Exobasidium vexans infection of Camellia sinensis increased 2,3-cis isomerisation and gallate esterification of proanthocyanidins

Infection of leaves of tea (Camellia sinensis (Kuntze) L, cv TRI 2025) which was susceptible to blister blight (Exobasidium vexans Massee), resulted in a shift of the proanthocyanidin stereochemistry away from 2,3-trans (e.g. catechin and gallocatechin) and towards 2,3-cis (e.g. epicatechin and epigallocatechin). Infection also resulted in increased gallic acid esterification of the initiating subunits of proanthocyanidins. This was shown by both mass spectroscopy and phloroglucinolysis. Proanthocyanidins isolated from healthy tissue had a predominantly 2,3-trans stereochemistry which accounted for 53% and 61% of the total initiating and extension units of proanthocyanidin, respectively. Conversely in infected tissue, proanthocyanidin subunits with a 2,3-trans stereochemistry accounted for 26% and 40% of the total initiating and extension units, respectively. Infection had little impact on the hydroxylation state of the B-rings of proanthocyanidins. The products of acid hydrolysis under oxidative conditions had a slight excess of di-hydroxylated B-rings with cyanidin accounting for 58.3+/-0.05% and 60.4+/-0.2% of the total anthocyanidin recovered following hydrolysis of proanthocyanidin isolated from infected and healthy leaves, respectively. Similar results were obtained by phloroglucinolysis.     

Development of a real time PCR Taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum

In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.     

Ruminococcin C, a new anti-Clostridium perfringens bacteriocin produced in the gut by the commensal bacterium Ruminococcus gnavus E1

When colonizing the digestive tract of mono-associated rats, Ruminococcus gnavus E1 - a bacterium isolated from human faeces - produced a trypsin-dependent anti-Clostridium perfringens substance collectively named Ruminococcin C (RumC). RumC was isolated from the caecal contents of E1-monocontaminated rats and found to consist of two antimicrobial fractions: a single peptide (RumCsp) of 4235 Da, and a mixture of two other peptides (RumCdp) with distinct molecular masses of 4324 Da and 4456 Da. Both RumCsp and RumCdp were as effective as metronidazole in combating C. perfringens and their activity spectra against different pathogens were established. Even if devoid of synergistic activity, the combination of RumCsp and RumCdp was observed to be much more resistant to acidic pH and high temperature than each fraction tested individually. N-terminal sequence analysis showed that the primary structures of these three peptides shared a high degree of homology, but were clearly distinct from previously reported amino acid sequences. Amino acid composition of the three RumC peptides did not highlight the presence of any Lanthionine residue. However, Edman degradation could not run beyond the 11th amino acid residue. Five genes encoding putative pre-RumC-like peptides were identified in the genome of strain E1, confirming that RumC was a bacteriocin. This is the first time that a bacteriocin produced in vivo by a human commensal bacterium was purified and characterized.     

Clostridium asparagiforme sp. nov., isolated from a human faecal sample

An obligatory anaerobic, Gram-positive, rod-shaped organism was isolated from faeces of a healthy human donor. It was characterized using biochemical, phenotypic and molecular taxonomic methods. The organism produced acetate, lactate, and ethanol as the major products of glucose fermentation. The G + C content was 53 mol%. Based on comparative 16S rRNA gene sequencing, the unidentified bacterium is a member of the Clostridium subphylum of the Gram-positive bacteria, and most closely related to species of the Clostridium coccoides cluster (rRNA cluster XIVa) [M.D. Collins et al., The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations, Int. J. Syst. Bacteriol. 44 (1994) 812-826]. Clostridium bolteae and Clostridium clostridioforme were identified as the most closely related described species. A 16S rRNA sequence divergence value of > 3% suggested that the isolate represents a new species. This was also supported by the gyrase-encoding gyrB gene sequences. Based on these findings, we propose the novel bacterium from human faeces to be classified as a new species, Clostridium asparagiforme. The type strain of C. asparagiforme is N6 (DSM 15981 and CCUG 48471).     

Antimalarial xanthones from Calophyllum caledonicum and Garcinia vieillardii

The antimalarial activity of 22 xanthones against chloroquino-resistant strains of Plasmodium falciparum was evaluated. Natural caloxanthone C (1), demethylcalabaxanthone (2), calothwaitesixanthone (3), calozeyloxanthone (4), dombakinaxanthone (5), macluraxanthone (6), and 6-deoxy-gamma-mangostin (7) were isolated from Calophyllum caledonicum. 1,6-dihydroxyxanthone (8), pancixanthone A (9), isocudraniaxanthone B (10), isocudraniaxanthone A (11), 2-deprenylrheediaxanthone B (12) and 1,4,5-trihydroxyxanthone (13) were isolated from Garcinia vieillardii. Moreover, synthetic compounds (14-22) are analogues or intermediates of xanthones purified from Calophyllum caledonicum (Oger J.M., Morel C., Helesbeux J.J., Litaudon M., Seraphin D., Dartiguelongue C., Larcher G., Richomme P., Duval O. 2003. First 2-Hydroxy-3-Methylbut-3-Enyl substituted xanthones isolated from Plants: structure elucidation, synthesis and antifungal activity. Natural Product Research 17(3), 195-199; Helesbeux J.J., Duval O., Dartiguelongue C., Seraphin D., Oger J.M., Richomme P., 2004. Synthesis of 2-hydroxy-3-methylbut-3-enyl substituted coumarins and xanthones as natural products. Application of the Schenck ene reaction of singlet oxygen with ortho-prenylphenol precursors. Tetrahedron 60(10), 2293-2300). The relationship between antimalarial activity and molecular structure of xanthones has also been explored. The most potent xanthones (2), (3) and (7) (IC50 = c.a. 1.0 microg/mL) are 1,3,7 trioxygenated and prenylated on the positions 2 and 8.     

The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells

Orally ingested botulinum toxin enters the circulatory system and eventually reaches the peripheral nerves, where it elicits a response of neurological dysfunction. In this study, we report the important findings concerning the mechanism of Clostridium botulinum type C progenitor toxin (C16S) endocytic mechanism. C16S toxin bound to high molecular weight proteins on the surface of human colon carcinoma HT-29 cells and was internalized, but not if the cells were pretreated with neuraminidase. Benzyl-GalNAc which inhibited O-glycosylation of glycoproteins also interfered in the toxin's ability to bind the cell surface. On the other hand, the toxin was internalized in spite of pretreatment of the cells with PPMP, an inhibitor of ganglioside synthesis. These results suggest that the glycoproteins, like mucin, fulfill the important roles of receptor and transporter of C16S toxin.     

Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.     

Refinement of the structures of cell-wall glucans of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy

Alkali extraction and methylation analyses in the 1970s revealed that the cell walls of the yeast Schizosaccharomyces pombe contain a (1-->3)-alpha-d-glucan, a (1-->3)-beta-d-glucan, a (1-->6)-beta-d-glucan, and a alpha-galactomannan. To refine the structures of these polysaccharides, cell-wall glucans of S. pombe were extracted, fractionated, and analyzed by NMR spectroscopy. S. pombe cells were treated with 3% NaOH, and alkali-soluble and insoluble fractions were prepared. The alkali-insoluble fraction was treated with 0.5M acetic acid or Zymolyase 100T to yield an alkali-insoluble, acetic acid-insoluble fraction, an alkali-insoluble, Zymolyase-insoluble fraction, and an alkali-insoluble, Zymolyase-soluble fraction. (13)C NMR and 2D-NMR spectra disclosed that the cell wall of S. pombe is composed of three types of glucans, specifically, a (1-->3)-alpha-d-glucan, a (1-->3)-beta-d-glucan, which may either be linear or slightly branched, and a highly branched (1-->6)-beta-d-glucan, in addition to alpha-galactomannan. The highly branched (1-->6)-beta-d-glucan was identified by selective periodate degradation of side-chain glucose as a highly (1-->3)-beta-branched (1-->6)-beta-d-glucan with more branches than that of Saccharomyces cerevisiae. Flexibility of these polysaccharides in the cell wall was analyzed by (13)C NMR spectra in D(2)O. The data collectively indicate that (1-->3)-alpha- and (1-->3)-beta-d-glucans are rigid and contribute to the cell shape, while the highly branched (1-->6)-beta-d-glucan and alpha-galactomannan are flexible.     

Isolation and partial characterization of trehalose 6-phosphate synthase aggregates from Selaginella lepidophylla plants

Trehalose 6-phosphate synthase was purified from Selaginella lepidophylla plants and three aggregates of the enzyme were found by molecular exclusion chromatography, ion exchange chromatography and electrophoresis. Molecular exclusion chromatography showed four activity peaks with molecular weights of 624, 434, 224 and 115 kDa. Ion exchange chromatography allowed three fractions to be separated with TPS activity which eluted at 0.35, 0.7 and 1 M KCl. Native PAGE of each pool had three protein bands with apparent M(r) 660, 440 and 200 kDa. Western blot results showed that anti-TPS antibody interacted with 115 and 67 kDa polypeptides; these polypeptides share peptide sequences as indicated by internal sequence data. The effects of pH and temperature on enzyme stability and activity were studied. For fractions eluted at 0.35 and 1.0 M KCl, the optimum pH is 5.5, while an optimum pH of 7.5 for 0.7 M fraction was found. The three fractions eluted from ion exchange chromatography were stable in a pH 5-11 range. Optimal temperatures were 25, 45 and 55 degrees C for 0.7, 0.35 and 1.0 M fractions, respectively. The 0.7 M KCl fraction showed highest stability in a temperature range of 25-60 degrees C, whereas the 0.35 M KCl fraction had the lowest in the same temperature range.     

p-Cresol formation by cell-free extracts of Clostridium difficile

Cell-free extracts of Clostridium difficile were shown to form p-cresol by decarboxylation of p-hydroxyphenylacetic acid. This activity required both high and low molecular weight fractions. The active component of the low molecular weight fraction had properties of an amino acid and could be replaced by serine, threonine or the corresponding alpha keto acids. Pyruvate was shown to function catalytically. Since the high molecular weight fraction was O₂-sensitive and since dithionite was as effective as pyruvate with some high molecular weight fractions, the alpha keto acids probably serve as low potential reducing agents in this system. Because of instability, the p-cresolforming enzyme could not be purified.     

Structural characterization of laminaran and galactofucan extracted from the brown seaweed Saccharina longicruris

Brown seaweed contains several polysaccharides like laminaran, fucoidan and alginate. Laminaran is a β-glucan that has shown anti-apoptotic and anti-tumoral activities, while galactofucan (fucoidan) is a sulfated polysaccharide that has displayed anticoagulant, anti-tumor, anti-thrombosis, anti-inflammatory and antiviral properties. In this study, crude laminaran and galactofucan (fucoidan) were extracted from the brown seaweed Saccharina longicruris at four harvest periods (M05, A05, N05 and J06). The galactofucan M05 and N05 fractions were depolymerized (RDP) over 2 or 4 h to give 4 RDP fractions (M05 RDP 2H, M05 RDP 4H, N05 RDP 2H and N05 RDP 4H) whose molecular weights, monosaccharide compositions and glycosidic linkages were determined by GC-MS. The laminaran fraction gave a molecular weight range from 2900 to 3300 Da and contained between 50.6% and 68.6% d-glucose and an average of 1.3% d-mannitol. The presence of a β-(1,3) linkage between d-glucose in the main chain was observed, with branching at positions 6 and 2. The M05 fraction contained less branching than other laminaran fractions, which might have influenced its conformation in solution and thus its activity. The crude galactofucan fractions displayed a molecular weight range from 638 to 1529 kDa, whereas the RDP fractions had molecular weights <30 kDa. The structure of the galactofucan fractions remained complex after depolymerization, with these also being more sulfated (30-39%) than the crude fractions (13-20%). The crude and RDP fractions contained 3-linked fucopyranose 4-sulfate and 6-linked galactopyranose 3-sulfate moieties, although the galactofucans isolated from M05 and J06 contained less 6-linked galactopyranose 3-sulfate than the A05 and N05 fractions.     

Antibacterial and antifungal lysozyme-type activity in Cameraria ohridella pupae

Lysozyme-type antibacterial and antifungal activity in pupae of Cameraria ohridella was studied. Activity against Micrococcus luteus and Bacillus megaterium was detected in pupae extract. Also antifungal activity from C. ohridella pupae extract directed against Saccharomyces cerevisiae strain W 303 was shown. During immunoblotting two bands in pupae extract, with molecular mass of about 15 and 28 kDa were recognized by antibodies directed against HEWL. After acid electrophoresis followed by bioautography of the extract, two lytic zones showing lysozyme-type activity against M. luteus were observed. Two bacteria: Gram-positive Aerococcus viridans and Gram-negative Aeromonas salmonicida ssp. masoucida were isolated from pupae of C. ohridella. Their activity against M. luteus, B. megaterium, and S. cerevisiae W303 was detected. After immunoblotting with antibodies against HEWL, also two proteins from bacterial suspensions of A. viridans and A. salmonicida were detected, about 15 and 28 kDa.     

Prevention of the cytopathic effect induced by Clostridium difficile Toxin B by active Rac1

Clostridium difficile Toxin B (TcdB) glucosylates low molecular weight GTP-binding proteins of the Rho subfamily and thereby causes actin re-organization (cell rounding). This "cytopathic effect" has been generally attributed to RhoA inactivation. Here we show that cells expressing non-glucosylatable Rac1-Q61L are protected from the cytopathic effect of TcdB. In contrast, cells expressing RhoA-Q63L or mock-transfected cells are fully susceptible for the cytopathic effect of TcdB. These findings are extended to the Rac1/RhoG mimic IpgB1 and the RhoA mimic IpgB2 from Shigella. Ectopic expression of IpgB1, but not IpgB2, counteracts the cytopathic effect of TcdB. These data strongly suggest that Rac1 rather than RhoA glucosylation is critical for the cytopathic effect of TcdB.     

Selection of herbal therapeutics against deltatoxin mediated Clostridial infections

Clostridium perfringens (a versatile pathogenic bacterium) secretes enterotoxins (the deltatoxin, virulent factor) and causes food borne gastroenteritis and gasgangrene. The organism was isolated and characterized from improperly cooked meat and poultry samples. The isolated organism showed multiple drug resistance indicating that the treatment is challenging. Hence, there is need for improved therapeutic agents. The rational design of improved therapeutics requires the crystal structure for the toxin. However, the structure for the toxin is not yet available in its native form. Thus, we modeled the toxin structure using α- hemolysin of Staphylococcus aureus (PDB: 3M4D chain A) as template. The docking of the toxin with the herbal extract curcumin (1,7-bis(4-hydroxy-3- methoxyphenyl)hepta-1,6-diene-3,5-dione) showed a binding energy of -8.6 Kcal/mol, in comparison to the known antibiotic Linezolid with binding energy of -6.1 Kcal/mol. This data finds application in the design and development of novel compounds against the deltatoxin from Clostridium perfringens.     

Genotoxic and antigenotoxic effects of catechin and tannins from the bark of Hamamelis virginiana L. in metabolically competent,human hepatoma cells (Hep G2) using single cell gel electrophoresis

The genotoxic and antigenotoxic activities of catechin, hamamelitannin and two proanthocyanidin fractions prepared from the bark of Hamamelis virginiana L. were investigated in a human derived, metabolically competent hepatoma cell line (Hep G2) using single cell gel electrophoresis (SCGE) for the detection of DNA-damage. DNA-migration was calculated as Olive tail moment (OTM). Catechin and a low-molecular weight proanthocyandin fraction (W(M)) caused only slight increases of OTM up to concentrations of 166 microg/ml whereas hamamelitannin and the proanthocyandin fraction with higher molecular weight (W(A)) led to a two-fold enhancement of OTM at the same concentrations. These effects were dose-independent. Treatment of the cells with the test compounds in a dose-range of 2-166 microg/ml prior to the exposure to benzo(a)pyrene (B(a)P, 10 microM, 2.5 microg/ml) led to a significant reduction of induced DNA damage which was dose-dependent for all test compounds, except for hamamelitannin. The inhibitory effects of proanthocyanidins were stronger than those of catechin and hamamelitannin; the lowest effective concentrations were about 2 microg/ml. In order to clarify the mechanisms of protection, possible effects of the test compounds on enzymes involved in toxification and detoxification of B(a)P were investigated. While B(a)P toxification by cytochrome P450 was not inhibited by the test compounds, detoxification by glutathion-S-transferase (GST) was induced by catechin and W(M). Combination experiments with the ultimate metabolite of B(a)P, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE; 5 microM, 1.5 microg/ml), revealed strong inhibitory effects, indicating that the observed protective effects were caused by scavenging of the ultimate mutagen by the test compounds. Exposure of Hep G2 cells to the test compounds after B(a)P treatment did not influence B(a)P induced DNA damage, demonstrating that repair mechanisms were not affected.     

Leishmanicidal cycloartane-type triterpene glycosides from Astragalus oleifolius

Two new cycloartane-type glycosides oleifoliosides A (1) and B (2) were isolated from the lower stem parts of Astragalus oleifolius. Their structures were identified as 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-xylopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane and 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-glucopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane, respectively, by means of spectroscopic methods (IR, 1D and 2D NMR, ESI-MS). Three known cycloartane glycosides cyclocanthoside E (3), astragaloside II (4) and astragaloside IV (5) were also isolated and characterized. All five compounds were evaluated for in vitro trypanocidal, leishmanicidal and antiplasmodial activities as well as their cytotoxic potential on primary mammalian (L6) cells. Except for the compound 5, all compounds showed notable growth inhibitory activity against Leishmania donovani with IC50 values ranging from 13.2 to 21.3 microg/ml. Only weak activity against Trypanosoma brucei rhodesiense was observed with the known compounds astragaloside II (4, IC50 66.6 microg/ml) and cyclocanthoside E (3, IC50 85.2 microg/ml), while all compounds were inactive against Trypanosoma cruzi and Plasmodium falciparum. None of the compounds were toxic to mammalian cells (IC50's > 90 microg/ml). This is the first report of leishmanicidal and trypanocidal activity of cycloartane-type triterpene glycosides.     

Expression and stability of the nontoxic component of the botulinum toxin complex

Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex associated with nontoxic-nonhemagglutinin (NTNHA) and/or hemagglutinin components. In the present study, high-level expression of full-length (1197 amino acids) rNTNHA from C. botulinum serotype D strain 4947 (D-4947) was achieved in an Escherichia coli system. Spontaneous nicking of the rNTNHA at a specific site was observed during long-term incubation in the presence of protease inhibitors; this was also observed in natural NTNHA. The rNTNHA assembled with isolated D-4947 BoNT with molar ratio 1:1 to form a toxin complex. The reconstituted toxin complex exhibited dramatic resistance to proteolysis by pepsin or trypsin at high concentrations, despite the fact that the isolated BoNT and rNTNHA proteins were both easily degraded. We provide definitive evidence that NTNHA plays a crucial role in protecting BoNT, which is an oral toxin, from digestion by proteases common in the stomach and intestine.     

Protein-proanthocyanidin interactions during extraction of scots pine needles

Successful protein extractions from Scots pine needles in different buffers (pH 5–9)with PVP-10 and Tween 80 suggest that both hydrogen bonds and hydrophobic interactions are responsible for the formation of insoluble protein-tannin complexes. Gel filtrations did not permit a separation of tannins from solubilized proteins. Contamination of the protein fractions was attributed to proanthocyanidins, both as such and when attached to the proteins. The ability of the soluble protein-proanthocyanidin complexes to adsorb on both Polyclar AT through hydrogen bonding and on Phenyl-Sepharose CL-4B through hydrophobic interactions suggests an amphiphilic capacity on the part of the proanthocyanidin. The factors causing persistent binding between proteins and proanthocyanidins through hydrophobic interactions are discussed. Although the vanillin test confirmed the presence of high molecular weight tannins, they occurred independently of the proteins.     

Evaluation of sulfated fungal beta-glucans from the sclerotium of Pleurotus tuber-regium as a potential water-soluble anti-viral agent

Six water-insoluble fractions of fungal beta-glucans extracted by hot alkali (TM8-1 to TM8-6) from the sclerotia of Pleurotus tuber-regium (PTR) having different molecular weights (M(w)) were sulfated to give their corresponding water-soluble derivatives (S-TM8-1 to S-TM8-6) with the degree of sulfation (DS) ranging from 1.14 to 1.74. The in vitro anti-viral activities of the native beta-glucans (TM8s) and their sulfated derivatives (S-TM8s) were evaluated by the cytopathic effect assay (CPE) and the plaque reduction assay (PRA) against four kinds of viruses, including herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), respiratory syncytial virus (RSV), and influenza A virus (Flu A). Although TM8s were inactive in inhibiting the viral replication in cell cultures, the S-TM8 fractions with the defined M(w) range had potent anti-viral activity against HSV-1 and HSV-2 as shown by the CPE assay. The PRA results suggested that S-TM8 fractions seemed to exert their anti-viral effect by binding to the viral particles, preventing the latter from infecting the host cells. It was plausible that the negative charges on the polymer chain of S-TM8 could interact with the positively charged glycoproteins on the surface of HSV, minimizing the interaction between the HSV and the negatively charged host cells. The anti-viral activity of the S-TM8s might also be explained by their more extended chain conformation in solution due to an increase in one of their molecular parameter, persistence length (q), as compared to the native TM8s. The potential use of S-TM8s as a water-soluble anti-HSV agent is discussed.     

In vitro antioxidant and antitumor activities of polysaccharides extracted from Asparagus officinalis

Ultrasonic circulating extraction technology was applied for the polysaccharide extraction from Asparagus officinalis. The crude polysaccharides were deproteinized by Sevag method and three main polysaccharide fractions, AOP-4, AOP-6 and AOP-8 were obtained by fractional precipitation with gradient concentrations of ethanol (40%, 60% and 80%). The in vitro antitumor and antioxidant activities of the polysaccharide fractions were evaluated by MTT assay and free radical-scavenging assay, respectively. Deproteinized AOPs showed higher antioxidant and antitumor activities than crude AOP. AOP-4 with molecular weight 5.75 × 10⁴ Da showed significant function of scavenging hydroxyl radical. Three AOP fractions had significant antitumor activity against HeLa and BEL-7404 cells in a dose dependent manner. Furthermore, the inhibit activity of AOP-4 against HeLa cells was higher than those of other AOPs and the inhibition rate reached 83.96% at the concentration of 10 mg/mL. These results indicated that the AOP might be useful for developing natural safe antitumor drugs or health food.