Size-exclusion Chromatography Combined With Laser Light Scattering Research Articles

Isolation and Structural Characterization of a Second Polysaccharide from Bulbs of Lanzhou Lily.

In this study, a second water-soluble polysaccharide (designated as LDP-2) was isolated from the bulbs of Lanzhou Lily (Lilium davidii var. unicolor). Based on monosaccharide composition and methylation analysis, its structural features were investigated using a variety of characterizations, such as size-exclusion chromatography combined with laser light scattering (SEC-LLS), gas chromatography-mass spectroscopy (GC-MS), infrared (IR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. The results demonstrate that LDP-2 is a homogeneous heteropolysaccharide (molecular weight 6.2 × 104Da), which includes four kinds of monosaccharides (Lyx, Man, Glc, and Gal in an approximate weight ratio of 6.74: 6.28: 76.50: 10.48). Furthermore, its morphology, thermal behavior, and preliminary hypoglycemic activities were also investigated.

Optimization of reaction conditions by RSM and structure characterization of sulfated locust bean gum

Sulfated derivatives of galactomannan from locust bean gum (LBG) with the degree of substitution (DS) of 0.34–1.07 were synthesized using chlorosulfonic acid/pyridine (CSA/Py) method. Box–Behnken design (BBD) of response surface methodology (RSM) was employed to optimize the reaction conditions. Results of FT-IR and X-ray photoelectron spectroscopy (XPS) indicated that SO3H groups were widely present in sulfated LBG (SLBG). 13C NMR result revealed that sulfation had occurred and C-6 substitution was predominant in SLBG. All sulfated samples showed a decrease in Mw and more broad molar mass distribution in size exclusion chromatography combined with laser light scattering (SEC-LLS) analysis. Results of MW – 〈S2〉z1/2 showed a decrease in fractal dimension (df) value. Laser light scattering results also showed a conformation transition from a compact chain conformation of branched clusters to a random coil conformation of SLBG. Compared to LBG and SLBG with low DS and molecular weight, SLBG2 exhibited an internal structure of random coil with a DS of 1.07. DS and molecular weight had great influence on its conformation in aqueous solution. Our results confirmed that the degradation of polysaccharide and SO3H groups improved significantly the stiffness of the chains due to the electrostatic effect.

Determination of molecular size and shape of hyperbranched polysaccharide in solution

The chemical structure of a water-soluble polysaccharide, coded as TM3b, extracted from sclerotia of Pleurotus tuber-rigium was analyzed to be a hyperbranched beta-D-glucan with beta-(1-->6), beta-(1-->4), and beta-(1-->3)-linked residues, with degree of branching (DB) of 57.6%. The results from size-exclusion chromatography combined with laser light scattering (SEC-LLS) revealed that the hyperbranched polysaccharide easily aggregated in 0.15 M aqueous NaCl, whereas it dispersed as individual chains in DMSO. The weight-average molecular weight (M(w)), radius of gyration, intrinsic viscosity, and chain density of TM3b in DMSO and in 0.15 M aqueous NaCl were measured with SEC-LLS, LLS, and viscometry. The results indicated that single chains and aggregates with aggregation number of 12 coexisted in the aqueous solution, whereas individual molecules of TM3b occurred in DMSO. In view of the molecular parameters, the aggregates in aqueous solution exhibited more compact chain structure than the individual molecules in DMSO. Furthermore, transmission electron microscopy and atomic force microscopy showed that all of the aggregates and individual molecules exhibited spherical particles in the solutions. This work provided the valuable information of chain conformation and molecular morphology of the hyperbranched polysaccharide in different solvents.

Chain conformation of carboxymethylated derivatives of (1 → 3)-β- d-glucan from Poria cocos sclerotium

A water-insoluble (1 → 3)-β- d-glucan (PCSG) isolated from the fresh sclerotium of Poria cocos was carboxymethylated to afford a water-soluble derivative coded as C-PCSG. The carboxymethylated (1 → 3)-β- d-glucan was fractionated to obtain eight fractions according to the nonsolvent addition method. The weight-average molecular mass ( M w), radius of gyration ( 〈 s 2 〉 z 1 / 2 ) and intrinsic viscosity ([ η]) of the fractions were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS) and viscometry in 0.2 M NaCl aqueous solution at 25 °C. The dependences of [ η] and 〈 s 2 〉 z 1 / 2 on M w for C-PCSG were found to be [ η ] = 1.49 × 10 - 2 M w 0.75 ( mL g - 1 ) , and 〈 s 2 〉 z 1 / 2 = 3.65 × 10 - 2 M w 0.56 (nm), respectively. Analysis of M w and [ η] in terms of the known theories for wormlike chain model yielded 633 nm −1 for molar mass per unit contour length ( M L), 5.5 nm for persistence length ( q), and 20.2 for characteristic ratio ( C ∞). These results indicated that C-PCSG exists as a relatively extended flexible chain in 0.2 M NaCl aqueous solution. Therefore, the introduction of the carboxymethyl groups into the β-glucan improved significantly the water solubility and enhanced the stiffness of the chains.

Antitumor Activities ofO-Sulfonated Derivatives of (1→3)-α-D-Glucan from DifferentLentinus edodes

Four water-insoluble (1-->3)-alpha-D-glucans, coded L-II1, L-II2, L-II3 and L-II4, with different molecular weights were isolated from four kinds of fruiting bodies of Lentinus Edodes. The four alpha-D-glucans were O-sulfonated to obtain derivatives (SL-II) having degrees of substitution (DS) from 0.9 to 2.1 respectively. The structure of the samples was analyzed by infrared spectra, elemental analysis, and 13C NMR. The weight-average molecular weight (Mw), radii of gyration (<s2>z1/2) and intrinsic viscosity ([eta]) of the native alpha-D-glucans and O-sulfonated derivatives were measured by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in 0.2 M aqueous NaCl and in dimethyl sulfoxide (DMSO) containing 0.25 M LiCl at 25 degrees C respectively. The Mw values of the O-sulfonated derivatives were much lower than those of the native alpha-D-glucans. The experimental results indicate that the O-sulfonated derivatives are water-soluble and exist as an expanded flexible chain in aqueous solution owing to intramolecular hydrogen bonding or interaction between charge groups. The in vivo and in vitro antitumor activities of the native alpha-D-glucans and their O-sulfonated derivatives against solid tumor Sarcoma 180 cells were evaluated and compared. Interestingly, all of the O-sulfonated derivatives exhibited higher antitumor activities than those of the native glucans. The results reveal that the effect of O-sulfonation of the alpha-D-glucan on the improvement of their antitumor activities was considerable.

Solution properties of (1→3)‐α‐D‐glucan and its sulfated derivative from Poria cocos mycelia via fermentation tank

From Poria cocos mycelia yielded via a pilot scale facility-fermentation tank, a water-insoluble (1-->3)-alpha-D-glucan coded as Pi-PCM3-I was isolated by extraction with 0.5 M NaOH/0.01 M NaBH(4) aqueous solution. Nine fractions from F1 to F9 with a weight-average molecular mass (M(w)) range from 7.75 x 10(4) to 57.3 x 10(4) were prepared from the Pi-PCM3-I sample by a nonsolvent addition method. The fractions were reacted with chlorosulfonic acid-pyridine complex to product water-soluble sulfated derivatives coded as S1 to S8 with M(w) from 2.36 x 10(4) to 14.5 x 10(4) and degree of substitution (DS) of 0.86-1.38. M(w), z-average radius of gyration (s(2) (z) (1/2)), the second virial coefficient (A(2)), and the intrinsic viscosity ([eta]) of the native and sulfated Pi-PCM3-I were measured by laser light scattering (LLS), size-exclusion chromatography combined with LLS (SEC-LLS), and viscometry at 25 degrees C. The Mark-Houwink equations for Pi-PCM3-I in 0.25 M LiCl/dimethylsulfoxide (DMSO) (Me(2)SO) and for its sulfated derivative in 0.15 M NaCl aqueous solution at 25 degrees C were established to be [eta] = 1.33 x 10(-2) M(w) (0.75+/-0.01) (mL g(-1)) and [eta] = 1.46 x 10(-4) M(w) (1.13+/-0.01) (mL g(-1)), respectively. On the basis of theories for a wormlike cylinder model, the conformational parameters of the native and sulfated Pi-PCM3-I were calculated to be 760 +/- 50 and 1060 +/- 30 nm(-1) for the molar mass per unit contour length (M(L)), 6.3 +/- 0.5 and 13.1 +/- 1 nm for the persistence length (q), and 14.9 +/- 0.2 and 31.8 +/- 1 for the characteristic ratio (C( proportional, variant)), respectively. The results revealed that Pi-PCM3-I existed as an extended flexible chain in 0.25 M LiCl/Me(2)SO, and its sulfated derivative existed as a semistiff chain in 0.15 M NaCl aqueous solution. Furthermore, Pi-PCM3-I possessed similar structure and molecular parameters to wc-PCM3-I from a rotary shaker; this suggests promising industrialization of Poria cocos polysaccharides.

Molecular architectures of four‐arm star‐shaped styrene‐butadiene copolymer

AbstractTo understand the molecular architectures of styrene‐butadiene four‐arm star (SBS) copolymers, a size exclusion chromatography combined with laser light scattering (SEC‐LLS) has been used to determine their weight‐average molecular weight (Mw) and radius of gyration (〈S2〉1/2), and a new method for the establishment of the Mark‐Houwink equation from one sample has been developed. Based on the Flory viscosity theory, we successfully have reduced the 〈S2〉1/2 values of numberless fractions estimated from many experimental points in the SEC chromatogram to intrinsic viscosities ([η]). For the first time, the dependences of 〈S2〉1/2 and [η] on Mw for the four‐arm star SBS in tetrahydrofuran at 25°C were found, respectively, to be 〈S2〉1/2 = 2.62 × 10−2 M (nm) and [η] = 3.68 × 10−2 M (mL/g) in the Mw range from 1.4 × 105 to 3.0 × 105. From data of [η] and 〈S2〉1/2 for linear and star SBS, we have obtained the information about the branching, namely, the ratios (g and g′) of 〈S2〉 and [η] for star SBS to that of the linear SBS of the same molecular weight, which agree with theoretical predictions. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 961–965, 2005

Effect of Molecular Mass on Antitumor Activity of Heteropolysaccharide fromPoria cocos

A water-soluble heteropolysaccharide ac-PCM0 from Poria cocos was successfully fractionated using a preparative size exclusion chromatography (SEC) column, and their weight-average molecular mass (M(w)) was characterized by analytical SEC combined with laser light scattering (SEC-LLS). The results indicate that the fractions having relatively high M(w) exhibited higher inhibition ratio in vivo antitumor activity than those having M(w) below 3.29 x 10(4). However, the relatively low molecular mass was beneficial to the in vitro antitumor activity. Moreover, ac-PCM0 has a significantly higher enhancement ratio of the body weight than 5-fluorouracil, and its 50% lethal dose is above 1250 mg/kg, indicating a nontoxic nature.

Structure and antitumor activities of the water-soluble polysaccharides from Ganoderma tsugae mycelium

Six water-soluble polysaccharides coded as GTM1 to GTM6 were extracted sequentially from the mycelium of Ganoderma tsugae with 0.2 M sodium phosphate buffer solution at 25, 40, and 80 °C, water at 120 °C, 0.5 M sodium hydroxide at 25 and 65 °C. The chemical structures were determined by using IR, EA, GC and 13C NMR. The weight-average molecular mass ( M w) was characterized by size exclusion chromatography combined with laser light scattering (SEC-LLS). The results indicated that the samples GTM1 and GTM2 were heteropolysaccharide–protein complexes with the protein content of 13.5 and 20.1%, and apparent-mean M w of 62.8×10 4, 81.8×10 4, respectively. GTM3 and GTM4 contained (1→3)-β- d-glucans and (1→4)-α- d-glucans, while GTM5 and GTM6 were mainly a (1→6)-branched (1→3)-β- d-glucan. Two peaks were found in the SEC patterns of the four samples GTM1 to GTM4, representing fractions 1 composed of branched (1→4)-α- d-glucan with high M w and fraction 2 composed of galactose, glucose and mannose with relatively low M w. With the progress of isolation, the content of fraction 2 decreased from 90.2 to 57.1%, accompanying with enhancing antitumor activity. The polysaccharides GTM1, GTM2 and GTM3 had significantly higher antitumor activity against solid tumor Sarcoma 180 with the inhibition ratio beyond 50%. The results suggested that the effects of the moderate content of galactose and bound protein, as well as relatively lower M w, on the improvement of antitumor activities of polysaccharides could not be negligible.

Comparison on chain stiffness of a water-insoluble (1→3)-α- d-glucan isolated from Poria cocos mycelia and its sulfated derivative

A water-insoluble (1→3)-α- d-glucan (ab-PCM3-I) isolated from the Poria cocos mycelia by extracting with 0.5 sodium hydroxide was fractionated into ten fractions by non-solvent addition method in 0.25 M lithium chloride in dimethylsulfoxide. Seven fractions were treated with chlorosulfonic acid-pyridine complex to synthesis water-soluble sulfated derivatives (S-ab-PCM3-I) with degrees of substitution in the range 0.35–0.81. The solution behaviours of the α- d-glucan and its derivatives were studied by laser light scattering (LLS), size exclusion chromatography combined with laser light scattering (SEC-LLS) and viscometry at 25 °C. The dependences of intrinsic viscosity ([ η]) on weight-average molecular mass ( M w) for the native glucan and the sulfated derivatives were found to be [ η]=9.92×10 −3 M w 0.77 (cm 3 g −1) in the range of M w from 1.08 to 23.1×10 4 and [ η]=2.92×10 −4 M w 1.09 (cm 3 g −1) in the range of M w from 0.74 to 5.80×10 4, respectively. On the basis of current theories for a wormlike chain model, the conformation parameters of the samples ab-PCM3-I and S-ab-PCM3-I were calculated to be 584 and 834 nm −1 for the molar mass per unit contour length ( M L), 3.7 and 6.8 nm for the persistence length ( q), 11.6 and 18.1 for characteristic ratio ( C ∝), respectively. The results indicated that the α- d-glucan ab-PCM3-I exists as an extended random coil chain in 0.25 M lithium chloride in dimethylsulfoxide, and that the sulfated derivatives S-ab-PCM3-I has good water-solubility and exist as semi-stiff chains in 0.9% w/v aqueous sodium chloride, owing to the steric hindrance and electrostatic repulsion of the sulfate groups.

Correlation of structure to antitumor activities of five derivatives of a β-glucan from Poria cocos sclerotium

A water-insoluble (1→3)-β- d-glucan isolated from fresh sclerotium of Poria cocos was, respectively, sulfated, carboxymethylated, methylated, hydroxyethylated, and hydroxypropylated, to afford five water-soluble derivatives. Their weight-average molecular masses ( M w) and intrinsic viscosities ([ η]) were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in phosphate buffer solution (PBS) at 37 °C. The antitumor activities, against Sarcoma 180 tumor cell (S-180) and gastric carcinoma cell strain (MKN-45 and SGC-7901) of the native β-glucan and the five derivatives, were tested in vitro and in vivo. The M w values of the five derivatives in PBS were determined to be 3.8 × 10 4, 18.9 × 10 4, 16.0 × 10 4, 76.8 × 10 4, and 224.3 × 10 4, respectively. The high M w values of the hydroxyethylated and hydroxypropylated derivatives in aqueous solution resulted from aggregation, and their true M w values obtained in dimethyl sulfoxide were 20.1 × 10 4 and 19.1 × 10 4. The sulfated and carboxymethylated derivatives having DS of 1.0–1.3 show good water solubility, and exist as relatively expanded chains in aqueous solution. Interestingly, the native β-glucan did not show antitumor activity, whereas the sulfated and carboxymethylated derivatives exhibit significant antitumor activities against S-180 and gastric carcinoma tumor cells. This work showed that good water solubility, relatively high chain stiffness, and moderate molecular mass of the derivatives in aqueous solution contribute beneficial to enhancement of antitumor activity.

Chemical components and molecular mass of six polysaccharides isolated from the sclerotium of Poria cocos

Six polysaccharides were extracted sequentially from the fresh sclerotium of Poria cocos cultivated in China using 0.9% NaCl (PCS1), hot water (PCS2), 0.5 M NaOH (PCS3-I and PCS3-II), and 88% formic acid (PCS4-I and PCS4-II). Their chemical and physical characteristics were determined using infrared spectroscopy (IR), gas chromatography (GC), GC–MS methylation analysis, 13C NMR spectroscopy, elementary analysis (EA), protein analysis, size exclusion chromatography combined with laser light scattering (SEC-LLS), light scattering (LS), and viscometry. The results indicated that the polysaccharides PCS1, PCS2, and PCS3-I were heteropolysaccharides containing d-glucose, d-galactose, d-mannose, d-fucose, and d-xylose; the predominant monosaccharide was d-glucose except for PCS1 where it was d-galactose. PCS3-II, the main component of the sclerotium of P. cocos, was a linear (1 → 3)-β- d-glucan of high purity. PCS4-I consisted of (1 → 3)-β- d-glucan with some β-(1 → 6) linked branches. PCS4-II was mainly composed of (1 → 3)-β- d-glucan containing some glucose branches. The M w values of the six polysaccharides PCS1, PCS2, PCS3-I, PCS4-I in 0.2 M NaCl aqueous solution, PCS3-II, and PCS4-II in dimethyl sulfoxide (Me 2SO) were determined to be 11.6 × 10 4, 20.8 × 10 4, 17.1 × 10 4, 9.1 × 10 4, 12.3 × 10 4, and 21.1 × 10 4, respectively. The six polysaccharides in aqueous solution or Me 2SO exist as flexible chains.

Chain conformation of sulfated derivatives of β-glucan from sclerotia of Pleurotus tuber-regium

Six water-insoluble (1→3)-β- d-glucan fractions TM8-1 to TM8-6 with weight-average molecular mass M w ranging from 5.76 to 77.4×10 4 obtained from the sclerotia of Pleurotus tuber-regium were sulfated to produce the water-soluble fractions S-TM8-1 to S-TM8-6 with M w from 6.0 to 64.8×10 4. The degree of substitution (DS) of S-TM8 fractions was analyzed by elemental analysis (EA) to be 1.14–1.74. The 13C NMR results indicated that the C-6 was fully substituted, and C-2, C-4 were partially substituted by the sulfo-groups. The M w and the intrinsic viscosity [ η] of the S-TM8 fractions were measured, respectively, by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS and viscometry in phosphate buffer solution (PBS) at 37 °C. The dependences of [ η] and radius of gyration 〈 s 2〉 z 1/2 on M w for the S-TM8 samples were found to be [ η]=1.89×10 −2 M w 0.70 (cm 3/g) and 〈 s 2〉 z 1/2=1.12×10 −4 M w 0.81 (nm) in the M w range tested. Based on current theories for a wormlike chain model, the molar mass per unit contour length M L and persistence length q of the S-TM8 were calculated to be 990 nm −1 and 8.5 nm, respectively. The relatively higher q value suggested a more expanded flexible chain of S-TM8 in PBS. The water-solubility and relatively expanded chain conformation of the STM8 fractions were considered to be significant to their antiviral activity.

Structure and antitumor activity of extracellular polysaccharides from mycelium

Two heteropolysaccharides coded as EPF1 and EPF2 were obtained from the crude extracellular polysaccharide (CEP) of Ganoderma tsugae mycelium by elution with DEAE-Sepharose CL-6B column. Their chemical structures and molecular mass were characterized by infrared, gas chromatography, 13C NMR and size exclusion chromatography combined with laser light scattering (SEC-LLS). The results indicated that they were mainly composed with mannose, fucose, xylose, galactose and glucosamine. EPF2 was confirmed to be mainly a β- d-galacto-α- d-mannan. The weight-average molecular mass of the samples EPF1, EPF2 and CEP were 92.0×10 4, 8.35×10 4 and 23.8×10 4, respectively. The EPF1 existed as a compact coil chain in 0.2 M NaCl aqueous solution at 25 °C, while EPF2 as a flexible chain. The antitumor activities against Sarcoma 180 were tested both in vitro and in vivo. All the samples exhibited high inhibition ratio against Sarcoma 180 in mice, and the CEP has higher inhibition effect both in vivo and in vitro than EPF1 and EPF2. In view of these results, the presence of bound protein and mannose, and relatively expanded chain would be helpful to the enhancement of antitumor activities. This study suggested that the extracellular polysaccharides had a potential application as natural antitumor drugs.

Characterization of a polysaccharide–protein complex from Ganoderma tsugae mycelium by size-exclusion chromatography combined with laser light scattering

A water-soluble polysaccharide–protein complex (GM3) extracted from the mycelium of Ganoderma tsugae was characterized using size-exclusion chromatography combined with laser light scattering (SEC-LLS). Two peaks coded as fractions I and II appeared in the SEC pattern of GM3 in 0.5 M NaCl aqueous solution, corresponding to the weight-average molecular mass ( M w) of 355×10 4 and 6.3×10 4, respectively. The relationship between the radius of gyration (< s 2> z 1/2) and M w showed that molecules of fraction I exhibited more compact coil conformation than that of fraction II in 0.5 M NaCl aqueous solution at 25 °C. To clarify the component of polysaccharide and protein in each fraction, the sample GM3 was treated with 0.2 M NaOH aqueous solution to degrade polysaccharide and trypsin to hydrolyze protein. The obtained products were analyzed by SEC combined with detectors such as UV, differential refractive index (DRI) and LLS. The results indicated that both the fractions I and II were protein-bound polysaccharide, but had different protein content and degree of branching, resulting in the difference of the chain conformation.

Fractionation and characterization of a Polysaccharide from the sclerotia of Pleurotus tuber-regium by preparative size-exclusion chromatography

A kind of regenerated cellulose gel (RCG) particles were treated by toluene to obtain particles with smaller mean pore size, then was mixed with the cellulose gel with pore size of 370 and 525 nm. A preparative size-exclusion chromatography (SEC) column (700×20 mm) packed with three gel particles was used to fractionate water-soluble polysaccharide (WEP) extracted from the sclerotia of Pleurotus tuber-regium by aqueous solution. The exclusion limit and fractionation range of the stationary phase of the preparative SEC were molecular mass 8×10 5 and 5×10 3 to 8×10 5, respectively. The calibration curve of the preparative SEC was represented as: log M=13.96–0.53 Ve. The WEP sample (weight-average molecular mass M w=2.2×10 4, polydispersity=2.4) was divided into three fractions with M w ranging from 1.4×10 4 to 3.4×10 4 by the preparative SEC column, and the fractions were characterized by gas chromatography GC, SEC combined with laser light scattering (SEC-LLS) and viscometry. The unfractionated WEP exhibited triple peaks due to different molecular mass, but each fraction exhibited single peak with the polydispersity of 1.1–1.8 in the SEC patterns. The results indicated that the preparative SEC was efficient for fractionation of polysaccharides having low molecular weight and for determination of their molecular mass.