the lowest M r regions and some oligogalacturonides were characterized by methylation analysis, FAB-mass spectrometry and NMR.