Enzyme-resistant Fraction Research Articles

In vitro colonic fermentation characteristics of barley-koji differ from those of barley.

Barley-koji is prepared by inoculating barley, a beneficial prebiotic source, with the fungi Aspergillus luchuensis mut. kawachii. In this study, the prebiotic effects of barley-koji on human colonic microbiota were evaluated in vitro compared with barley, using pig feces. The enzyme-resistant fraction of the following sample groups each was added to respective fermenters: cellulose, barley (Commander and β104), and barley-koji (Commander-koji and β104-koji). Short-chain fatty acid and ammonia-nitrogen production increased and decreased, respectively, in barley-koji and barley groups. Furthermore, the propionate concentration increased in the barley group, showing a positive correlation with the abundance of the genus Dialister. In the barley-koji group, however, acetate and n-butyrate concentrations increased during the early stages of incubation, and the relative abundance of the genus Megasphaera was higher than those of the other genera. Therefore, this study demonstrated that barley-koji might possess beneficial physiological properties for colonic fermentation, which differ from those of barley.

New Insights into the Structure of Kappa/Beta-Carrageenan: A Novel Potential Inhibitor of HIV-1.

New insights into the structure of the hybrid κ/β-carrageenan (κ/β-CRG) of the red alga Tichocarpus crinitus have been obtained. Carrageenan oligosaccharides were prepared through the chemical and enzymatic depolymerization of κ/β-CRG with κ-carrageenase and its the enzyme-resistant fraction. The composition and distribution of the repetition units of κ/β- CRG were investigated by using the negative ion tandem MALDI-TOFMS and ESIMS method, which made it possible to prove and characterize the hybrid structure of this polysaccharide. An analysis revealed the blockwise distribution of the long β-blocks along the polysaccharide chain, with the inclusion of κ/β, μ/ν-blocks and some ι-blocks. Furthermore, the desulfated κ/β-CRG was shown to contain of –G–D– repeating units up to 3.5 kDa. Previous studies have demonstrated that CRGs suppress the replication of several viruses. Here, we established that κ/β-CRG and its oligosaccharides significantly inhibit the transduction efficiency of replication-defective lentiviral particles pseudotyped with the envelope proteins of three different viruses. We found that the polysaccharide and its oligosaccharides strongly reduced the transduction efficiency of lentiviral particles pseudotyped with GP160—the envelope protein of the human immunodeficiency virus HIV-1—when added to T-lymphocyte Jurkat cells. The CRG oligosaccharides displayed significantly higher antiviral activity.

Characterization of resistant waxy maize dextrins prepared by simultaneous debranching and crystallization followed by acidic or enzymatic hydrolysis

Resistant dextrins (RD) were prepared by simultaneous debranching and crystallization with waxy maize starch at 50 °C for 7 days, followed by the hydrolysis using an acid (3.16 M sulfuric acid) or ɑ-amylase (5% based on starch solids) for 24 h. The crystallinity, thermal transition, morphology, and in vitro digestibility of the hydrolyzed RDs were analyzed. The RD hydrolysates showed typical A-type crystalline patterns under X-ray diffraction with increases in crystallinity and thermal stability compared to the unhydrolyzed counterparts. The RD crystals were obtained as nano-sized particles by the hydrolysis, which readily aggregated while drying. Based on the in vitro digestibility test, the enzyme-resistant fraction of the hydrolyzed RDs was greater than the unhydrolyzed one by 5–10%. Heating in a boiling water-bath (15 min) decreased the enzyme-resistant fraction to less than 50%, while the hydrolysis raised the fraction by 10–15%. The hydrolysis could be used in the preparation of nano-sized RD with increased crystallinity and resistance to the digestion.

Molecular-structure evolution during in vitro fermentation of granular high-amylose wheat starch is different to in vitro digestion

This study investigates the evolution of the distributions of whole molecular size and of chain length of granular wheat starches (37 ~ 93% amylose content), subjected to in vitro fermentation with a porcine faecal inoculum or digestion with pancreatic enzymes. The results showed that the molecular structures of high-amylose starch (HAS) unfermented residues largely remained unchanged during the fermentation process, while wild-type starch (37% amylose content) showed a preferential degradation of the amylopectin fraction. In contrast, under simulated digestion conditions, the undigested residues of HAS showed structural changes, including a decrease in amylose content, a shift of amylose peak position towards lower degrees of polymerisation, and an enzyme-resistant fraction. These changes of starch structure are likely to be dependent on the different starch-degrading enzyme activities present in pancreatic vs. microbial systems. Molecular changes in response to fermentation metabolism revealed by size-exclusion chromatography can help understand the microbial utilization of resistant starch.

Enzyme-Resistant Dextrin from Chinese Yam Starch for Potential Application in Beverage Industry: Preparation, Physicochemical Properties and In Vitro Digestion

Preparation conditions, physicochemical properties and in vitro digestibility of enzyme-resistant dextrin from Chinese yam starches, which are native, easily accessible and cheap raw materials in China, were investigated in the current study. The results showed that the enzyme-resistant fraction content in enzyme-resistant dextrin increased and the whiteness of the enzyme-resistant dextrin decreased with the increasing concentrations of hydrochloric and citric acids and the rising heating temperatures, and the prolonged heating times. Considering the manufacturing cost and the beneficial usage, the enzyme-resistant dextrin produced by heating of Chinese yam starch in the presence of hydrochloric (0.11% dsb) and citric (0.2% dsb) acids at 150°C for 60min was selected. Enzyme-resistant dextrin had a low molecular weight of about 6800 Da and rich “non-digestible” α-1,2 glycosidic bond and was well-soluble in water, stable at pH 3.0–7.0 or after heating at 85°C for 0.5 h showed, high thermal stability and high resistant to the artificial gastric juice and small intestinal fluids. These characteristics make the selected enzyme-resistant dextrin suitable for use in the soft drink industry as the soluble dietary fibres and prebiotics in the beverages. This would provide an excellent opportunity to reduce the caloric value of the beverages and exert a beneficial effect on the intestinal microflora of people consuming beverages enriched with enzyme-resistant dextrins.

Structure elucidation of an immunostimulatory arabinoxylan-type polysaccharide prepared from young barley leaves (Hordeum vulgare L.)

We recently isolated an immunostimulating polysaccharide (BLE-P-I), which possesses a large proportion of arabinoxylan, from young barley leaves. In the present study, to elucidate the structural details of BLE-P-I, it was fractionated into enzyme-resistant fraction (BLE-P-I-X1) and arabinoxylan-rich oligosaccharide fraction (BLE-P-I-X2) after endo-xylanase hydrolysis. Commercial wheat arabinoxylan was also fractionated into WAX-XI and WAX-X2 after the same treatment. BLE-P-I-X2 consisted of arabinose and xylose, including 11 types of neutral glycosidic linkages, whereas WAX-X2 had less, with 6 types of linkages. Mass spectrometric results indicated that WAX-X2 was composed of a xylan backbone bearing only arabinose. In contrast, BLE-P-I-X2 consisted of a xylan backbone with acetate, arabinose, galactose, glucuronic acid, and 4-O-methylglucuronic acid, resembling structural characteristics as glucuronoarabinoxylan. Macrophage-stimulatory activity showed that BLE-P-I-X2 has a role in the expression of the activity. These results indicate that the structural complexity of arabinoxylan seems to be responsible for the immunomodulatory activity in barley leaf.

Structural characterization of an intestinal immune system-modulating arabino-3,6-galactan-like polysaccharide from the above-ground part of Astragalus membranaceus (Bunge)

Arabino-3,6-galactan (AMA-1-b-PS2), an intestinal immunomodulatory compound, was purified from the above-ground portion of Astragalus membranaceus (Bunge). Its structure was characterized using sequential enzymatic digestion with exo-α-l-arabinofuranosidase (AFase) and exo-β-d-(1→3)-galactanase (GNase), producing small amounts of intermediate-sized and shorter oligosaccharide (AF-PS2-G2 and AF-PS2-G3) fractions, and a large GNase-resistant fraction (AF-PS2-G1). Simultaneous AFase and GNase digestion of the enzyme-resistant fraction produced two long fragments (AF3-PS2-G1-1-1 and AF3-PS2-G1-1-2). Products of GNase digestion of the upper fractions showed decreased intestinal immunomodulatory activity; the GNase-resistant fraction (AF-PS2-G1) retained significant activity. Sugar component, methylation, and FAB-MS analyses indicated that the oligosaccharides consisted of hexosyl tri- to hexa-decasaccharides and hexosyl di- to hepta-saccharides mainly comprising 6-linked Gal(f) and Gal(p); some were partially mono- or di-arabinosylated. These oligosaccharide fractions were attached to the non-reducing terminus of the β-d-(1→3)-galactan backbone as side chains at position 6. AMA-1-b-PS2 likely modulates both the systemic and gastric mucosal immune systems.

In vitro fermentation of spent turmeric powder with a mixed culture of pig faecal bacteria.

The fermentation potential of spent turmeric was studied in in vitro swine faecal batch cultures. The spent turmeric residue (the enzyme-resistant fraction from spent turmeric, EST) was obtained through the use of the digestive enzymes amyloglucosidase and pancreatin and compared to cellulose and high-amylose starch (HAS) as carbon sources. EST showed significant increases in total anaerobes, bifidobacteria, lactobacilli and lactic acid bacteria populations compared to cellulose at 12, 24 and 48 h, and the total anaerobic level in the HAS group was significantly higher than that in the cellulose group at 24 and 48 h. However, a significant decrease in the coliform population was only found in the HAS group compared to the cellulose group at 48 h. The total short-chain fatty acid (SCFA) concentrations in the EST and HAS groups were significantly higher than that in the cellulose group at 12 h and 48 h. However, there was no significant difference in the total SCFA concentration between the EST and HAS groups at 12 h and 48 h. Ammonia and pH levels in the EST and HAS groups were significantly lower than those in the cellulose group at 24 and 48 h, but there was no significant difference between the EST and HAS groups. These results indicate that the fermentation potential of the enzyme-resistant fraction from spent turmeric is comparable to that of commercially established resistant starch.

Mechanism for Starch Granule Ghost Formation Deduced from Structural and Enzyme Digestion Properties

After heating in excess water under little or no shear, starch granules do not dissolve completely but persist as highly swollen fragile forms, commonly termed granule "ghosts". The macromolecular architecture of these ghosts has not been defined, despite their importance in determining characteristic properties of starches. In this study, amylase digestion of isolated granule ghosts from maize and potato starches is used as a probe to study the mechanism of ghost formation, through microstructural, mesoscopic, and molecular scale analyses of structure before and after digestion. Digestion profiles showed that neither integral nor surface proteins/lipids were crucial for control of either ghost digestion or integrity. On the basis of the molecular composition and conformation of enzyme-resistant fractions, it was concluded that the condensed polymeric surface structure of ghost particles is mainly composed of nonordered but entangled amylopectin (and some amylose) molecules, with limited reinforcement through partially ordered enzyme-resistant structures based on amylose (for maize starch; V-type order) or amylopectin (for potato starch; B-type order). The high level of branching and large molecular size of amylopectin is proposed to be the origin for the unusual stability of a solid structure based primarily on temporary entanglements.

Synergistic and Antagonistic Effects of α-Amylase and Amyloglucosidase on Starch Digestion

The influence of biomacromolecular physical structure on the kinetics of degradation with exo-acting or a mixture of endo- and exo-acting enzymes was studied using three physical forms of maize and potato starch, amyloglucosidase (exo-acting) and α-amylase (endo-acting) as exemplars. For starch in granular form, there was synergism between the enzymes in the production of glucose. In contrast, endo- and exo-acting enzymes showed antagonistic effects in digestion of cooked starches. Antagonism was ascribed to the rapid production of low molecular weight oligomers by α-amylase, which are less efficiently digested by amyloglucosidase than polymeric substrates. The rates of digestion of swollen granule ghosts cooked under low shear conditions were slower than starches cooked under high shear conditions that prevent granule ghost formation. There was also an enzyme-resistant fraction present in granule ghosts, in contrast to high shear cooked starches that were fully digested under the conditions used.

Laboratory Process Development and Physicochemical Characterization of a Low Amylose and Hydrothermally Treated Ready-to-Eat Rice Product Requiring No Cooking

A low amylose and hydrothermally treated ready-to-eat rice product that requires no cooking was prepared in the laboratory. Hot soaking for 1–3 min with subsequent variable steaming at open and under pressures remarkably altered the kernel and flour properties. Increase in water absorption and lowering of cooking time with extent of steaming were prominent. Soaking of the product at 50 °C for 20 min gave texture values more similar to cooked samples. The viscosity parameters of hot soaking alone were in between those of hot soaking with open steaming and pressure steaming. Pressure steamed samples exhibited almost constantly increasing slurry viscosity throughout the heating and cooling phases of the rapid viscosity analyzer profile. Steaming variably altered the native A-type X-ray diffraction pattern. Pressure steaming of samples with 3 min hot soaking caused complete loss of the A-type conformation with feeble peaks for B- and V-type patterns. The open steamed samples showed peaks for all A-, B-, and V-type patterns. No endotherms for amylose-lipid complexes were however found in the differential scanning calorimetry of the pressure steamed samples. The raw rice flour was highly resistant to α-amylolysis. In open steamed samples, steaming severity decreased the hydrolysis rate indicating formation of enzyme-resistant fractions, while pressure steamed samples showed higher digestibility with treatment severity.

Comparison of the structures of hybrid κ-/β-carrageenans extracted from Furcellaria lumbricalis and Tichocarpus crinitus

Abstract κ-/β-Carrageenans extracted from Furcellaria lumbricalis and Tichocarpus crinitus are hybrid polysaccharides that have roughly similar κ- and β-carrabiose contents. The distributions of these carrabiose moieties were compared using gel permeation chromatography to analyse the end-products obtained after degradation with Pseudoalteromonas carrageenovora κ-carrageenase. Three fractions were obtained: standard oligo-κ-carrageenans, hybrid oligo-κ-/β-carrageenans and an enzyme-resistant fraction. The structure of the most abundant hybrid oligo-κ-/β-carrageenans and the composition of the κ-/β-fractions resistant to degradation were determined by 1H NMR. Altogether, the results suggest that the κ- and β-carrabiose moieties in F. lumbricalis are more randomly distributed than in T. crinitus carrageenan, which has a more regular block-type distribution.

Hybridity of carrageenans water- and alkali-extracted from Chondracanthus chamissoi, Mazzaella laminarioides, Sarcothalia crispata, and Sarcothalia radula

Carrageenans water- and alkali-extracted from Chondracanthus chamissoi, Mazzaella laminarioides, Sarcothalia crispata, and Sarcothalia radula were degraded by Pseudoalteromonas carrageenovora κ-carrageenase and Alteromonas fortis ι-carrageenase. The composition of the high molecular weight fraction (i.e., enzyme-resistant fraction) as well as the standard and hybrid oligosaccharide content highlighted differences between the species-specific carrageenans in terms of composition and distribution of the carrabiose moieties. Inspection of the distribution of μ- and ν-carrabiose, the biosynthetic precursors of κ- and ι-carrabiose, revealed their localization in κ-carrabiose- or ι-carrabiose-rich segments, respectively.

Starch Digestion Mechanistic Information from the Time Evolution of Molecular Size Distributions

Size-exclusion chromatography [SEC, also termed gel permeation chromatography (GPC)] is used to measure the time evolution of the distributions of molecular size and of branch length as starch is subjected to in vitro digestion, including studying the development of enzyme-resistant starch. The method is applied to maize starches with varying amylose contents; the starches were extruded so as to provide an analogue for processed food. The initial rates of digestion of amylose and amylopectin components were found to be the same for high-amylose starches. A small starch species, not present in the original starting material, was formed during the digestion process; this new species has a slower digestion rate and is probably formed by retrogradation of longer branches of amylose and amylopectin as they are partially or wholly liberated from their parent starch molecule during the digestion process. The data suggest that the well-known connection between high amylose content and resistant starch arises from the greater number of longer branches, which can form the small retrograded species. The method is useful for the purpose of comparisons between different starches undergoing the process of digestion, by observing the changes in their molecular structures, as an adjunct to detailed studies of the enzyme-resistant fraction.

New insights into the structure of hybrid κ-/μ-carrageenan and its alkaline conversion

Hybrid κ-/μ-carrageenan, water-extracted from Kappaphycus alvarezzi, was incubated with Pseudoalteromonas carrageenovora κ-carrageenase. The five most abundant hybrid κ-/μ-carrageenan oligosaccharides were purified and their structure was determined by NMR and mass spectrometry. Since the enzyme-resistant fraction was small, these five oligosaccharides were representative of the distribution of the μ-carrabiose moieties along the carrageenan chain. Three main distributions of μ-carrabiose were found: -μ-, -μ-μ- and -μ-μ-μ-. Interestingly, the chemical shifts of the anomeric proton and carbon were strongly influenced by the identity of the adjacent carrabiose, which explains the asymmetrical D6S-H1 NMR signal that we observed. Although μ-carrabiose chemical shifts are usually attributed to blocks of μ-carrabiose as expected in ideal μ-carrageenan, we demonstrate that they are in fact due to μ-carrabiose units positioned between κ-carrabiose units. Using NMR and chromatography to monitor the alkaline conversion of μ- to κ-carrabiose, we found that the μ-carrabiose distribution (-μ-, -μ-μ-, -μ-μ-μ-) did not influence the conversion rate.

In Vitro Study of the Proteolytic Degradation of Antheraea pernyi Silk Fibroin

In this study, Antheraea pernyi silk fibroin (Ap-SF) films were incubated with Protease Type XXI from Streptomyces griseus, at 37 degrees C, to investigate the degradation behavior in an in vitro model system. The enzyme-resistant fractions of Ap-SF films and the soluble peptides formed by proteolytic degradation were collected at specified times, from 1 to 17 days, and analyzed by high performance liquid chromatography, differential scanning calorimetry, FT-Raman, and FT-IR spectroscopy. Proteolysis resulted in extensive weight loss and progressive fragmentation of films, especially at long degradation times. A range of soluble peptides was formed by proteolysis. By high performance-size exclusion chromatography it was found that their average molecular weight changed with the time of incubation. The chemical analysis of the enzyme-resistant fraction of Ap-SF films at different times of degradation indicated that the proteolytic attack preferentially occurred in the less ordered Gly rich sequences and that the contribution of the Ala rich crystalline regions to the composition of biodegraded films became progressively larger. Accordingly, DSC and spectroscopic results showed an enhancement of the crystalline character of the biodegraded films. From the behavior of the most important thermal transitions, it was deduced that the alpha-helix domains probably represent the most enzyme-resistant fraction. The in vitro approach used in the present study seems to be a valid tool for studying the rate and mechanism of degradation of Ap-SF films and of other biopolymers of potential biomedical utility.

Enzyme-resistant fractions of beans lowered serum cholesterol and increased sterol excretions and hepatic mRNA levels in rats.

Feeding rats beans with resistant starch reduces their serum cholesterol concentration; however, the mechanism by which this occurs is not fully understood. We examined the effects of enzyme-resistant fractions of adzuki (Vigna angularis) and tebou (Phaseolus vulgaris, var.) beans on serum cholesterol and hepatic mRNA in rats. Rats were fed a cholesterol-free diet with 50 g of cellulose powder (CP)/kg, 50 g of an enzyme-resistant fraction of adzuki starch (AS)/kg, or 50 g of an enzyme-resistant fraction of tebou starch (TS)/kg diet for 4 wk. There were no significant differences in body weight, liver weight, and cecum contents among the groups, nor was there a significant difference in food intake among the groups. The levels of serum total cholesterol, VLDL + intermediate density lipoprotein + LDL-cholesterol, and HDL cholesterol in the AS and TS groups were significantly (P < 0.05) lower than in the CP group throughout the feeding period. Total hepatic cholesterol in the CP group was significantly (P < 0.05) lower than in the AS and TS groups, fecal cholesterol excretion in the TS group was significantly (P < 0.05) greater than in the CP and AS groups, and the fecal total bile acid concentrations in the AS and TS groups were significantly (P < 0.05) higher than in the CP group. Cecal acetate, propionate, and n-butyrate concentrations in the AS and TS groups were significantly (P < 0.05) higher than in the CP group. The level of hepatic scavenger receptor class B1 (SR-B1) mRNA in the TS group was significantly (P < 0.05) higher than in the CP group, and the levels of hepatic cholesterol 7alpha-hydroxylase mRNA in the AS and TS groups were significantly (P < 0.05) higher than in the CP group. These results suggest that AS and TS have a serum cholesterol-lowering function due to the enhanced levels of hepatic SR-B1 and cholesterol 7alpha-hydroxylase mRNA.

Structural characterization of intestinal immune system modulating new arabino-3, 6-galactan from rhizomes of Atractylodes lancea DC.

Intestinal immune system modulating arabino-3,6-galactan (ALR-5IIa-1-1) has been purified from rhizomes of Atractylodes lancea DC. In order to characterize the structure of ALR-5IIa-1-1 sequential enzymic digestion using exo-α- l-arabinofuranosidase and exo-β- d-(1→3)-galactanase was employed. After ALR-5IIa-1-1 was digested with the arabinofuranosidase to give AF-ALR-5IIa-1-1, the galactanase digestion cleaved only 10% and 23% of 3-linked and 3,6-branched Gal in AF-ALR-5Iia-1-1, respectively, and gave small amounts of intermediate size (AF-GN-2) and shorter oligosaccharide (AF-GN-3) fractions in addition to a large amount of the galactanase-resistant fraction (AF-GN-1). When AF-GN-1 was re-digested gradually with the arabinofuranosidase and galactanase, it released trace amounts of oligosaccharides in addition to a large amount of the enzyme-resistant fraction. When the final enzyme-resistant fraction from AF-GN-1 was digested simultaneously with both arabinofuranosidase and galactanase, the resistant fraction was significantly degraded into two long fragments (AF 3-GN 3-1 and 2). Mixture of digestion products from the first galactanase digestion of AF-ALR-5IIa-1-1 showed decreased intestinal immune system modulating activity which was correspond to about 30% of the activity of ALR-5IIa-1-1, but the galactanase resistant fraction (AF-GN-1) still had significant activity. Although the second gradual enzymic digestion of AF-GN-1 caused little decrease in activity, the resulting fragments (AF 3-GN 3-1 and 2) from the final enzymic digestion lost most of the activity. Component sugar, methylation and MALDI-TOF-MS analyses indicated that the oligosaccharides released from AF-ALR-5IIa-1-1 by the first galactanase digestion consisted of galactosyl di- to hexadecasaccharides mainly comprising 6-linked Gal p and Gal f some of which were partially mono- or diarabinosylated. These oligosaccharides were attached to the non-reducing terminal side of β- d-(1→3)-galactan backbone as side chains. The final fragments (AF 3-GN 3-1 and 2) were located in the inner core of ALR-5IIa-1-1, and AF 3-GN 3-2 was composed mainly of terminal, 3-linked and 6-linked Gal whereas AF 3-GN 3-1 was suggested to have a complex structure

Characterization of antigenic epitopes in anti-ulcer pectic polysaccharides from Bupleurum falcatum L. using several carbohydrases

A polyclonal antibody (anti-bupleuran 2IIc/PG-1-IgG) against the “ramified” region (PG-1) of an anti-ulcer pectic polysaccharide was prepared and its antigenic epitopes were analyzed by using several carbohydrases. Enzymatic removal of arabinosyl residues from PG-1 by endo-(1→5)- α- l-arabinanase (from Aspergillus niger) did not reduce the binding ability of anti-bupleuran 2IIc/PG-1-IgG to PG-1. When the endo-(1→5)- α- l-arabinanase-resistant fraction (EA-1) was digested with rhamnogalacturonase A (rRGase A from A. aculeatus), a high-molecular-mass fragment fraction (RA-1) and an oligosaccharide fraction (RA-3) were obtained. RA-3 contained at least four kinds of oligosaccharides liberated from the rhamnogalacturonan core. This partial removal of the rhamnogalacturonan core in EA-1 also did not reduce the binding of the antibody to the polysaccharide. Further digestion of RA-1 with exo-(1→3)- β- d-galactanase (from Irpex lacteus), gave a high-molecular-mass fragment (EXG-1) and a trace of oligosaccharides (EXG-3). Methylation and FABMS analyses indicated that EXG-3 contained mono- and di-galactosyl oligosaccharides possessing terminal GlcA or GlcA4Me. Removal of the EXG-3 fraction from RA-1 by exo-(1→3)- β- d-galactanase significantly reduced the ability of the binding of the antibody to the polysaccharide. When PG-1 was digested with endo-(1→6)- β- d-galactanase (from Trichoderma viride) or β- d-glucuronidase (from A. niger), the reactivities of both enzyme-resistant fractions to the antibody were decreased in comparison with that of PG-1. Both radish arabinogalactan (containing GlcA4Me) and β- d-Glc pA-(1→6)- β- d-Gal p-(1→6)- d-Gal p were shown to inhibit the reactivity of PG-1 to the antibody by competitive ELISA. These results suggest that 6-linked galactosyl chains containing terminal GlcA or GlcA4Me attached to (1→3)- β- d-galactosyl chains, are important sugar residues in the antigenic epitopes of the “ramified” region of bupleuran 2IIc.

Existence of a Rhamnogalacturonan II-like Region in Bioactive Pectins from Medicinal Herbs

Unusual component sugars such as 2-methylfucose (2-Me-Fuc), 2-methylxylose (2-Me-Xyl), apiose (Api), and aceric acid (AceA) are contained in the bioactive pectins from Bupleurum falcatum, Glycyrrhiza uralensis, and Angelica acutiloba, but not in the other bioactive pectic heteroglycans and arabinogalactans from Chinese and Japanese herbs tested. Each pectin was digested with endo-alpha-(1-->4)-polygalacturonase, and gave two enzyme-resistant fractions, PG-1 (rhamnogalacturonan core with neutral sugar side-chains) and PG-2, and an oligogalacturonide fraction (PG-3) by gel filtration on Bio-gel P-30. The PG-2 fractions commonly consisted of unusual sugars such as 2-Me-Fuc, 2-Me-Xyl, Api, AceA, 3-deoxy-D-lyxo-heptulosaric acid (Dha), and 3-deoxy-D-manno-2-octulosonic acid (Kdo) in addition to Rha, Fuc, Ara, Xyl, Man, Gal, Glc, GalA, and GlcA. Lithium degradation of each PG-2 gave a pentosyl-->6-deoxyhexosyl-->6-deoxyhexosyl-->pentitol fragment as a major oligosaccharide in addition to some neutral di- to trisaccharide alditols. Methylation analysis of the lithium degradation products from each PG-2 also indicated that these oligosaccharide alditols mainly consisted of terminal Rha, Araf, Fuc, Xyl, and Gal, 4-linked Rha, 3-linked Fuc, and 3'-linked Api. HPLC analysis showed that PG-2 had molecular heterogeneity. These results indicate that the bioactive pectins from medicinal herbs commonly consist of the minor KDO-containing region which resembles the rhamnogalacturonan II in plant cell walls.