Isolation, Structural Analysis and Anti-Inflammatory Activity of a Polysaccharide from Ilex cornuta Fruits.

In the present study, a polysaccharide from Ilex cornuta fruits (LCFP-3) was obtained by hot water extraction, Diethyaminoethyl cellulose-52 (DEAE-52) chromatography column and Sephadex G-100 gel column purification. Its structural characteristics were further explored using high performance anion exchange chromatography (HPAEC), gas chromatography and mass spectrometry (GC/MS), scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Monosaccharide composition analysis revealed LCFP-3 contained mainly Galactose (31.92 %), Arabinose (25.87 %) and Galacturonic acid (23.35 %) while small percentage of Rhamnose, Glucose, Mannose and Xylose. Chemical composition analysis showed that the total sugar content of LCFP-3 was 90.31 % and the protein content was 0.246 %. Gel permeation chromatography (GPC) analysis showed that its average molecular weight was 41.199 kDa. Structural analysis showed that LCFP-3 may be composed of residues, T-α-Arap, T-α-Rhap, 1,3-α-Arap, 1,4-α-Arap, T-ß-Galp, 1,4-α-GalpA(OMe), 1,4-ß-Glcp, 1,3-ß-Galp, 1,3,6-ß-Manp, 1,6-ß-Galp, 1,3,4-ß-GalpA, 1,4,6-ß-Manp, 1,3,6-ß-Glcp, 1,2,3,4-α-Xylp. The anti-inflammatory activity of LCFP-3 was evaluated using lipopolysaccharide (LPS)-induced RAW246.7 macrophages. The results showed that 1-200 µg/mL LCFP-3 could dose-dependently protect against LPS-induced toxicity and 1 µg/mL LCFP-3 could significantly inhibit LPS-induced NO production. Therefore, LCFP-3 exerted an anti-inflammatory activity and has great potential as a functional ingredient.

Diversity in Fruit Morphology and Nutritional Composition of Juglans mandshurica Maxim in Northeast China.

Although Manchurian walnut (Juglans mandshurica Maxim) is widely distributed in northeast China, very few studies had been reported on its diversity among different populations. We surveyed 12 J. mandshurica populations in their native habitats across the northeast region of China and profiled 13 fruit morphological traits. We found a large degree of variations for these traits, especially for fruit weight (coefficient of variation, or CV of 22.00%), nut weight (CV of 19.42%), and kernel weight (CV of 19.89%). Statistical analysis showed that a large portion of the total variation can be attributed to within-population variation (66.64%), followed by random error (20.96%). We also comprehensively quantified the nutritional composition including fatty acids, amino acids, vitamins, and micronutrients. Similar to fruit morphological traits, we found large variation for most kernel components, which mostly can be explained by within-population variation. Further correlation analysis revealed the dependence of some morphological and nutritional traits on key geographical and ecological factors such as latitude, accumulated temperature, and day length. For instance, a significant positive correlation was found between fruit dimensions and equivalent latitude and precipitation, indicating that such factors should be considered for breeding. Taken together, our data provided a rich dataset for characterizing the variation among J. mandshurica populations and a foundation for selective breeding.

Chemical characterization of a new sulfated polysaccharide from Gracilaria chouae and its activation effects on RAW264.7 macrophages.

This study aimed to characterize the chemical composition of a new sulfated polysaccharide from the red alga Gracilaria chouae and evaluate its activation effects on RAW264.7 macrophages. It showed that the obtained G. chouae polysaccharide (GCP-3A) was a sulfated acidic polysaccharide with a molecular weight of 11.87 kDa. GCP-3A was composed of xylose, galactose, glucose, and mannose with a molar ratio of 3.00:29.28:0.63:0.45, and it contained α,ß|-glycosidic linkages. Scanning electron microscopy (SEM) and a Congo red test showed that it was a heterogeneous polysaccharide with irregular interwoven sheets and rods, and did not have a triple-helix conform|ation. Furthermore, GCP-3A significantly promoted the proliferation of RAW264.7 macrophages and the secretion of nitric oxide (NO) in tests of 3-|(4,|5-dimethylthiahiazo-2-yl)|-2,|5-diphenytetrazoliumromide(MTT) and NO.

Seed Oil Quality and Cultivation of Sambucus williamsii Hance as a New Oil Crop.

Natural edible oil derived from wild non-cultivated oil crops contributed to human daily nutritional diversity and disease prevention. It was important to investigate the nutritional value of these oils and the feasibility of crop cultivation. The present study focused on the assessment of seed oil quality of Sambucus williamsii Hance (SWH) and its molecular breeding. Wild SWH seed oil was extracted by supercritical CO2 technology and the composition of the oil was determined by using gas chromatography mass spectrometry (GC-MS) analysis. The oil content of SWH seeds reaches around 40%. Its seed oil was found to be rich in unsaturated fatty acids, such as 24.24% of linolenic acid and 50.56% of linoleic acid, and vitamin E (25.92 mg kg-1). The cytotoxicity and heavy metal analysis showed SWH seed oil was safe for consumption. In addition, the SWH strains with excellent characteristics were screened out for cultivation according to genetic diversity and morphological analysis. Amplified fragment length polymorphism (AFLP) markers were used to evaluate the genetic diversity of 28 accessions of wild SWH seeds and 5 accessions were selected to cultivate. Among them, two strains of SWH (sample 3 and 6) with high yielding (275.7 and 266.8 area yield kg-1) were suitable for dense planting and could be used to establish the raw material forest of SWH seed oil. The results of this study indicated the potential of development of selected SWH as novel oil crops and their wide cultivation.

Characterization and anti-inflammatory effects of sulfated polysaccharide from the red seaweed Gelidium pacificum Okamura.

In the present study, crude polysaccharides were extracted from Gelidium pacificum Okamura, and further purified to obtain the sulfated polysaccharide with molecular weight of 28,807 Da. Its monosaccharide composition mainly consisted of xylose (7.1%), galactose (59.7%) and galacturonic acid (19.76%). And the sulfate ester content of the sulfated polysaccharide was estimated as 8.8%. Structure analysis showed that the sulfated polysaccharide comprised of 1,4-linked-α-D-Galp3S, 1,2-linked-α-D-Xylp and 1,3-linked-ß-D-GalpA residues, respectively. Its anti-inflammatory effects were investigated in LPS-stimulated human monocytic (THP-1) cells. The sulfated polysaccharide at a concentration of 5 µg/mL fully protected the THP-1 cells against LPS-stimulated cytotoxicity. Furthermore, the addition of sulfated polysaccharide resulted in a significant reduction of NO production in LPS-treated cells, and this effect appeared to be dose-related. The sulfated polysaccharide (5 µg/mL) significantly suppressed the mRNA and protein expression of toll-like receptor-4 (TLR-4), myeloid differentiation factor (MyD88) and tumor necrosis factor receptor-associated factor-6 (TRAF-6) in LPS-stimulated THP-1 cells. These results showed the sulfated polysaccharide not only provided a good protection against LPS-induced cell toxicity, but also exerted an anti-inflammatory effect via the TLR4 signaling pathway.

Genotyping of Multiple Clinical Samples with a Combined Direct PCR and Magnetic Lateral Flow Assay.

Developing a sensitive, low-cost, and easy-to-use point-of-care testing system for genotyping is important for informing treatment decisions and predicting the risk of underlying diseases. Conventional methods normally require complex operational procedures as well as expensive and sophisticated instruments. Here, we report a general approach that enables us to detect the genotype of multiple sample types directly without DNA purification. Moreover, the PCR results can be further quantitatively analyzed based on a magnetic lateral flow assay (MLFA) system, which avoids multiple steps needed for conventional nucleic acid biosensors. As a demonstration, we show that three genotypes of aldehyde dehydrogenase 2 (ALDH2) can be identified using a small volume of sample with an accuracy of 100% and a sensitivity of 1.0 × 102 cells/µL, which are better than those of the gold standard methods. We believe that the direct PCR-MLFA system represents a significant advance toward the development of portable, sensitive biomedical platforms.

A direct isothermal amplification system adapted for rapid SNP genotyping of multifarious sample types.

Genotyping of single nucleotide polymorphisms (SNPs) in point-of-care (POC) settings could be further improved through simplifying the treatment of samples. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on direct loop-mediated isothermal amplification (LAMP) without DNA extraction, known as Direct-LAMP. Samples from various sources (including whole blood, dried blood spot, buccal swab and saliva), treated with NaOH, can be used directly in amplification. The turnaround time was about 30 min from sample collection to provision of results. The accuracy was evaluated by assessing the polymorphisms of methylenetetrahydrofolate reductase (MTHFR) C677T and aldehyde dehydrogenase-2 (ALDH2) Glu504Lys, which are better known for their critical role in folate and ethanol metabolism, respectively. Completely consistent genotyping results reveal that Direct-LAMP is generally concordant with sequencing. This system can serve as a very promising platform in the fields of disease predisposition, drug metabolism and personalized medicine.

Multiple SNPs Detection Based on Lateral Flow Assay for Phenylketonuria Diagnostic.

Single nucleotide polymorphisms (SNPs) are closely related to genetic diseases, but current SNP detection methods, such as DNA microarrays that include tedious procedures and expensive, sophisticated instruments, are unable to perform rapid SNPs detection in clinical practice, especially for those multiple SNPs related to genetic diseases. In this study, we report a sensitive, low cost, and easy-to-use point-of-care testing (POCT) system formed by combining amplification refractory mutation system (ARMS) polymerase chain reaction with gold magnetic nanoparticles (GMNPs) and lateral flow assay (LFA) noted as the ARMS-LFA system, which allow us to use a uniform condition for multiple SNPs detection simultaneously. The genotyping results can be explained by a magnetic reader automatically or through visual interpretation according to the captured GMNPs probes on the test and control lines of the LFA device. The high sensitivity (the detection limit of 0.04 pg/µL with plasmid) and specificity of this testing system were found through genotyping seven pathogenic SNPs in phenylalanine hydroxylase gene ( PAH, the etiological factor of phenylketonuria). This system can also be applied in DNA quantification with a linear range from 0.02 to 2 pg/µL of plasmid. Furthermore, this ARMS-LFA system was applied to clinical trials for screening the seven pathogenic SNPs in PAH of 23 families including 69 individuals. The concordance rate of the genotyping results detected by the ARMS-LFA system was up to 97.8% compared with the DNA sequencing results. This method is a very promising POCT in the detection of multiple SNPs caused by genetic diseases.

Coupling of a bifunctional peptide R13 to OTMCS-PEI copolymer as a gene vector increases transfection efficiency and tumor targeting.

BACKGROUND: A degradable polyethylenimine (PEI) derivative coupled to a bifunctional peptide R13 was developed to solve the transfection efficiency versus cytotoxicity and tumor-targeting problems of PEI when used as a gene vector. METHODS: We crossed-linked low molecular weight PEI with N-octyl-N-quaternary chitosan (OTMCS) to synthesize a degradable PEI derivative (OTMCS-PEI), and then used a bifunctional peptide, RGDC-TAT (49-57) called R13 to modify OTMCS-PEI so as to prepare a new gene vector, OTMCS-PEI-R13. This new gene vector was characterized by various physicochemical methods. Its cytotoxicity and gene transfection efficiency were also determined both in vitro and in vivo. RESULTS: The vector showed controlled degradation and excellent buffering capacity. The particle size of the OTMCS-PEI-R13/DNA complexes was around 150-250 nm and the zeta potential ranged from 10 mV to 30 mV. The polymer could protect plasmid DNA from being digested by DNase I at a concentration of 23.5 U DNase I/µg DNA. Further, the polymer was resistant to dissociation induced by 50% fetal bovine serum and 400 µg/mL sodium heparin. Compared with PEI 25 kDa, the OTMCS-PEI-R13/DNA complexes showed higher transfection efficiency both in vitro and in vivo. Further, compared with OTMCS-PEI, distribution of OTMCS-PEI-R13 at tumor sites was markedly enhanced, indicating the tumor-targeting specificity of R13. CONCLUSION: OTMCS-PEI-R13 could be a potential candidate as a safe and efficient gene delivery carrier for gene therapy.

[Identification of six species of Cassia seeds by capillary electrophoresis].

OBJECTIVE: To identify Cassia seeds of six species by capillary electrophoresis. METHOD: The water-soluble extracts of Cassia seeds of six species were analyzed by capillary electrophoresis. The running buffer was 0.1 mol.L-1 borate, 0.1 mol.L-1 SDS, pH 8.5. The separation voltage was 25 kV. RESULT: Four common peaks could be found in the electropherograms of six species Cassia seeds, and the characteristic peaks could also be observed. CONCLUSION: Fingerprints of the six species of Cassia seeds show significant differences, which can be used for their identification.