Advancement of Research Progress on Synthesis Mechanism of Cannabidiol (CBD).

Cannabis sativa L. is a multipurpose crop with high value for food, textiles, and other industries. Its secondary metabolites, including cannabidiol (CBD), have potential for broad application in medicine. With the CBD market expanding, traditional production may not be sufficient. Here we review the potential for the production of CBD using biotechnology. We describe the chemical and biological synthesis of cannabinoids, the associated enzymes, and the application of metabolic engineering, synthetic biology, and heterologous expression to increasing production of CBD.

Immunostimulatory effects of a polysaccharide from Pimpinella anisum seeds on RAW264.7 and NK-92 cells.

Polysaccharides from Pimpinella anisum were isolated using water at elevated temperature and DEAE Sepharose FF chromatography to examine their chemical structure and activation capacity on immune cells. P. anisum fractions (PAF1, PAF2 and PAF3) were mainly composed of neutral sugars (84.0-98.2%) and uronic acids (2.1-11.8%) with weight average molecular weight (Mw) ranging from 186.6 to 5474.5 × 103 g/mol. Polysaccharides induced a significant inflammatory response in RAW264.7 murine macrophage cells releasing nitric oxide and expressing TNF-α, IL-1ß, IL-6 and IL-10 cytokines. The induction of NK-92 natural killer cells resulted in TNF-α and IFN-γ production and activation of GrB/perforin-, NKG2D- and FasL-mediated cytotoxicity. Polysaccharides triggered the phosphorylation of NF-κB, ERK, JNK and p38 proteins in RAW264.7 and NK-92 cells indicating the involvement of NF-κB and MAPKs signaling pathways. The most active polysaccharide was a galactoarabinan with complex structure.

Structural Elucidation and Immunostimulatory Activities of Quinoa Non-starch Polysaccharide Before and After Deproteinization.

Non-starch polysaccharides derived from natural resources play a significant role in the field of food science and human health due to their extensive distribution in nature and less toxicity. In this order, the immunostimulatory activity of a non-starch polysaccharide (CQNP) from Chenopodium quinoa was examined before and after deproteination in murine macrophage RAW 264.7 cells. The chemical composition of CQNP and deproteinated-CQNP (D-CQNP) were spectrometrically analysed that revealed the presence of carbohydrate (22.7 ± 0.8% and 39.5 ± 0.8%), protein (41.4 ± 0.5% and 20.8 ± 0.5%) and uronic acid (8.7 ± 0.3% and 6.7 ± 0.2%). The monosaccharide composition results exposed that CQNP possesses a high amount of arabinose (34.5 ± 0.3) followed by galactose (26.5 ± 0.2), glucose (21.9 ± 0.3), rhamnose (7.0 ± 0.1), mannose (6.0 ± 0.1) and xylose (4.2 ± 0.2). However, after deproteination, a difference was found in the order of the monosaccharide components, with galactose (41.1 ± 0.5) as a major unit followed by arabinose (34.7 ± 0.5), rhamnose (10.9 ± 0.2), glucose (6.6 ± 0.2), mannose (3.4 ± 0.2) and xylose (3.2 ± 0.2). Further, D-CQNP potentially stimulate the RAW 264.7 cells through the production of nitric oxide (NO), upregulating inducible nitric oxide synthase (iNOS) and various pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α). Moreover, stimulation of RAW 264.7 cells by D-CQNP takes place along the NF-κB and the MAPKs signaling pathways through the expression of cluster of differentiation 40 (CD40). This results demonstrate that RAW 264.7 cells are effectively stimulated after removal of the protein content in C. quinoa non-starch polysaccharides, which could be useful for develop a new immunostimulant agent.

Purification, characterization and immunostimulatory effects of polysaccharides from Anemarrhena asphodeloides rhizomes.

The crude polysaccharide was extracted from A. asphodeloides rhizomes and further purified to produce two fractions F1 (50.0%) and F2 (19.6%). The chemical constitutions of the polysaccharides were neutral sugars (51.4%-89.7%), uronic acids (1.0%-30.2%) and sulfate esters (3.4%-8.1%), with various ratios of monosaccharides including rhamnose (1.4%-6.1%), arabinose (7.1%-21.2%), xylose (0.2%-4.8%), mannose (39.9%-79.0%), glucose (6.0%-11.1%) and galactose (2.6%-22.0%). The molecular properties of the polysaccharides were investigated by the HPSEC-UV-MALLS-RI system, revealing the Mw 130.0 × 103-576.5 × 103 g/moL, Rg 87.6-382.6 nm and SVg 0.3-54.3 cm3/g. The polysaccharides stimulated RAW264.7 cells to produce considerable amounts of NO and up-regulate the expression of TNF-α, IL-1 and COX-2 genes. Polysaccharides exhibited the growth inhibitory effects on cancer cells lines of AGS, MKN-28 and MKN-45, in which F2 fraction exhibited prominent bioactivities. The AGS cells treated with F2 experienced condensed cytoplasm, shrinkage of nucleus and chromatin marginalization with the highest number of cells at early-stage apoptosis reaching 54.6%. The inhibitory effect of F2 polysaccharide on AGS cells was through MAPKs and STAT3 signaling pathways. The backbone of the F2 was mainly linked by (1 â†’ 4)-linked mannopyranosyl and (1 â†’ 3)-linked galactopyranosyl. Taken together, the polysaccharide from A. asphodeloides rhizomes could be utilized as medicinal, pharmacological and functional food ingredients.

Extraction, structural elucidation and immunostimulating properties of water-soluble polysaccharides from wheat bran.

A water-soluble polysaccharide was extracted from wheat bran (WBP) and investigate their structural characteristics and immunostimulatory activities. The chemical composition of WBP and purified fraction (WBP-F) mainly consists of neutral sugars (91.2 ± 1.2 and 98.7 ± 1.2%), proteins (8.6 ± 0.3 and 0.2 ± 0.1%) and uronic acids (0.7 ± 0.1 and 0.6 ± 0.1%). The molecular weight (Mw ) of WBP and WBP-F was calculated as 911.7 and 510.2 × 103  g/mol, respectively. The WBP-F stimulates the RAW264.7 cells through the production of nitric oxide and various cytokines. The treatment of WBP-F facilitated the phosphorylation of P38, JNK, ERK, and NF-ƘB in RAW264.7 cells suggesting that they might stimulate RAW264.7 cells through the activation of NF-ƙB and MAPKs pathways. Furthermore, the structural details of WBP-F were studied by GC-MS and NMR spectrum, which confirms that the main backbone consists of 4-α-D-linked glucopyranosyl residues with branching points at C-6. PRACTICAL APPLICATIONS: Wheat bran is a potential source of health-promoting compounds. It has been reported that polysaccharides of wheat bran containing numerous beneficial activities. In this study, the wheat bran polysaccharide was extracted, fractionated and investigated their immunostimulatory activities. The results found in this study revealed that the purified polysaccharide from wheat bran potentially enhanced the RAW264.7 cells activation. Hence, these polysaccharides could be utilized as a potent immunity-enhancing agent in food and pharmaceutical industries.

The activation of NF-κB and MAPKs signaling pathways of RAW264.7 murine macrophages and natural killer cells by fucoidan from Nizamuddinia zanardinii.

Polysaccharides from Nizamuddinia zanardinii were extracted using water at elevated temperature and fractionated by a DEAE Sepharose FF column yielding four fractions (F1-F4). Crude and fractions were composed of neutral sugars (50.8-57.4%), proteins (10.8-18.1%), sulfates (7.5-17.3%) and uronic acids (3.5-7.7%). Various levels of galactose (13.4-44.4%), fucose (34.1-40.1%), mannose (14.1-33.2%) and xylose (7.4-15.2%) formed the building blocks of the polysaccharide structures. The weight average molecular weights (Mw) of polysaccharides varied between 40.3 and 1254.4 × 103 g/mol. F3 polysaccharide was the most active fraction stimulating RAW264.7 murine macrophage cells to secrete NO, TNF-α, IL-1ß and IL-6, and activating NK cells to release TNF-α, INF-γ, granzyme-B, perforin, NKG2D and FasL through NF-κB and MAPKs signaling pathways. Highly-branched F3 polysaccharide mainly consisted of (1 â†’ 2)-Fucp, (1 â†’ 2,3)-Manp, (1 â†’ 3)-Galp, (1 â†’ 2)-Manp, (1 â†’ 3)-Manp, (1 â†’ 2,3,4)-Manp and (1 â†’ 2,3,6)-Manp residues with great amount of (→1)-Fucp and (→1)-Xylp. Sulfates substituted at C-2 of fucose and galactose residues. Overall, fucoidan from N. zanardinii showed immense potency in boosting immune system through macrophages and NK cells activations and therefore suitable for further exploration in immune-mediated biomedical applications.

Structural characterization of immunostimulating protein-sulfated fucan complex extracted from the body wall of a sea cucumber, Stichopus japonicus.

The water soluble protein-sulfated fucan (PSF) complex isolated from the body wall of Stichopus japonicus was fractionated using an anion-exchange chromatography to obtain four purified fractions (F1, F2, F3, and F4) and to investigate their structural characteristics and immuno-enhancing activities. The crude PSF and fractions mostly consisted of neutral sugars, proteins and sulfates in various proportions. Fucose was their major monosaccharide unit with small portion of mannose, glucose and galactose. The molecular weight (Mw) of the crude PSF and fractions ranged from 323.6×103 to 3630.0×103g/mol. The crude PSF, F2, and F3 were potent stimulator of RAW264.7 cells inducing marked nitric oxide (NO) and cytokines production. The glycosidic linkage of polysaccharides was evaluated using GC-MS and confirmed by 2D-NMR. The main backbone of highly immunostimulating F3 fraction was (1→3)-α-l-linked fucosyl residue with sulfation at C-2 and/or C-4.

Water soluble sulfated-fucans with immune-enhancing properties from Ecklonia cava.

Water-soluble sulfated fucans isolated from Ecklonia cava were fractionated using an anion-exchange chromatography to investigate their molecular characteristics and immunomodulating activities. The crude fucoidan extract and purified fractions (EF1, EF2, and EF3) consisted mostly of different ratios of neutral sugars, proteins, sulfates, uronic acids, and their monosaccharide compositions were also significantly different. The backbone of the most immunoenhancing fraction, EF2, was mainly linked by (1→3)-linked fucopyranosyl and (1→4)-linked mannopyranosyl residues with sulfates at C-4 of fucopyranosyl units. The molecular weights of the crude fucoidan extract and purified fractions ranged from 8.3×10(3) to 442.6×10(3)g/mol. The crude extract, EF1 and EF2 stimulated RAW264.7 cells to produce considerable amounts of nitric oxide and cytokines. The treatment of cells with the sulfated fucans induced the degradation of Iκ-B and the phosphorylation of MAPK in RAW264.7 cells, implying that they might stimulate RAW264.7 cells through the activation of NF-κB and MAPK pathways.

Structural Effects of Sulfated-Glycoproteins from Stichopus japonicus on the Nitric Oxide Secretion Ability of RAW 264.7 Cells.

The effect of various levels of proteins, sulfates, and molecular weight (M w) of a sulfated-glycoprotein (NF3) from a sea cucumber, Stichopus japonicus, on nitric oxide (NO) releasing capacity from RAW 264.7 cells was investigated. The NF3 derivatives had various amounts of proteins (4.8~11.2%) and sulfates (6.8~25.2%) as well as different M w (640.3×10(3)~109.2×10(3) g/mol). NF3 was able to stimulate RAW 264.7 cells to release NO with lower protein contents, indicating that the protein moiety was not an important factor to stimulate macrophages. On the other hand, the NO inducing capacity was significantly reduced with decreased levels of sulfates and M w, implying that sulfates and M w played a pivotal role in activating RAW 264.7 cells. It was not clear why sulfates and a certain range of M w were essential for stimulating macrophages. It appeared that certain levels of sulfates and M w of sulfated-glycoproteins were required to bind to the surface receptors on RAW 264.7 cells.