Anti-complementary Polysaccharides Research Articles

Structural characterization and anti-inflammatory activities of anti-complementary polysaccharides from Rhododendron principis.

Rhododendron principis leaves have been used as "Dama", a Traditional Tibetan Medicine for treating inflammatory diseases. R. principis crude polysaccharides (CZND) with anti-complementary activity demonstrated promising anti-inflammatory effects on acute lung injury (ALI) induced by lipopolysaccharide (LPS). CZND significantly decreased the levels of TNF-α and IL-6 in both serum and BALF (blood and bronchoalveolar lavage fluid) in LPS-induced ALI mice by intragastric administration (100 mg/kg). A heteropolysaccharide, ZNDHP, was obtained from CZND with successive anti-complementary activity-guided separation. ZNDHP was characterized as a branched neutral polysaccharide with a backbone composed of →2)-β-Glcp-(1→, →2,6)-α-Glcp-(1→, →6,3)-β-Galp-(1→, →2,6)-α-Galp-(1→, →6,2)-β-Glcp-(1→, →4)-α-Glcp-(1→, →5)-β-Araf-(1→, →3,5)-α-Araf-(1→, and →4,6)-β-Manp-(1→, and the backbone structure was further confirmed by partial acid hydrolysis. In addition to anti-complementary and antioxidant activities, ZNDHP exhibited potent anti-inflammatory activity by significantly inhibiting the secretion of NO, TNF-α, IL-6, and IL-1β of LPS treated RAW264.7 cells. However, all of these activities decreased greatly after partially hydrolyzing, indicating the importance of the multi-branched structure for its bioactivity. Therefore, ZNDHP might be an important component of R. principis for treating inflammation.

Beneficial effects of Houttuynia cordata polysaccharides on “two-hit” acute lung injury and endotoxic fever in rats associated with anti-complementary activities

Houttuynia cordata Thunb. is a traditional herb used for clearing heat and eliminating toxins, and has also been used for the treatment of severe acute respiratory syndrome (SARS). In vitro, the crude H. cordata polysaccharides (CHCP) exhibited potent anti-complementary activity through both the classical and alternative pathways by acting on components C3 and C4 of the complement system without interfering with the coagulation system. This study was to investigate the preventive effects of CHCP on acute lung injury (ALI) induced by hemorrhagic shock plus lipopolysaccharide (LPS) instillation (two-hit) and LPS-induced fever in rats. CHCP significantly attenuated pulmonary injury in the “two-hit” ALI model by reducing pulmonary edema and protein exudation in bronchoalveolar lavage fluid (BALF). In addition, it reduced the deposit of complement activation products in the lung and improved oxidant-antioxidant imbalance. Moreover, CHCP administration inhibited fever in rats, reduced the number of leukocytes and restored serum complement levels. The inhibition on the inappropriate activation of complement system by CHCP may play an important role in its beneficial effects on inflammatory diseases. The anti-complementary polysaccharides are likely to be among the key substances for the heat-clearing function of H. cordata.

Removal of lipopolysaccharides from anti-complementary crude polysaccharides of Houttuynia Herba

An Affi-Prep Polymyxin column was combined with a Phenyl Sepharose column and a Sephacryl S-300 column, respectively, to remove the lipopolysaccharides(LPS) in the anti-complementary crude polysaccharides of Houttuynia Herba. The contents of LPS in the polysaccharides were determined by chromogenic tachypleus amebocyte lysate(TAL)method during the procedure of purifying. The anti-complementary activities of the polysaccharides were also compared before and after the removal of LPS. Less remanent LPS was detected after purified using Penyl Sepharose combined with polymyxin column, with the clearance rate of 42.85%. All the columns had no effect on the anti-complementary activity of the polysaccharides. Penyl Sepharose combined with polymyxin column would be sound for LPS removal of the anti-complementary polysaccharides without reducing their bioactivity.

Isolation of an anti-complementary polysaccharide from the root of Bupleurum chinense and identification of its targets in complement activation cascade

Isolation of an anti-complementary polysaccharide from the root of Bupleurum chinense and identification of its targets in complement activation cascade

Isolation and Chemical Analysis of Potent Anti-Complementary Polysaccharides from Fruiting Bodies of the Fomes fomentarius

The five anti-complementary polysaccharides (MFKF-NP, MFKF-AP1, , and MFKF-AP2, ) were separated from hot water extracts of fruiting bodies of Fomes fomentarius by two subsequent column chromatography using DEAE-sepharose FF and Concanavalin A-sepharose 4B. The order of anti-complementary activity was MFKF-AP1 > MFKF-AP1 > MFKF-AP2 > MFKF-AP2 > MFKF-NP > Polysaccharide Krestine (PSK). Especially, MFKF-AP1 among those showed the most excellent anti-complementary activity (70% of ITCH50 value at ). The monosaccharide composition analysis by gas chromatography indicates that MFKF-AP1 and are a kind of homoxylan consisted mainly of xylose above 97%. Molecular weight of MFKF-AP1, major anti-complementary polysaccharide, was estimated to be about 12,000 by high performance liquid chromatography (HPLC). After the incubation of the serum with MFKF-AP1 in the presence or absence of and ions, its anti-complementary activity was investigated. This result indicated that MFKF-AP1 seems to be activator both on the classical and the alternative pathway of complement activation.

An anti-complementary polysaccharide from the roots of Bupleurum chinense

Bioactivity-guided fractionation of hot-water extract from the roots of Bupleurum chinense DC led to the isolation of a homogeneous polysaccharide BC-PS1 composed of Gal, GalA, Glc, Ara and Man in the ratio of 1.6:1.1:1.8:1.7:1.0, along with trace of Rha, Fuc and Xyl. The main linkages of the residues of BC-PS1 include terminal, 1,4-linked and 1,3-linked Galp (GalA), terminal and 1,4-linked Glcp, terminal and 1,5-linked Araf, 1,4,6-linked Manp. Bioassays revealed that the concentrations of BC-PS1 that resulted in 50% hemolytic inhibition on classical complement pathway (CH50) and alternative complement pathway (AP50) were 0.199±0.025mg/mL and 0.371±0.017mg/mL, respectively. Preliminary mechanism study indicated that BC-PS1 interacted with C1q, C2, C5 and C9.

Isolation of an anti-complementary polysaccharide from the root of Bupleurum chinense and identification of its targets in complement activation cascade

AimTo isolate and characterize the anti-complementary polysaccharide from the root of Bupleurum chinense. MethodsBioactivity-guided fractionation and purification was used to obtain the anti-complementary polysaccharide from the hot-water extract of the root of Bupleurum chinense. The polysaccharide was characterized by various chemical and spectral analyses. The anti-complementary activities were evaluated by hemolytic assay in vitro. The action targets were identified in the system with individual complement-depleted sera. ResultsA homogeneous polysaccharide BC-PS2 was isolated as an anti-complementary agent. It was identified as a branched polysaccharide with an average molecular weight about 2 000 KDa, composed of Glc, Ara, Gal, and Man in the ratio 3.5 : 2.4 : 2.0 : 1.0, respectively, along with a trace of Rha and Xyl, and only 1.11% of protein. The main linkages of the residues of BC-PS2 include terminal, 1, 6-linked, 1, 3-linked and 1, 3, 6-linked Glcp, terminal and 1, 5-linked Araf, terminal, 1, 4-linked, 1, 6-linked and 1, 4, 6-linked Galp, terminal, and, 1, 4-linked and 1, 4, 6-linked Manp. The bioassay experiments revealed that BC-PS2 inhibited complement activation on both the classical and alternative pathways, with CH50 and AP50 of (0.222 ± 0.013) and (0.356 ± 0.032) mg·mL−1, respectively. Preliminary mechanism studies indicated that BC-PS2 interacted with C1q, C2, and C9 components. ConclusionThe results demonstrated that BC-PS2 is an anti-complementary polysaccharide, and should be important constituent of the root of Bupleurum chinense for its application in the treatment of diseases associated with the excessive activation of complement system.

Preparation procedures of anti-complementary polysaccharides from Houttuynia cordata

To establish and optimize the preparation procedures of the anti-complementary polysaccharides from Houttuynia cordata. Based on the yield and anti-complementary activity in vitro, the conditions of extraction and alcohol precipitating process were optimized by orthogonal tests. The optimal condition of deproteinization was determined according to the results of protein removed and polysaccharide maintained. The best decoloring method was also optimized by orthogonal experimental design. The optimized preparation procedures were given as follows: extract the coarse powder 3 times with 50 times volume of water at 90 degrees C for 2 hours every time, combine the extracts and concentrate appropriately, equivalent to 0.12 g of H. cordata per milliliter. Add 4 times volume of 90% ethanol to the extract, allow to stand for 24 hours to precipitate totally, filter and the precipitate was successfully washed with anhydrous alcohol, acetone and anhydrous ether. Resolve the residue with water, add trichloroacetic acid (TCA) to a concentration of 20% to remove protein. Decoloration was at a concentration of 3% with activated carbon at pH 3.0, 50 degrees C for 50 min. The above procedures above were tested 3 times, resulting in the average yield of polysaccharides at 4.03% (RSD 0.96%), the average concentrations of polysaccharides and protein at 80.97% (RSD 1.5%) and 2.02% (RSD 2.3%), and average CH50 at 0.079 g x L-(-1) (RSD 3.6%). The established and optimized procedures are repeatable and reliable to prepare the anti-complementary polysaccharides with high quality and activity from H. cordata.

상황버섯(Phellinus linteus) 균사체로부터 항보체 활성 다당류의 정제 및 특성

식용버섯으로부터 항보체 활성물질을 탐색하여 새로운 소재화 연구의 일환으로 상황버섯(Phellinus linteus) 균사체로부터 항보체 활성 물질을 분리, 정제를 하였으며 화학적 특성을 검토하였다. 정제는 상황버섯의 열수추출한 후, 열수추출물(PL-0)를 한외여과하여 분자량별로 5개의 획분을 얻었다. 이 중, 300kDa 이상의 획분(PL-5)을 DEAE-Sepharose FF 이온교환 크로마토그래피, Sepharose CL-4B 겔여과 크로마토그래피를 실시하여 PL-5-IIIa 획분을 얻었다. PL-5-IIIa 획분을 HPLC를 행한 결과 단일 피크로 나타났으며 정제다당 PL-5-IIIa의 분자량은 800,000Da으로 측정되었다. 정제다당 PL-5IIIa는 64.2%의 당과 29.6%의 단백질로 구성되었으며 구성당은 galactose가 43.1%, mannose가 40.6%, fucose가 15.8%로 나타났다. We have isolated an anti-complementary polysaccharide from the hot water extracts of Phellinus linteus mycelia. Anti-complementary polysaccharide, PL-5-IIIa, was purified by ultrafiltration, gel permeation chromatography using Sepharose CL-4B. GPC (Sepharose CL-4B) and its homogenicity was demonstrated by HPLC. Using gel permeation chromatography with standard dextrans, its molecular weight was determined as about 800,000 dalton. The purified PL-5-IIIa was identified as a protein bound polysaccharide comprising of 29.6% protein and 64.2% carbohydrate which was composed of fucose(15.8%), galactose(43.1%) and mannose(40.6%).

Immunomodulatory effects of chlorogenic acid and ethyl acetate fraction from Lonicera japonica on cytokine gene expression profiles in spleen and thymus

ABSTRACT Objective：Lonicera japonica contains anti complementary polysaccharides and polyphenolic c ompound. Among these polyphenolic substances, chlorogenic acid is the major active component of this plant. However, the immunological mechanisms for these activities, have not been elucidated, nor the active components. To clarify immunomodulatory effects of those we examined the relationship between the activity of CD8+ T cell-mediated lysis and the frequency of cytokine profiles in spleen, thymus (especially IFN-γ, IL-4, GM-CSF etc.) expressing CD8+ T cells activated by IL-2. Methods：To study immunomodulatory effects ethyl acetate fraction from Lonicera japonica, chlorogenic acid on cytokine gene expression from spleen, thymus cells, RT-PCR was performed after quantitative normalization for each gene by a densitometry using β-actin gene expression. A mo dified standard 51 Cr-release assay was used to measure cytotoxic activities of cytotoxic T cells. Spleen, t hymus cells from NOD mice were stained with CD3, CD4, CD44, CD69 in staining buffer and analyzed by two color flow cytometry.Results：We showed that ethyl acetate fraction from Lonicera japonica in combination with IL-2 resulted in a significant enhancement of PCR products for IFN-γ, IL-4, IL-10, GM-CSF, IL-6 and cytotoxtic CD8+ T cell proportion in spleen and thymus T cells in NOD mice. This sugge sts that IFN-γ, IL-6 like IL-4 may be acting as a regulatory rather than proinflammatory cytokine.Conclusions：In conclusion, based on the results of the present study which showed that ethyl acetate fraction from Lonicera japonica and chlorogenic acid upregulating cytokine gene expression in s pleen and thymus, we are tempted to speculate that some of the therapeutic efficacies such as anti-diabetic activity of Lonicera japonica are due to the immunomodulatory its ethylacetate fraction and chlorogenic acid.Key words：Immunomodulation, Lonicera japonica thumb., chlorogenic acid, c ytokines, CD8+

Isolation and characterization of an anti-complementary polysaccharide D3-S1 from the roots of Bupleurum smithii

The preliminary data from hemolytic assays indicated that the hot-water extract of the roots of Bupleurum smithii had anti-complementary activity. Further bioactivity-guided fractionation led to the isolation of D3-S1, a homogeneous form of acidic polysaccharide. D3-S1 was a branched polysaccharide with average molecular weight about 2,000,000 Da, composed of Ara, Gal and GalA in the ratio of 2.6:1.0:1.2, along with trace of Rha, Glc, Xyl and Man. Methylation analysis and NMR identified the linkages of the residues of D3-S1. Functional analysis showed that D3-S1 inhibited complement activation on both the classic and alternative pathways with CH 50 value of 0.34 ± 0.02 mg/ml and AP 50 value of 0.081 ± 0.003 mg/ml, respectively. Preliminary mechanism studies by using complement component depleted-sera indicated that D3-S1 selectively interacts with C1s, C3 and C4, but not C1q, C1r, C2, C5 and C9. The results suggested that D3-S1 could be of potential benefits in treatment of the complement-associated diseases.

포도주에 함유된 항보체 활성 다당류

포도주에 함유된 항보체 활성 다당류를 규명 및 분리하기 위하여 포도와 관련된 과실주로부터 조다당 획분을 시판되고 있는 적포도주, 백포도주 및 머루주로부터 농축과 에탄올 침전을 통하여 조제하였다. 항보체 활성의 검토 결과, 적포도주의 조다당 획분(RW-0)이 백포도주의 획분(WW-0)보다 더 높은 활성을 보여주었다. 항보체 활성을 갖는 포도주의 조다당 획분 중 Sephadex G-75의 gel permeation chro matography를 통하여 고분자 획분으로 RW-1과 WW-1으로, 저분자 획분으로 RW-2와 WW-2로 분획되었으며 머루주의 경우에는 분획되지 않았다. 분획된 획분 중 RW-1이 가장 높은 활성을 보여주었으며 91.3%의 당이 함유되어 있음을 확인할 수 있었다. 적포도주의 항보체 활성이 백포도주의 획분보다 높은 활성을 갖는 것은 펙틴과 헤미셀룰로오즈 등이 함유된 포도껍질로부터 활성 다당이 기인하는 것으로 보이며, 높은 당 함량과 수율을 보이는 고분자 획분, RW-1과 WW-1이 저분자 획분보다 활성이 높음을 알 수 있었다.

An anti-complementary polysaccharide from the roots of Bupleurum smithii and its mechanism of inhibition of the complement activation cascade

An anti-complementary polysaccharide from the roots of Bupleurum smithii and its mechanism of inhibition of the complement activation cascade

Biochemical properties of polysaccharides from black pepper.

The purified polysaccharides from Piper nigrum were prepared as follows: a hot water extract of pepper seeds was fractionated by ultrafiltration with a 5-kDa-membrane cartridge. A fraction with 5 kDa or bigger molecules was successively purified by open column chromatography on DEAE-Toyopearl 650C and Bio-gel P-60 with each active fraction, resulting in PN-Ib and PN-IIa, purified anti-complementary polysaccharides. None of the anti-complementary activity of any polysaccharide was changed by pronase digestion or polymyxin B treatment, but they were decreased by periodate oxidation. Analysis of component sugar and molecular mass determination of the anti-complementary polysaccharides indicated that PN-Ib with an average molecular mass of 21 kDa contained 88.5% glucose and other negligible minor monosaccharides, while PN-IIa showed a different monosaccharide composition, which contained a significant proportion of galactose, arabinose, galacturonic acid and rhamnose. The molar ratio of galactose and arabinose of PN-IIa (48 kDa) was 1.93:1. PN-1 did not react with beta-glucosyl Yariv reagent, however, PN-IIa did react, which indicated that PN-IIa might be an arabinogalactan. Based upon these results, the usefulness of purified anti-complementary polysaccharides from Piper nigrum is suggested as a supplement for immune enhancement.

Purification and characterization of anti-complementary polysaccharide from leaves of Thymus vulgaris L.

For the purification of the anti-complementary polysaccharide from Thymus vulgaris L., the hot-water extract of thyme leaves was successively fractionated by ethanol precipitation and ultra-filtration. The retentate with 300-kDa membrane cartridge showed a potent anti-complementary activity. It was further purified by open column chromatographies on DEAE-Toyopearl 650C, Butyl-Toyopearl 650M and Sephadex G-100, obtaining TV3-IIA-I, the purified anti-complementary polysaccharide. The anti-complementary polysaccharide exhibited the anti-complementary activity via both classical and alternative pathways. Based upon the methylation analysis and the reaction with beta-glucosyl Yariv reagent, the anti-complementary polysaccharide from thyme might contain an arabinogalactan moiety, at least in part.

Fractionation and chemical properties of immunomodulating polysaccharides from roots of Dipsacus asperoides.

A crude polysaccharide fraction (DAP-1) was prepared from roots of Dipsacus asperoides by hot water extraction and EtOH precipitation, and tested for anti-complementary activity, mitogenic activity of lymphocytes, and effects on acid phosphatase and phagocytic activities of macrophages. DAP-1 showed not only anti-complementary activity but also a stimulating effect on the mitogenic activity of lymphocytes. DAP-1 also significantly suppressed the phagocytic activity of macrophages. Although DAP-1 directly stimulated the mitogenecity of lymphocytes, it had no effect on lipopolysaccharide- or concanavalin A-induced mitogenic activity of lymphocytes. Periodate oxidation and pronase digestion suggested that the polysaccharide moiety in DAP-1 contributed to the expression of its anti-complementary and mitogenic activities and that the protein moiety in DAP-1 was responsible for its effect on phagocytosis. DAP-1 gave three polysaccharide fractions (DAP-2, 3, and 4) by fractionation using cetyltrimethylammonium bromide. All the fractions had potent anti-complementary activity, but showed different stimulating or suppressive effects on the proliferation of lymphocytes, phagocytosis, or acid phosphatase activity. Three potent anti-complementary polysaccharides (DAP-4I-1a, DAP-4I-1b, and DAP-4IIa-1) were purified from DAP-4 by anion-exchange chromatography, gel filtration, and HPLC. DAP-4I-1a, I-1b, and IIa-1 consisted of Ara, Rha, Xyl, Gal, Glc and GlcA in a molar ratio of 1.0:0.7:1.0:18.6:22.2:nil; 1.0:0.1:0.3:19.3:26.8:nil; and 3.7:trace:0.6:26.3:5.5:1.0; respectively. Among the polysaccharides, only DAP-4IIa-1 reacted with beta-glucosyl-Yariv antigen. Methylation analysis indicated that DAP-4I-1a mainly comprised 4-linked Gal and 3-, 4-, and 6-linked Glc, whereas DAP-4IIa-1 consisted mainly of terminal Araf, 3-linked Glc, and 3,6-branched Gal.

Fractionation and characterization of anticomplementary and mitogenic substances from Panax ginseng extract G‐115

AbstractPanax ginseng extract G‐115 had anticomplementary and mitogenic activities. Incubation with artificial gastric juice slightly decreased the anticomplementary activity, whereas incubation with artificial intestinal juice or with artificial gastric juice followed by artificial intestinal juice slightly increased the activity. Incubations with artificial gastric and intestinal juices slightly decreased mitogenic activity similar to that with water. The G‐115 was fractionated in order to characterize the active substances for anticomplementary and mitogenic activities. The most potent anticomplementary activity was observed in the crude polysaccharide fraction, G‐115G, whereas the water‐soluble dialysable fraction, G‐115E showed the most potent mitogenic activity. Although pronase digestion did not change the anticomplementary activity of G‐115G, G‐115G decreased the anticomplementary activity after the treatments with periodate and endo‐polygalacturonase indicating that a carbohydrate moiety containing polygalacturonan may contribute to the activity. G‐115G was further purified by precipitation with cetyltrimethylammonium bromide, anion‐exchange chromatography on DEAE‐Sepharose and gel filtration on Sepharose CL‐4B, and major potent anticomplementary polysaccharide, G‐115I1‐IIa‐2‐3 was obtained. G‐115I1‐IIa‐2‐3 was homogeneous, and its molecular weight was estimated to be 3.68 × 105. G‐115I1‐IIa‐2‐3 consisted mainly of arabinose, galactose and glucose in addition to small amounts of galacturonic acid, glucuronic acid and rhamnose.

Characterization of in vitro IL‐2 production enhancing and anticomplementary pectic polysaccharides from kampo (Japanese herbal) medicine ‘Juzen‐Taiho‐To’

AbstractA crude polysaccharide fraction (F‐5) from ‘Juzen‐Taiho‐To’ (TJ‐48), a Kampo (Japanese herbal) medicine prepared by decocting a prescription of 10 herbs, enhanced the production of interleukin‐2 (IL‐2) by mouse spleen cells stimulated with concanavalin‐A (Con‐A). When F‐5 was fractionated by cetavlon precipitation, the neutral polysaccharide fraction (F‐5‐5) enhanced IL‐2 production, and also showed anticomplementary activity. Anion‐exchange chromatography and gel filtration indicated that F‐5‐5 contained at least seven kinds of polysaccharides with anticomplementary activity or IL‐2 production enhancing activity. Among these polysaccharides relatively higher molecular weight polysaccharides (F‐5‐5IIa‐1‐1, IIa‐2‐1, IIb‐1 and IIc‐1) showed only anticomplementary activity, whereas relatively lower molecular weight polysaccharides (F‐5‐5IIb‐3A, IIb‐3C and IIc‐2A) enhanced only IL‐2 production of the spleen cells. These immunomodulating polysaccharides consisted of neutral sugars such as arabinose, galactose and galacturonic acid, and the IL‐2 production enhancing polysaccharides also contained a relatively large proportion of protein. Pronase digestion of F‐5‐5 failed to decrease the enhancement of IL‐2 production, whereas the activity significantly decreased by chemical degradation of the carbohydrate moiety or uronic acid with periodate or lithium. Methylation analysis before and after base‐catalysed β‐elimination suggested that the anticomplementary polysaccharides were grouped into pectic polysaccharides, and that the most potent IL‐2 production enhancing polysaccharide (F‐5‐5IIc‐2A) contained an arabinogalactan moiety in addition to (1→4)‐linked galacturonic acid.

Structure of the complement-activating proteoglycan from the pilose antler of Cervus nippon Temminck

An anti-complementary polysaccharide, DWA-2, isolated from an unossified pilose, antler of C. nippon Temminck by digestion with pronase, gel filtration, and affinity chromatography, consisted mainly of GalNAc, GlcA, IdoA, and sulfate in the molar ratios 1.0:0.6:0.3:0.8, and small proportions of Man, Gal, GlcNAc, and protein (4.5%). Methylation analysis, NMR spectroscopy, and degradation with enzymes indicated that DWA-2 contained chondroitin sulfate A-, B-, and C-like moieties. DWA-2 showed potent anti-complementary activity, and crossed immunoelectrophoresis indicated that it cleaved complement C3 in the absence of Ca 2+ ion. Digestion of DWA-2 with chondroitinase ABC or ACI reduced the anti-complementary activity to a low level, but digestion with chondroitinase B reduced the activity by ∼ 40% and the enzyme-resistant fraction still showed a significant activity.

Heterogeneity and characterisation of mitogenic and anti-complementary pectic polysaccharides from the roots of Glycyrrhiza uralensis Fisch et D.C.

Two anti-complementary polysaccharide fractions (GR-2IIa and GR-2IIb), isolated from the roots of Glycyrrhiza uralensis Fisch et D.C., each gave five anti-complementary polysaccharides (GR-2IIa-1–5 and GR-2IIb-1–5) on h.p.l.c; likewise, GR-2IIc gave two anti-complementary and mitogenic polysaccharides (GR-2IIc-1–2A and -2IIc-2) by gel filtration and h.p.l.c. GR-2IIc-1–2A showed the most potent anti-complementary activity. GR-2IIa-1–5 and GR-2IIb-1–5 contained 40–85% and 50–90% of GalA, respectively, in addition to Rha, Ara, and Gal. GR-2IIc-1–2A and -2IIc-2 mainly comprised Glc, Gal, GalA, and GlcA in addition to Rha, Fuc, Xyl, Ara, and Man. Methylation analysis and digestion with endo-α-(1→4)-polygalacturonase indicated that all of the polysaccharides contained polygalacturonan regions which were frequently methyl-esterified. GR-2II-a, -2IIb, and -2IIc gave enzyme-resistant fractions of large and intermediate sizes, in addition to oligogalacturonides. Each large fraction from GR-2IIa and -2IIb consistent mainly of Ara, Gal, and GalA, whereas the intermediate fractions were composed of small proportions of 2-Me-Fuc, 2-Me-Xyl, and apiose (Api), in addition to Rha, Ara, Gal, and GalA. The large fraction from GR-2IIc mainly contained Rha, Man, Gal, and GalA in addition to Fuc, Ara, Xyl, and Glc, whereas the intermediate fraction consisted of 2-Me-Fuc, 2-Me-Xyl, and Api, in addition to Rha, Ara, GalA, Fuc, Xyl, Man, Gal, and Glc. Base-catalysed β-elimination followed by ethylation indicated that all the polysaccharides except GR-2IIc-2 contained a 4-linked uronic acid attached to position 2 of 2,4-linked Rha. Single radical gel diffusion, using the β- d-glucosyl-Yariv antigen, indicated that GR-2IIa-1 and GR-2IIc-2 contained relatively large proportions of (1→3,6)-β- d-galactan moieties. The anti-complementary activities of GR-2IIa-3, GR-2IIa-4, and GR-2IIb-4 decreased after de-esterification followed by digestion with endo-α-(1→4)-polygalacturonase. The large fractions from GR-2IIa-2IIc showed more potent anti-complementary activities than the original polysaccharide fractions, whereas the intermediate fractions and oligogalacturonides were inactive. The large fraction from GR-2IIc had more potent mitogenic activity than GR-2IIc, whereas the intermediate fraction and oligogalacturonides from GR-2IIc were inactive.