DEAE-cellulose Column Research Articles

Griseococcin (1) from Bovistella radicata (Mont.) Pat and antifungal activity

BackgroundTo evaluate the antimicrobial and microbicidel activity of B. radicata fermentation broth, the broth was purified by DEAE-cellulose and sephadex LC-20 column. The compounds were submitted to spectral analyses (HPLC, FT-IR, 1D and 2D NMR etc.).ResultsThe purified compounds were identified as the Griseococcin(s) which were naphthoquinone derivatives, the Chemical formula and MW of Griseococcin (1) was determined as C37O10H43N and 661 Da. only Griseococcin (1) has good antimicrobial activity among the Griseococcin(s). The zone of inhibition (ZOI), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) of Griseococcin (1) were used to investigate the antimicrobial activity. Antifungal activity of Griseococcin (1) was significant, especially for main pathogenic fungus Trichophyton rubrum and Trichophyton mentagrophytes, MFC/MIC of Griseococcin (1) was 1, while MFC/MIC of postive control was greater than 4, the fungicidal effect of Griseococcin (1) was better than that of positive control.ConclusionsIn this paper, the secondary metabolite compound Griseococcin (1) from B. radicata was purified. The purified compound can restrain main pathogens (T. rubrum and T. mentagrophytes) leading to tinea pedis. The antifungal activity of Griseococcin (1) was similar to that of the positive control and the fungicidal effect of Griseococcin (1) was better than that of positive control, it might be suitable for pharmaceutical industries.

Glucose- lowering effect of Trametes orientalis polysaccharides in hyperglycemic and hyperlipidemic mice

To explore the glucose-lowering effect of the polysaccharide fractions of Trametes orientalis. The crude polysaccharides of T. orientalis were chromatographed on DEAE cellulose column using H2O and 0.5 mol/L NaCl solution as the eluent and DEAE-water and DEAE-salt with high polysaccharide contents were collected. The two fractions were separated using Sephadex G-100 gel column to obtain 4 polysaccharide fractions TOPW-1, TOPW-2, TOPS-1, and TOPS-2. The anti-oxidation activity of the polysaccharide fractions was investigated with ABTS method. The fractions TOPW-1 and TOPS-1 with consistent UV detection signals were collected and HPGPC was used to determine their relative molecular mass. The monosaccharide composition in homogeneous TOPW-1 was determined by acid hydrolysis combined with HPLC. The in vitro inhibitory activities of TOPW-1 and TOPS-1 against DPP4, adipocyte glucose intake and lipase activity were tested to preliminarily assess their glucose-lowering effect. In a mouse model of hyperglycemic and hyperlipidemic, the glucose-lowering effect of TOPS-1 (0.1, 0.2, and 0.4 g/kg) and its effect on blood lipid metabolism were investigated in comparison with Xiaoke pills (5 pills/kg) and Danshen tablets (0.5 g/kg). TOPW-1 was a homogeneous polysaccharide composed mainly of D-mannose, D-glucose, D-galactose, and D-fucose, with weak antioxidant and hypoglycemic effects. TOPS-1 was not a single polysaccharide and at the concentration of 500 μg/mL showed an high ABTS clearance rate (90.15%). In the mouse model of hyperglycemia and hyperlipidemia, treatment with TOPS-1 (0.2 g/kg) enhanced the activity of lipase and significantly reduced fasting glucose level and serum contents of total cholesterol, triacylglycerol, and low-density lipoprotein cholesterol without causing death in the mice. The glucose-lowering effects of TOPS-1 was not significant at the low (0.1 g/kg) or high (0.4 g/kg) dose, and a high dose tended to increase the mortality of the mice. T. orientalis polysaccharides have anti-oxidation, glucose-lowering and lipid-lowering effects in mice, and their glucose-lowering effect is probably medicated by reducing oxidative stress and ameliorating lipid metabolism disorder.

Optimization, characterization, rheological study and immune activities of polysaccharide from Sagittaria sagittifolia L.

To improve the extraction efficiency of polysaccharides from Sagittaria sagittifolia L. (SPU) by ultrasonic assisted extraction (UAE), the optimal extraction conditions were optimized as follows: extraction temperature of 85℃, extraction time of 15 min and ratio of liquid to raw material 43 mL/g, under these conditions, the yield of SPU increased by about 168 % compared with hot water extraction (HWE). After separation and purification by DEAE-52 cellulose column and Sephadex G-50 column, the pure polysaccharide fraction (SPU70-W1) was obtained, and its structure, rheology and immune activity were analyzed. The results indicated that SPU70-W1 (7.70 kDa) contained mannose, glucose and galactose in the molar ratio of 2.06:93.58:4.36 with typical pseudoplasticity fluids behavior and possessed the backbone of →2,6)-α-D-Glcp-(1→, →3,6)-α-D-Glcp-(1→, α-D-Glcp-(1→ and 6)-α-D-Glcp-(1→. In addition, SPU70-W1 exhibited remarkable immunomodulatory activity. Thus, SPU70-W1 could contribute to the food, medicine, cosmetics as a functional additive.

Biochemical Isolation and Characterization of Hyaluronidase Enzyme from Venom of Egyptian Honey Bee Apis Mellifera Lamarckii

Abstract The hyaluronidase enzyme has been used in many such fields of medicine as ophthalmology, orthopaedia, internal medicine, gynecology, surgery, oncology and dermatology. In this study, the hyaluronidase enzyme was purified and characterized for the first time from Egyptian bee venom homogeneously using DEAE-cellulose and Sephacryl S-300 columns. Bee venom hyaluronidase specific activity was 411.7 units/mg protein with 49.9% yield and 3.23-fold purification. The molecular weight of the purified bee venom hyaluronidase native form was 37 kDa. The purified enzyme was found homogeneous on native PAGE and SDS-PAGE, with two congruent subunits of 18.4 kDa and isoelectric point (pI) of 8.6–8.8. The enzyme was found to be stable over a wide range of temperature (20–60°C) and pH (4.5–6.5), and its optimum activity at 37°C, pH 5.4 and 0.15 M NaCl. K m for bee venom hyaluronidase was 0.029 mg/ml hyaluronic acid and its activity was elevated in presence of MgCl2 and ZnCl2 and lowered in presence of FeCl2. Heparin inhibited the hyaluronidase enzyme noncompetitively with a Ki value of 2.9 units heparin and one binding site on the enzyme molecule.

Extraction and analysis of polysaccharide from Momordica charantia

Momordica charantia is a kind of medicine and food homologous fruit. The M. charantia polysaccharide has the physiological functions of clearing away heat and detoxification, lowering blood sugar, improving immunity and so on. It is an ideal health care product.MP3 from M. charantia polysaccharides were isolated and purified by DEAE cellulose column chromatography and gel column chromatography. The composition of polysaccharides was determined by thin layer chromatography (TLC) and gas chromatography, and the relative molecular weight was determined by gel filtration. The results showed there were typical absorption peaks of polysaccharide in its infrared spectrum, and there were no characteristic absorption peaks of nucleic acids and proteins in its ultraviolet scanning. MP3 contained rhamnose, xylose, galactose and arabinose, and its average molecular weight was 9.57 × 104. This method was easy to operate and laid a foundation for the development of M. charantia polysaccharide products.

Antiviral activity against porcine epidemic diarrhea virus of Pogostemon cablin polysaccharide

Ethnopharmacological relevanceThe dry overground parts of Pogostemon cablin (Blanco) Benth. is widely used in China as a traditional Chinese medicine for the treatment of diarrhea, vomiting, nausea and fever. Polysaccharide is an important component of Pogostemon cablin (Blanco) Benth. but has not been studied. Pogostemon cablin (Blanco) Benth. is used to treat porcine epidemic diarrhea. But it is not known whether Pogostemon cablin polysaccharides (PCPs) has the antiviral activities against porcine epidemic diarrhea virus (PEDV). Aim of the studyThe purpose of present study is to investigate the structural characterization and the anti-PEDV activities of PCPs. Materials and methodsPCPs were prepared by water extraction and alcohol precipitation method and purified with DEAE-52 cellulose column and Sephadex G-100 column. Then, the structural characterization of the polysaccharides including the infrared spectrum, molecular weight and monosaccharide composition were analyzed. Afterwards, the antiviral effect of PCPs against PEDV on IPEC-J2 cells was studied by MTT method and real-time PCR method. Additionally, the effects of PCPs on PEDV adsorption, penetration and replication were analyzed by real-time PCR method. Furthermore, we also investigate whether the anti-oxidative effects of PCPs were important to the anti-PEDV activities. ResultsFour polysaccharides were obtained and named as PCP1.1 (31.3 kDa), PCP1.2 (3.5 kDa), PCP2.1 (9.1 kDa) and PCP2.2 (8.3 kDa). PCP1.1, PCP1.2 and PCP2.1 were composed of fucose, arabinose, galactose, glucose, mannose, galacturonic acid and glucuronic acid; and PCP2.2 was composed of arabinose, galactose, glucose, galacturonic acid and glucuronic acid. All PCPs showed anti-PEDV activities. PCP1.1 and PCP1.2 inhibited PEDV replication, while PCP2.1 and PCP2.2 inhibited PEDV penetration and replication. All PCPs showed anti-oxidative effects, which were important to the anti-PEDV activities. ConclusionsThe treatment effect of Pogostemon cablin (Blanco) Benth. on porcine epidemic diarrhea might be related to the anti-PEDV effect of PCPs. Furthermore, the anti-oxidative effects of PCPs play important roles in their antiviral activities against PEDV.

Preparation, structural characterization, and bioactivity of PHPD-IV-4 derived from Porphyra haitanensis

Polysaccharides are a major active component of Porphyra haitanensis, which is an important food source in many countries. Four different molecular-weight fractions, namely PHPD-I (329 kDa), PHPD-II (203 kDa), PHPD-III (128 kDa), and PHPD-IV (10 kDa), were obtained from P. haitanensis polysaccharides by degradation using the H2O2/ascorbic acid system. PHPD-IV elicited the highest level of antioxidant and immunostimulatory activity among the four fractions. PHPD-IV was purified by DEAE-cellulose column and five fractions were obtained, designated PHPD-IV-1–PHPD-IV-5. PHPD-IV-4 displayed the greatest biological activity by up-regulating the phosphorylation of MAPK signalling molecules. PHPD-IV-4 was further purified, and its structure was characterized by monosaccharide composition and 1/2D-NMR analysis. The result revealed that PHPD-IV-4 was repeated units of → 3) β-d-galactose (1 → 4) 3, 6-anhydro-α-l-galactose (1→, and → 3) β-d-galactose (1 → 4) α-l-galactose-6-S (1→. This study provides a theoretical basis for the utilisation and structure-activity assessment of P. haitanensis polysaccharides.

Analysis of the water-soluble polysaccharides from Camellia japonica pollen and their inhibitory effects on galectin-3 function

Pollen has been defined as dietary supplement used to supplement the diet in many countries, but the primary structure and activity of Camellia japonica pollen polysaccharide remain unclear. In this study, the water-soluble polysaccharide extracted from Camellia japonica pollen (WCPP) was fractionated into one neutral fraction (WCPP-N) and two acidic fractions (WCPP-A1 and WCPP-A2) by DEAE-cellulose column, and WCPP-A2 was further fractionated into two homogeneous sub-fractions (WCPP-A2a and WCPP-A2b) by Sepharose CL-6B column. Monosaccharide composition results showed that WCPP-N might mainly contain starch-like glucan as well as some arabinogalactan, while WCPP-A1, WCPP-A2 and its sub-fractions might mainly composed of rhamnogalacturonan I (RG-I) pectic polysaccharide domain backbone with some different types of side chains, including arabinan, galactan, and/or arabinogalactan. The primary structure analysis of WCPP-A2a by NMR spectra analysis suggested that WCPP-A2a was an RG-I-like pectic polysaccharide, branched at the O-4 of Rha residues in the backbone, with α-(1 → 3,5)-L-arabinan as well as type-II arabinogalactan side chain to which were attached. The results of galectin-3-mediated hemagglutination assay indicated that WCPP-A2a exhibited the strongest inhibitory effect on galectin-3 with MIC value around 0.27 μg/mL. These results suggested the potential use of Camellia japonica pollen polysaccharide as a galectin3 inhibitor in functional foods.

Cytotoxic and antioxidant activity of the polysaccharide isolated from the seeds of Strychnos potatorum

Abstract Nonconventional natural treatments have gained wide acceptance due to their promise of a cure with minimal or no side effects in cancer, but little scientific evidence exists. The polysaccharides were extracted from the seeds of Strychnos potatorum and were successively purified by chromatography on DEAE cellulose and Sephadex G-100 column. The purified polysaccharide was identified to be a heteropolysaccharide 1,4 β-D- galactomannan characterized using FTIR, NMR. The molecular weight was determined to be ∼ 1.28 X 10 4 Da through MALDI-TOF analysis. Presented, here is the first use of the polysaccharide isolated from the seeds of Strychnos potatorum exhibiting promising cytotoxic activity with IC50 of 365 μg/mL against human breast cancer cell line MCF-7. The antioxidant property of the polysaccharide was also evaluated and they possess potential scavenging activity for DPPH assay and hydroxy radical scavenging assay with increase in concentration.

Enzyme-assisted extraction of a cup plant (Silphium perfoliatum L.) Polysaccharide and its antioxidant and hypoglycemic activities

The optimum enzyme-assisted extraction conditions of cup plant (Silphium perfoliatum L.) polysaccharides (CPP) were obtained by response surface methodology (RSM), and were found to be an enzymatic hydrolysis time of 66 min, a liquid-solid ratio of 22 mL/g, pH 5.1, and an enzyme-complex dosage of 1.59 %, allowing a CPP yield of 13.69 %. DEAE-52 cellulose and Sepharose CL-6B columns were used to purify the CPP. A purified polysaccharide component, CPP-1.2, was obtained and further analyzed for its characteristics, antioxidant properties, and hypoglycemic activity. The monosaccharide composition of CPP-1.2 was mannose, galacturonic acid, glucose, galactose, and arabinose with a molar percentage ratio of 2.24:16.34:44.17:24.28:12.97. The 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2-Azino-bis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) radical scavenging abilities of CPP-1.2 (1.0 mg/mL) reached 95.20 % and 93.69 % that of ascorbic acid (1.0 mg/mL), respectively. Moreover, CPP-1.2 presented significantly higher inhibitory properties on the activities of α-amylase and α-glucosidase than acarbose at each concentration tested. These results suggest that CPP-1.2 possesses the potential to be used as an antioxidant and hypoglycemic agent.

Purification and characterization of chitinase from Paenibacillus sp. .

The chitinase-producing bacteria Paenibacillus sp. was isolated from soil samples. The chitinase was purified successively by ammonia sulfate fractional precipitation followed by chromatography on DEAE 52-cellulose column and then on SephadexG-75 column. The chitinase has a molecular weight of ca. 30 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Its optimum pH is 4.5, and its optimum temperature is 50°C with colloidal chitin as a substrate. The enzyme is stable below 45°C and in pH ranges between 4.5 and 5.5. It is activated by glucosamine, glucose, N-acetylglucosamine, and metal ions including Ca2+ , Fe2+ , Fe3+ , and Ni2+ . It is inhibited by SDS, H2 O2 , ascorbic acid, Cu2+ , Mg2+ , Ba2+ , Sn2+ , Cr3+ , and K+ . With colloidal chitin as substrate, the Km and the Vmax of the chitinase are 4.28 mg/mL and 14.29 μg/(Min·mL), respectively, whereas the end products of the enzymatic hydrolysis are 14.33% monomer and 85.67% dimer of N-acetylglucosamine. The viscosity of carboxymethylchitin decreased rapidly at the initial stages when subjected to chitinase hydrolysis, which indicates that the chitinase acts in an endosplitting pattern.

Extraction, purification, emulsifying property, hypoglycemic activity, and antioxidant activity of polysaccharides from comfrey

Performing the researches on extraction conditions, purification and activities of polysaccharides are of great significance to the development of comfrey (Symphytum officinale L.) resources. In this study, the optimum extraction conditions of polysaccharides from comfrey (CPs) were obtained by response surface methodology (RSM), which were liquid to solid ratio 25 mL/g, extraction temperature 89 °C, and extraction time 155 min. A CPs yield of 19.20 % was obtained under these conditions. DEAE-52 cellulose and Sepharose CL-6B columns were used to purify CPs, and the purified polysaccharide component CPs-1.2 was obtained. The characteristics, emulsifying property, and hypoglycemic activity of CPs-1.2 were analyzed. The monosaccharide compositions of CPs-1.2 were galacturonic acid, arabinose, glucose, and galactose with the molar ratio of 1.00:0.88:2.28:1.13. CPs-1.2 showed notable DPPH and ABTS radical scavenging abilities, which could reach 95.00 % and 96.71 % of those of ascorbic acid at the determination concentrations of 0.4 mg/mL and 1.5 mg/mL, respectively. The α-glucosidase inhibitory activity of CPs-1.2 (10 mg/mL) could reach 80.00 % of that of acarbose. In addition, CPs-1.2 showed good emulsifying properties for olive oil, benzene, xylene and methylbenzene. These results suggested that CPs possessed potential as a hypoglycemic agent, an antioxidant or a polysaccharide-based emulsifier.

Structural characterisation and immunomodulatory activity of exopolysaccharides from liquid fermentation of Monascus purpureus (Hong Qu)

As a traditional Chinese medicinal and edible fungus, Monascus has been widely studied for its secondary metabolites such as Monascus pigments and γ-aminobutyric acid. However, the exopolysaccharides have not been extensively studied and this has limited their food and industrial applications. In this study, exopolysaccharides isolated from the liquid fermentation of Monascus purpureus (Hong Qu) were successfully separated into two fractions (EPS-1 and EPS-2) by DEAE-Cellulose column chromatography. A possible pigment-exopolysaccharides complex was identified. The molecular structure of the polysaccharides portion was systematically investigated using the combined techniques of FT-IR, monosaccharide analysis, methylation analysis and 1D & 2D NMR. Monosaccharide compositional analysis showed that EPS-1 was composed of mannose, glucose and galactose in the molar ratio of 8:1:11, while EPS-2 consisted of six different monosaccharides, namely, rhamnose, arabinose, xylose, mannose, glucose and galactose, with the molar ratio of 2.2:1.9:1:5.6:1.9:7.7, respectively. Moreover, the backbone of EPS-1 mainly consisted of 5-β-D-Galf (31.74 wt%) and 2-β-D-Manp (17.48 wt%) with other sugar residues including 2,6-β-D-Galf, T-α-D-Manp, 6-α-D-Manp and 3,5-β-D-Galf also detected at small percentages. Furthermore, the immunomodulatory activity of EPS-1 was investigated by RAW 264.7 cell lines. The cell proliferation and pinocytosis activity, cytokines releasing, NO production and related mRNA levels all have been determined. The results indicated that EPS-1 could significantly promote cytokines secretion including IL-6, TNF-α, and IL-10 and improve expression levels of the related mRNA.

Effects of freeze drying and hot-air drying on the physicochemical properties and bioactivities of polysaccharides from Lentinula edodes

Fresh Lentinula edodes were dried using two process technologies—freeze drying (FD) and hot-air drying (HD). The physicochemical, antioxidant and immunomodulatory properties of purified polysaccharides from dried L. edodes (LEP) were then comparatively investigated. Two neutral polysaccharides (FLEP-1 and HLEP-1) and two acidic polysaccharides (FLEP-2 and HLEP-2) were obtained by DEAE-52 cellulose column. The HD treated LEP had higher levels of uronic acid than that of the FD treated LEP. The molar ratios of monosaccharides in FLEP-1, FLEP-2, HLEP-1 and HLEP-2 were different. Moreover, HD treated LEP had more galactose and less glucose. The (1 → 3)-α-glucan structure was dominant in the two neutral polysaccharides, whereas the (1 → 6)-β-glucan was dominant in the two acidic polysaccharides. Hot-air drying could thus promote the α-configuration in neutral polysaccharides while reducing the β-configuration in acidic polysaccharides. FLEP-1, FLEP-2, HLEP-1 and HLEP-2 had potential scavenging capacity against the ABTS+, whereas freeze-dried polysaccharides exhibited a stronger scavenging ability than that of hot-air dried polysaccharide. LEP could improve immunity by inducing the secretions of NO, TNF-α and IL-6, whereas hot-air drying improved the immunomodulatory activity of LEP. Our results suggested that freeze drying and hot air drying could be appropriately used to obtain functional polysaccharides from L. edodes.

Purification, characterization and anticancer activities of exopolysaccharide produced by Rhodococcus erythropolis HX-2

In the present study, an exopolysaccharide (EPS) producer Rhodococcus erythropolis HX-2 was isolated from Xinjiang oil field, China. The HX-2 EPS (name HPS) production reached 8.957 g/L by RSM in MSM medium. The HPS was purified by ethanol precipitation and fractionation by DEAE-Cellulose and Sepharose column, the yield of HPS was 3.736 g/L. HPS composed by glucose, galactose, fucose, mannose and glucuronic acid. FT-IR spectroscopy indicated the presence of a large amount of hydroxyl groups. NMR spectroscopy indicated the existence of both α and β-configuration for sugar moieties present in HPS. The degradation temperature (255.4 °C) of the HPS was determined by thermogravimetric analysis (TGA). A reticular structure of HPS was observed by SEM and the AFM analysis of the HPS revealed straight chains line. Meanwhile, the WSI and WHC of HPS were 92.15 ± 3.05% and 189.45 ± 5.65%, respectively. Finally, In vitro anticancer activity purified EPS was evaluated on L929 normal cells, A549 cancer cells, SMMC-7721 liver cancer cells and Hela cervical cancer cell. HPS inhibited the growth of cancer cells in a certain concentration without damage to normal cells. These characteristics indicate that its potential application value in food, industry and pharmaceutical application.

Physicochemical Characterization and Immunomodulatory Activity of a Novel Acid Polysaccharide from Solanum muricatum.

To investigate the structure and immunomodulatory activity of polysaccharide from Solanum muricatum, a novel acid polysaccharide named SMP-3a was purified from Solanum muricatum pulp through DEAE-52 cellulose column and Sephadex G-200 chromatography. Monosaccharide composition analysis showed that SMP-3a was mainly composed of rhamnose, arabinose, galactose, and galacturonic acid with the molar ratio of 1.09:2.64:1.54:1. The average molecular weight was found to be 227 kDa by high performance gel permeation chromatography (HPGPC). Thermal studies revealed the SMP-3a was a thermally stable polymer. Based on the results of methylation and NMR analysis, the backbone chain of SMP-3a was composed of →2)-α-l-Rhap-(1→, →4)-α-d-GalpA-(1→ and →4)-α-d-Galp-(1→. The side chain was consisted of α-l-Araf-(1→ and →5)-α-l-Araf-(1→. Immunomodulatory assay indicated that SMP-3a could significantly promote the proliferation of macrophages and stimulate the secretion of cytokines, including TNF-α, IL-1β, and IL-6. Our results suggested that SMP-3a could be used as a novel potential immunomodulatory agent in functional food.

Preparation of chondroitin sulfates with different molecular weights from bovine nasal cartilage and their antioxidant activities

Biological functions of chondroitin sulfate, including anti-oxidation and anti-inflammation, are associated with its molecular weight. This study aimed to evaluate the correlation between antioxidant activity and molecular weights of chondroitin sulfate derived from bovine nasal cartilage (BCS). BCS extracted by compound enzymatic method was further purified via DEAE-cellulose column separation to obtain BCS-II (129.4 kDa), which was further degraded by H2O2-Vc to obtain four subfractions: BCS-II-1 (92.7 kDa), BCS-II-2 (54.1 kDa), BCS-II-3 (26.3 kDa), and BCS-II-4 (19.7 kDa). Changes in the physicochemical properties of BCS-II before and after degradation were compared via FT-IR, NMR and monosaccharide composition analysis. Finally, antioxidant activities of BCS-II and its subfractions BCS-II-1–4 were compared. Our results showed that the H2O2-Vc system did not disrupt the primary functional group of BCS-II, with no significant change in sulfate content between BCS-II and its degraded fractions; however, uronic acid levels increased in degraded fractions when compared with BCS-II. In vitro, BCS-II-4 displayed the lowest molecular weight and had the strongest antioxidant activity. Therefore, the antioxidant activity of chondroitin sulfate in vitro is robustly associated with its molecular weight, and low-molecular-weight chondroitin sulfate can be used as an antioxidant in the food and pharmaceutical industries and other sectors.

Structure and anti-tumor activity of a polysaccharide from Bletilla ochracea Schltr.

The polysaccharide fraction of Bletilla ochracea (BOP) was isolated from its tubers, and purified by DEAE-52 Cellulose and Sephadex G-200 column chromatography. The structural features of BOP were analyzed by GC–MS, HPLP, FT-IR, methylation and NMR. The average molecular weight of BOP was approximately 4.9 × 105 Da, and the monosaccharide composition was 7.88:2.12 mix of mannose and glucose. The FT-IR and NMR analyses revealed the following linkage: [→(4-β-D-Man-1)4 → (4-β-D-Glc-1)2 → 4-2-O-acetyl-β-D-Man-1 → 4-β-D-Man-1 → 4-3-O-acetyl-β-D-Man-1 → 4-2-O-acetyl-β-D-Man-1→]. Although BOP had no effect on the proliferation rate of CT26 colon carcinoma cells, it significantly inhibited tumor xenograft growth in vivo by stimulating CD4+ T cell expansion in the spleen of the tumor-bearing mice. Taken together, BOP is a potent immunomodulatory agent that can be considered for anti-tumor therapy.

Extraction, isolation, immunoregulatory activity, and characterization of Alpiniae oxyphyllae fructus polysaccharides

For further applications of Alpiniae oxyphyllae fructus in modern clinical medicine, Alpiniae oxyphyllae fructus polysaccharide (AOFP) was studied in the present work. The extraction conditions of AOFP were optimized by the response surface method with a Box-Behnken design. The maximum extraction rate of AOFP was 3.18%. An anion-exchange DEAE-52 cellulose column and a Sephadex G-100 gel column were used to isolate the AOFP, and three polysaccharides (AOFP1, AOFP2, AOFP3) were obtained. All three polysaccharides possessed immunoregulatory activity, but the effects of AOFP1 were greater than the other two polysaccharides. AOFP1 significantly stimulated Th1- and Th2-type immune responses and specific immune responses. Meanwhile, the characterization of AOFP1 was studied. AOFP1 was composed of arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid at a molar ratio of 16.46:12.7:4.9:17.11:4.35:6.52:6 with an average molecular weight of 43.4 kDa. These results suggest that AOFP1 can be developed as a natural immunomodulatory drug.

Two alanine aminopeptidase isoenzymes from the developing embryos of the camel tick Hyalomma dromedarii, Koch 1884 (Acari: Ixodidae)

ABSTRACTThe specific activity of alanine aminopeptidase was monitored during embryonic development of the camel tick H. dromedarii and a lag period was observed during the first six days. The specific activity was increased gradually until days 15–16 and the maximum level was recorded at age 24–25 days. Two isoenzymes of alanine aminopeptidase were resolved and designated AAP1 and AAP2 and were purified from 24–25 day old eggs of H. dromedarii by chromatography on a DEAE cellulose column and gel filtration on a Sephacryl S-300 column. The purification of AAP1 and AAP2 were increased 9.8-fold and 8.75-fold over the crude extract with recovery percentages of 30.54% and 18.0%, respectively. The molecular weight of both isoenzymes was detected to be 75 ± 2 kDa and the optimum activity of both isoenzymes was found at pH 8. MgCl2 activated AAP1, while MnCl2 and CoCl2 activated AAP2, whereas Bestatin and DTT strongly inhibited AAP1 and AAP2, respectively. Two different isoelectric points were detected at pH 8.2–8.4 and 6.6–6.8 for AAP1 and AAP2, respectively. Both AAP1 and AAP2 isoenzymes did not hydrolyse the synthetic and natural substrates of endopeptidases, indicating the absence of endopeptidase activity. Although both isoenzymes are similar, the activators, inhibitors, and isoelectric point values distinguish them.