Phenol-sulfuric Acid Assay Research Articles

A method for the quantitative analysis of Lycium barbarum polysaccharides (LBPs) using Fourier-transform infrared spectroscopy (FTIR): From theoretical computation to experimental application

Lycium barbarum polysaccharides (LBPs) is one of the most important active substances in Lycium barbarum (LB). It is a challenge to quantitatively determine the content due to their complex structures and lack of suitable reference standard in practice. In this study, a quantitative analysis method of LBPs in LB was established based on Fourier-transform infrared spectroscopy (FTIR). The stretching vibration of CO on the pyranose ring of saccharide at 921 cm−1 was selected as the characteristic absorption band by theoretical calculation, which can’t be impacted by the preparation methods and interfered by the component monosaccharides. The molecular weight CRM of dextran (Mw 63.3 kDa) served as the reference standard. The introducing internal standard (KSCN) can obtain a good precision (RSD = 1.10 %) and effectively compensate for the analysis errors caused by the environment, quality loss and uneven distribution during the tablet pressing processes. The methodological verification suggested that the method had good accuracy according to the recovery rate (96.61 %–105.45 %) and the blank recovery (92.39 %–99.37 %), respectively. The LOD and LOQ of CRMD were 0.10 mg and 0.32 mg, respectively. The polysaccharide content of LB from 24 different regions (0.50–2.54 %) and 10 batches of LB extracts (7.09–10.56 %) determined by the developed method less than the ones using phenol–sulfuric acid assay (1.95 %–4.83 % for LB and 9.83–15.53 % for extracts, respectively). The established method based on FTIR could be served as a supplement to phenol–sulfuric acid assay and a rapid quantitative assay for polysaccharides products. In additional, this study provided a new idea for the quantitative analysis of plant polysaccharides.

An easy-to-use platform for colorimetric determination of dextran: A potential application for the sugar industry

A user-friendly platform based on foam sheets and silica solid support was developed for colorimetric sensing of dextran via phenol–sulfuric acid assay. The chemical reagents, sulfuric acid and phenol were separately preserved by silica gel. The brown color product of the phenol–sulfuric acid reaction occurred on the silica solid support when sample solutions were added. The color intensity of brown products was easily obtained by a smartphone and color processing software. Subsequently, a broad concentration range of dextran could be determined up to 10,000 mg/L dextran, with a detection limit of 360 mg/L. Furthermore, a precision study, including inter-day and intra-day studies, presented a satisfactory performance for dextran detection. The developed platform was successfully applied for the sugar industry's dextran determination of syrup samples.

Effect of titanium dioxide on Streptococcus mutans biofilm.

Streptococcus mutans (S. mutans) participates in the dental caries process. Titanium dioxide (TiO2) nanoparticles produce reactive oxygen species capable of disrupting bacterial DNA synthesis by creating pores in cell walls and membranes. The objective of this study was to determine the effect of TiO2 on the disruption of S. mutans biofilm. This study was conducted in four phases involving a TiO2-containing toothbrush and TiO2 nanoparticles. Each phase was completed using 24 h established S. mutans biofilm growth. Phase one data was collected through a bacterial plating study, assessing biofilm viability. Biofilm mass was evaluated in phase two of the study by measuring S. mutans biofilm grown on microtiter plates following crystal violet staining. The third phase of the study involved a generalized oxygen radical assay to determine the relative amount of oxygen radicals released intracellularly. Phase four of the study included the measurement of insoluble glucan/extracellular polysaccharide (EPS) synthesis using a phenol-sulfuric acid assay. Both exposure time and time intervals had a significant effect on bacterial viability counts (p = 0.0323 and p = 0.0014, respectively). Bacterial counts after 6 min of exposure were significantly lower than after 2 min (p = 0.034), compared to the no treatment control (p = 0.0056). As exposure time increased, the amount of remaining biofilm mass was statistically lower than the no treatment control. Exposure time had a significant effect on oxygen radical production. Both the 30 and 100 nm TiO2 nanoparticles had a significant effect on bacterial mass. The silver nanoparticles and the 30 and 100 nm TiO2 nanoparticles significantly inhibited EPS production. The TiO2-containing toothbrush kills, disrupts, and produces oxygen radicals that disrupt established S. mutans biofilm. TiO2 and silver nanoparticles inhibit EPS production and reduce biofilm mass. The addition of TiO2 to dental products may be effective in reducing cariogenic dental biofilm.

Development of a high-throughput screening method for exopolysaccharide-producing Streptococcus thermophilus based on Congo red

As a thickening or stabilizing agent, exopolysaccharide (EPS) can significantly enhance food texture. However, the conventional analytical and screening methods of EPS have been unable to meet the current needs of industrial production, owing to low efficiency and heavy time-consumption. A simple and rapid qualitative/quantitative method is needed to accelerate the selection of high EPS-producing strains. Here, a high-throughput screening (HTS) program for EPS production was established in S. thermophilus by combining Congo red agar method (CRA, primary screening) and microplate colorimetric assay (MPC, secondary screening). The correlation coefficient (R2) between CRA/MPC and the phenol-sulfuric acid assay (a classical EPS quantification method) were 0.779 and 0.862, respectively, suggesting the feasibility of this HTS method. A mutant library (>300 colonies) of S. thermophilus was constructed using ribosome engineering strategy, and EPS-producing mutants with the titer from 50 mg/L to 200 mg/L were rapidly obtained by our developed HTS method, confirming that the HTS method is simple and efficient for the isolation of EPS-producing S. thermophilus. Taken together, this HTS method was developed for rapidly screening of EPS-producing S. thermophilus, which could be a valuable tool not only for the rapid detection of EPS production capabilities, but also for the screening of strain libraries from genetic engineering with desirable characteristics.

Characterization of Staphylococcus aureus biofilms via crystal violet binding and biochemical composition assays of isolates from hospitals, raw meat, and biofilm-associated gene mutants

Staphylococcus aureus (SA) is a gram-positive coccus and an opportunistic pathogen of humans. The ability of SA to form biofilms is an important virulence mechanism because biofilms are protected from host immune responses and antibiotic treatment. This study examines the relative biofilm strength of a variety of hospital and meat-associated strains of SA, using a crystal violet (CV) staining assay. Biofilms were treated with either DNase or proteinase K prior to CV staining, and compared to mock-treated results, to better understand the biochemical composition. Biofilm polysaccharide concentration was also measured using the phenol sulfuric-acid assay which was normalized to base biofilm strength. We found that hospital-associated isolates have biofilms that bind significantly more CV than for meat isolates and are significantly more protein and polysaccharide-based while meat isolates have significantly more DNA-based biofilms. This study also investigates the effects that biofilm-related genes have on biofilm formation and composition by analyzing specific transposon mutants of genes previously shown to play a role in biofilm development. agrA, atl, clfA, fnbA, purH, and sarA mutants produce significantly weaker biofilms (bind less CV) as compared to a wild-type control, whereas the acnA mutant produces a significantly stronger biofilm. Biofilms formed from these mutant strains were treated (or mock-treated) with DNase or proteinase K and tested with phenol and sulfuric acid to determine what role these genes play in biofilm composition. The acnA, clfA, fnbA, and purH mutants showed significant reduction in biofilm staining after either proteinase K or DNase treatment, agrA and sarA mutants showed significant biofilm reduction after only proteinase K treatment, and an atl mutant did not show significant biofilm reduction after either proteinase K or DNase treatment. These data suggest that biofilms that form without acnA, clfA, fnbA, and purH are DNA- and protein-based, that biofilms lacking agrA and sarA are mainly protein-based, and biofilms lacking atl are mainly polysaccharide-based. These results help to elucidate how these genes affect biofilm formation and demonstrate how mutating biofilm-related genes in SA can cause a change in biofilm composition.

Quantification of total sugars and reducing sugars of dragon fruit-derived sugar-samples by UV-Vis spectrophotometric method

In the present work, the phenol-sulfuric acid method and the 3,5-dinitrosalicylic acid (DNS) method were developed with the aim to quantitatively analyze total sugars and reducing sugars, respectively. In regard with the phenol-sulfuric acid assay, 1.0 mL of sample was treated with 1.0 mL of 5% phenol, 5.0 mL of concentrated H2SO4 and measured at 485 nm, with the linearity range of 10–100 ppm for total sugars. The DNS method was performed on 2.0 mL of sample, using 1.5 mL of DNS at 80 °C for 10 minutes and measured at 510 nm, with the linearity range of 50–400 ppm for reducing sugars. The sugar contents of white dragon fruit-derived sugar-samples (extracted from species in Binh Thuan province, Vietnam) were also estimated by the above measured methods, exhibiting the total sugars of above 90% and the reducing sugars of above 5%. The methods were well-performed with the acceptable relative standard deviations of repeatability in accordance with the Association of Official Analytical Chemists (AOAC).

Validation of a phenol-sulfuric acid method in a microplate format for the quantification of soluble sugars in ruminant feeds.

Soluble sugars in feeds are important for ruminant production; however, performing numerous sugar analyses within a short period is a laborious task. Here, we developed a phenol-sulfuric acid (PSA) assay in a microplate format to quantify soluble sugars in ruminant feeds. This method is easy and quick and requires only a small quantity of harmful reagents. We found that assay measurements were not affected by the representative organic acids and sugar alcohol contained in feeds. The treatment of activated charcoal with ethanol extract prior to the PSA assay was effective in removing interfering compounds for a more accurate determination of soluble sugars in certain feeds. Furthermore, the inter-day and intra-day repeatability of the present method was acceptable. Hence, we conclude that the method developed in this study is suitable for routine analysis of soluble sugars content in ruminant feeds.

Purification, characterization and utilization of polysaccharide of Araucaria heterophylla gum for the synthesis of curcumin loaded nanocarrier

In this study, gum of Araucaria heterophylla was collected. The collected gum was subjected for extraction of polysaccharide using solvent extraction system. Thus, extracted polysaccharide was further purified using solvent method and was characterized using UV–Vis spectroscopy, Phenol sulfuric acid assay, FTIR, TGA, TLC and GC–MS. The gum derived polysaccharide was found to have the following sugars Rhamnose, Allose, Glucosinolate, Threose, Idosan, Galactose and Arabinose. The extracted polysaccharide was tested for various in-vitro bioactive studies such as antibacterial activity, antioxidant activity and anticancer activity. The polysaccharide was found to have antioxidant and anticancer activity. Further, the polysaccharide was subjected for carboxymethylation to favor the nanocarrier synthesis, where it was chelated using Sodium Tri Meta Phosphate (STMP) to form nanocarriers. The nanocarriers so formed were loaded with curcumin and were characterized using FTIR, SEM, EDX and AFM. Both the loaded and unloaded nanocarriers were studied for its in-vitro cytotoxic effect against the MCF7 human breast cancer cell lines. The nanocarriers were found to deliver the drug efficiently against the cancer cell line used in this study.

Chemoenzymatic quantification for monitoring unpurified polysaccharide in rich medium.

The heparosan polysaccharide serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The previous quantification methods for heparosan rely on time-consuming purification or expensive instruments not readily available for many labs. Here, a chemoenzymatic approach is developed to monitor the production of heparosan in rich medium without purification. After removing the interfering small molecules by ultrafiltration, heparosan was decomposed into oligosaccharides using heparin lyase III. The oligosaccharides were separated from large molecules by ultrafiltration and quantitatively determined by the anthrone-sulfuric acid assay using a spectrophotometer. Based on the different substrate specificity of heparin lyases, the study showed that the concentration of heparosan and heparin in a mixture was discriminatively determined by the two-step chemoenzymatic assay. Furthermore, the anthrone-sulfuric acid assay was observed to be more reliable than the phenol-sulfuric acid assay under these conditions. Besides heparosan and heparin, the chemoenzymatic assay may be adapted to quantify other types of polysaccharides if the specific lyases were available.

Silver nano-particles: synthesis and characterization by using glucans extracted from Pleurotus ostreatus

The extensive use of antibiotics has led to drug-resistant bacteria, which is a major public health issue worldwide. Silver nano-particles have been recognized as efficient broad-spectrum antimicrobial agent. It uses mushroom glucans as reducing and capping agent to reduce its toxicity. In the present work, we performed the characterization of glucans and glucan–AgNPs. The crude glucans were extracted by hot water then fractionated and purified by anion exchange chromatography (DEAE-cellulose 52); the elution of glucans was monitored by phenol sulfuric acid assay. The major fractions were further purified with gel filtration chromatography (Sephadex-G200) then lyophilized. Green synthesis of silver nano-particles was performed by glucans from selected Pleurotus spps. Extracted glucans and synthesized glucan–AgNPs were confirmed by UV–Vis spectra at 190–250 nm and 380–420 nm, respectively. Size of glucan–AgNPs (100.1 nm) was determined by dynamic light scattering. The glucans were characterized by FT-IR, SEM and XRD while glucan–AgNPs were characterized by FT-IR, SEM–EDS, TEM and XRD. Based on FT-IR spectra, the functional groups associated with the glucans and glucan–AgNPs were determined. SEM analysis revealed that the structure of glucan was spongy with asymmetrical particle size while SEM–EDS/TEM analysis revealed the glucan–AgNPs were spherical with size range of 15–45 nm for Pleurotus ostreatus. XRD spectra of glucan showed diffraction peaks at 2θ = 5.9°, 10.1° and 19.6°. By SEM–EDS/TEM analysis, the silver nano-particles against Braggs reflection planes of (111), (200), (220) and (311) in X-ray diffraction, showed diffraction at 2θ = 38.2°, 44.5°, 64.7° and 77.5° in pattern.

Enhancing sensitivity of phenol-sulfuric acid assay using orthophosphoric acid as modifier

<p class="abstract"><strong>Background:</strong> Phenol-sulfuric acid assay has been widely used for quantification of sugars in biological fluid and industry. The conventional method originally proposed by Dubois et al, was modified several times for enhancing sensitivity of assay such as substituting phenol with other chromogens, by optimizing assay conditions and by adding phenol after dehydration reaction. Both conventional and modified assays have utilized acid catalysis property of sulphuric acid but effect of adding phosphoric acid has not been studied before.</p><p class="abstract"><strong>Methods:</strong> The present study was conducted in Department of Biochemistry, ANIIMS, Port Blair and IGIMS, Patna. The method being developed in our study consisted of adding orthophosphoric acid to the reaction mixture before addition of sulphuric acid. The method was optimized with different amounts of orthophosphoric acid. Statistical analysis was done using SPSS and Microsoft Excel software. </p><p class="abstract"><strong>Results:</strong> The current study found an enhancing effect (on sensitivity) of orthophosphoric acid in optimal concentration of 5.16 mmoles. Comparison among standard curves of methods that were compared showed that the curve was steepest for current study and average absorbance was 0.199±0.017 for conventional, 0.253±0.011 for method by Rasouli et al, and 0.290±0.013 for current study. Pooled serum analysis exhibited absorbance of 0.157±0.015 in conventional method while in modified conventional method absorbance was 0.234±0.010 and highest absorbance was observed in current study at 0.281±0.012.</p><p class="abstract"><strong>Conclusions:</strong> Our results suggest that orthophosphoric acid exerts a positively modifying effect on phenol sulphuric acid assay.</p><p class="abstract"> </p>

Selection of lactic acid bacteria for exopolysaccharide production

An important source of natural alternative to commercial additives that are commonly extracted from plants and animals is the exopolysaccharide (EPS) produced by lactic acid bacteria (LAB). A screening for EPS production by Lactobacillus delbrueckii, Lactobacillus rhamnosus NBRC 3425 and Weisella paramesenteroides was conducted to identify which among these three LAB would produce the highest yield of EPS. The test organisms were grown in a Semi-defined Medium (SDM) of Sanchez et al (2006) with some modifications. EPS production was confirmed by the formation of precipitate after mixing the broth medium with 95% absolute ethanol. Results of total sugar analysis by phenol-sulfuric acid assay revealed that estimated EPS yield of L. rhamnosus NBRC 3425 was significantly higher at p<0.05 than those of W. paramesenteroides and L. delbrueckii ssp. lactis with values of 0.1355g/L, 0.0652g/L and 0.0544g/L, respectively even though their viable count did not differ significantly from each other. Correspondingly, the pH of L. rhamnosus NBRC 3425 media was also significantly higher (pH 4.03) than L. delbrueckii (pH 3.60) and W. paramesenteroides (pH 3.83).

Protein N-Glycans: Incorporating Glycochemistry into the Undergraduate Laboratory Curriculum

Glycoscience continues to be an underrepresented topic in current undergraduate-biochemistry-laboratory curricula. Of the educational laboratories present in this subject area, few introduce students to cutting-edge methods and techniques related to carbohydrates. A multiweek series of experiments is described that highlights a recently published bleach-mediated-carbohydrate-cleavage protocol in the context of the isolation and identification of the soybean glycoprotein, β-conglycinin. Two different levels of undergraduate biochemistry courses (a first-year introductory course and an upper-level bioanalytical course) completed the experimental set. Although the same sequence of experiments took place for both levels, the teaching approaches to the material varied. The introductory course used an inquiry-type learning approach, whereas the upper-level students were taught with a guided-inquiry approach. Students in both courses were able to successfully isolate glycosylated β-conglycinin from inexpensive soy flour and then analyze the protein with SDS-PAGE and mass spectrometry. Once β-conglycinin was isolated, students were able to effectively adapt a recently published bleach-mediated-carbohydrate-cleavage protocol on this previously untested glycoprotein and quantitate the carbohydrate content with a colorimetric phenol–sulfuric acid assay. The effective execution of this biochemistry-laboratory series demonstrates an alternative undergraduate-laboratory curriculum that introduces students to traditional glycoscience and protein methods along with more cutting-edge techniques in these areas.

A new high-throughput assay for determining soluble sugar in sorghum internode-extracted juice.

A high-throughput method combining liquid handling system and 96-well microplate pipetting format was developed for total sugar determination. With this new method, we characterized diverse sugar accumulation in sorghum varieties. Sweet sorghum accumulates large amounts of sucrose in its stalk and, therefore, has emerged as one important bioenergy crop. The commonly used sugar measurement, Brix, limits the characterization of internode variation of the sugar concentrations due to its low throughput. Here we developed a low-cost, high-throughput method to determine profiles of total sugars in sorghum internodes with a liquid handling system-based sample preparation and a phenol-sulfuric acid assay in 96-well microplate format. The present method generates results highly correlated with commonly used Brix measurements (r = 0.922). The inter-assay coefficient of variation ranged from 4.8 to 7.6%. The present method can reliably estimate mixed sugars composed of 80% sucrose. We characterized the profiles of 35 sorghum accessions and identified 21 accessions with significantly different sugar concentrations between internodes either due to dried-up internodes or concentration differences. As a high-throughput alternative to Brix measurements, the new method makes it possible to phenotype total sugars from large numbers of internode samples and, therefore, will be useful for genetic and breeding purposes.

Determination of the Amount of Carbohydrates Adsorbed on Solid Substrates

Carbohydrates, for example in form of polysaccharides such as dextran are essential components of all living organisms and are involved in cell and bacterial adhesion. The latter can lead to biofilms which may either cause diseases or are beneficial in biofilm reactors. To utilize effects of bound carbohydrates it is required to know under which conditions they bind to solid surfaces such as dental materials or biofilm reactor walls. For this purpose one interesting question is to which amount the polysaccharides bind to the surface under a predefined condition. However, the quantification of bound carbohydrates is very difficult. A well‐known method to quantify carbohydrates in solution is the phenol‐sulfuric acid (PSA) assay. Here, this method is adapted for bound carbohydrates. This modified PSA assay delivers reproducible results and does only need a small amount of reagents and carbohydrates. In the case of dextran bound on titanium as biomaterial it delivers the expected results, that is that higher concentrations lead to a higher adsorbed amount.

Extraction of flocculants from a strain of Bacillus thuringiensis and analysis of their properties

In a preliminary screening study, our laboratory isolated from the biofloc in aquaculture waters a strain of Bacillus thuringiensis, which produced highly efficient bio-flocculants. In the present study, we extracted the crude flocculants from this strain and analyzed their properties. Distribution analysis indicated that the flocculants were mainly distributed in the supernatant of the fermentation liquid. The flocculants were extracted using an ethanol extraction method, and the chemical compositions and morphology of the crude flocculants were analyzed using the Molish reaction, Fehling reaction, ninhydrin reaction, biuret reaction, phenol-sulfuric acid assay, Coomassie brilliant blue staining, ultraviolet scanning, infrared scanning and scanning electron microscopy. The carbohydrate composition of the polysaccharides in the flocculants was analyzed with thin layer chromatography. The results indicated that flocculants were solid substances with an ivory white color and their texture was loose and soft. Visualization under scanning electron microscopy revealed that their ultra-morphology consisted of small, long and fiber-like shapes. Chemical and physical analyses indicated that polysaccharides accounted for 34.5% of the components in the crude flocculants. The monosaccharides present in crude flocculants included mainly glucose, galactose and mannitose.

Antioxidant enzyme activity and lipid peroxidation in gills of fish (Sparus aurata) upon exposure to swarms of Pelagia noctiluca

During the last decade, massive blooms of jellyfish have occurred in the Mediterranean basin, putting great concern on cultured fish in offshore cage. Current studies enter in this task, and they consist on examining the antioxidant responses as well as lipid peroxidation and mucous secretion in gills of the gilthead sea bream (Sparus aurata). This is done upon exposure to the scyphozoan jellyfish Pelagia noctiluca at environmentally realistic densities (3, 7 and 15 individus per tanks). This finding revealed that P. noctiluca was a highly toxic organism to the cultured fish. Morphological examination showed lesions of the secondary gill lamellae in fish exposed to jellyfish. Enzymatic activities were measured after 0, 1, 2, 7, 15 and 30 days after initiating the exposure. The set of analyzed activities included catalase (CAT) and glutathione reductase (GR) activities. Lipid peroxidation was measured by quantifying malondialdehyde (MDA). Estimation of mucus secretion was done by using the phenol sulfuric acid assay protocol. The results showed that jellyfish induced antioxidant enzyme activities (CAT, GR) at different densities. Gills MDA content was significantly increased in all treated groups versus the control groups throughout the exposure periods. Our results show also that fish exposed to jellyfish swarms showed mucus hypersecretion on gills. Based on the results of this study, we consider the scyphozoan jellyfish P. noctiluca to be harmful in aquaculture system. The observed changes in analyzed parameters (especially CAT and GR activities, MDA contents and mucous production) suggest that these parameters could be useful tools in environmental monitoring programs of the aquaculture where jellyfish blooms occur.

Synthesis of anthrose lipidic derivative as mimic of B. anthracis BclA glycoprotein for use in ELISA-like binding assays

ABSTRACTThe surfaces of Bacillus anthracis endospores expose anthrose-containing oligosaccharides, which have been considered for use as a target for specific detection of the spores. In this direction, we have developed an efficient and straightforward synthetic strategy toward anthrose lipidic derivate tetradecyl 4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-β-d-glucopyranoside 16 as a model target for B. anthracis spores. The ability of the prepared anthrose and glucose (for control purposes) lipidic derivatives to display on a multiwell plate was demonstrated by a colorimetric phenol-sulfuric acid assay and their potential utility in multiwell binding assays was assessed using fluorescein-labeled concanavalin A (ConA-FITC) and Aleuria aurantia (AAL-FITC).

Physicochemical Characterization and Antitumor Activity of Water-Soluble Polysaccharides from Bupleurum chinense

Bupleurum chinense belongs to the genus Bupleurum spp. and is one of the most important plant medicines in China used for over a thousand years. B. chinense is widely distributed in the northern region of China and is customarily called “Bei chaihu.” The clinical practice from ancient plant pharmacy experts showed that B. chinense possesses many pharmacological functions, such as balancing different organs and energies within the body, strengthening the action of the digestive tract, improving liver and circulatory system function, and relieving liver tension. In addition, contemporary pharmacological research indicated that B. chinense can effectively inhibit hepatoma and protect and promote hepatocyte regeneration. However, the active ingredients in B. chinense and possible mechanisms of its antihepatoma action are still unclear. The crude polysaccharide from the roots of Bupleurum chinense was extracted by hot water and ethanol precipitation with a yield of 12.4%. After deproteination by a combination of proteinase and the Sevag method, the supernatant was collected, dialyzed, and lyophilized to obtain water-soluble B. chinense polysaccharides (BCPs) 1 . BCPs appeared as a pale yellow powder and had a negative response to the Bradford assay 2 . In addition, no absorption at either 280 or 260 nm was detected by UV spectrophotometer, which indicated the absence of protein and nucleic acid. Results from phenol–sulfuric acid assay showed that BCPs contained 93.7% carbohydrate 3 and 5.4% uronic acid 4 . According to GC analysis, BCPs are composed of mannose, galactose, glucose, and galacturonic acid with the molar ratio 25:7.4:10:6.7 5 . We tested in vitro growth inhibition and cytotoxicity of BCPs against three tumor cell lines and two nontumorous cell lines. BCPs exhibited a significant cytotoxicity against H22 with the IC50 value of 103.3 g/mL. The IC50 values against B16 and S-180 were 289.3 and 371.2 g/mL, which showed that the inhibition effects of BCPs against B16 and S-180 were significantly lower than that against H22. The above results demonstrated that BCPs could specifically inhibit proliferation of tumor cells and had a discriminate antiproliferative activity against different types of tumor cells. In addition, BCPs exhibited extremely lower cytotoxicity to mouse embryonic fibroblast cell (NIH 3T3) and mouse macrophage cell (RAW 264.7) with high IC50 values over 1700 g/mL, implying that BCPs had no direct cytotoxicity to noncancerous cells and was safer than other chemotherapeutic drugs, having no negative side effects in inhibiting proliferation of nontumorous cells. We further evaluated the in vivo antitumor activity of BCPs on H22 solid tumor-bearing mice. Due to the fast growth of H22 tumor cells, the transplanted tumor model mice gradually exhibited a series of weakness symptoms such as loss of appetite and reduced body weight with dull hairs. After oral treatment with BCPs (50, 100, and 200 mg/kg), the growth of H22 tumors in the model mice was significantly suppressed compared with the negative control group (p < 0.05), and the tumor inhibition ratios were 22.7, 35.1, and 51.1% at concentrations of 50, 100, and 200 mg/kg body weight, respectively. Furthermore, the body weights of the BCP-treated group were increased significantly when compared with the negative control group and the CTX-treated group during the 15-day experimental period (data not shown).

A rapid and accurate method for the quantitative estimation of natural polysaccharides and their fractions using high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector

In this study, a rapid and accurate method for quantitative analysis of natural polysaccharides and their different fractions was developed. Firstly, high performance size exclusion chromatography (HPSEC) was utilized to separate natural polysaccharides. And then the molecular masses of their fractions were determined by multi-angle laser light scattering (MALLS). Finally, quantification of polysaccharides or their fractions was performed based on their response to refractive index detector (RID) and their universal refractive index increment (dn/dc). Accuracy of the developed method for the quantification of individual and mixed polysaccharide standards, including konjac glucomannan, CM-arabinan, xyloglucan, larch arabinogalactan, oat β-glucan, dextran (410, 270, and 25kDa), mixed xyloglucan and CM-arabinan, and mixed dextran 270K and CM-arabinan was determined, and their average recoveries were between 90.6% and 98.3%. The limits of detection (LOD) and quantification (LOQ) were ranging from 10.68 to 20.25μg/mL, and 42.70 to 68.85μg/mL, respectively. Comparing to the conventional phenol sulfuric acid assay and HPSEC coupled with evaporative light scattering detection (HPSEC-ELSD) analysis, the developed HPSEC-MALLS-RID method based on universal dn/dc for the quantification of polysaccharides and their fractions is much more simple, rapid, and accurate with no need of individual polysaccharide standard, as well as free of calibration curve. The developed method was also successfully utilized for quantitative analysis of polysaccharides and their different fractions from three medicinal plants of Panax genus, Panax ginseng, Panax notoginseng and Panax quinquefolius. The results suggested that the HPSEC-MALLS-RID method based on universal dn/dc could be used as a routine technique for the quantification of polysaccharides and their fractions in natural resources.