Isolation, structure modification, and anti-rheumatoid arthritis activity of isopimarane-type diterpenoids from Orthosiphon aristatus.

Orthosiphon aristatus is a well-known folkloric medicine and herb for Guangdong soup for the treatment of rheumatism in China. Eight isopimarane-type and migrated pimarane-type diterpenoids (1-8), including a new one with a rarely occurring α,ß-unsaturated diketone C-ring, were isolated from O. aristatus. Their structures were determined by spectroscopic methods and quantum chemical calculations. Furthermore, the most abundant compound, orthosiphol K, was structurally modified by modern synthetic techniques to give seven new derivatives (9-15). The anti-rheumatoid arthritis activity of these diterpenoids were evaluated on a TNF-α induced MH7A human rheumatoid fibroblast-like synoviocyte model. Compound 10 showed the most potent activity among these compounds. Based on their inhibitory effects on the release levels of IL-1ß, the preliminary structure-activity relationships were concluded. Furthermore, western blot analysis revealed that 10 could increase the expression of IκBα and decrease the expression of NF-κB p65, and the expression levels of COX-2 and NLRP3 proteins were consequently down-regulated.

Lignan glucosides from Gentiana macrophylla with potential anti-arthritis and hepatoprotective activities.

Ten lignans, including six previously undescribed phenolic ester glycosyl lignans (1-6), were isolated from a well-known traditional Chinese medicine, Qin-Jiao, which is the dry root of Gentiana macrophylla Pall. (Gentianaceae). Their structures were determined by spectroscopic and chemical methods, especially 2D NMR techniques. Quantum chemical calculations of theoretical ECD spectra allowed the determination of their absolute configurations. Refer to its traditional applications for the treatment of rheumatic arthralgia and hepatopathy, these compounds were evaluated on a TNF-α induced MH7A human synoviocyte inflammation model and a D-GalN induced AML12 hepatocyte injury model. Compounds 1, 2, 5, and 6 significantly reduced the release of proinflammatory cytokine IL-1ß in MH7A cells at 15 µM and they also could strongly protect AML12 cells against D-GalN injury at 30 µM. Flow cytometry and Western blot analysis showed that compound 5 ameliorated D-GalN induced AML12 cell apoptosis by upregulating the expression of anti-apoptotic Bcl-2 protein and down-regulating the expression of pro-apoptotic Bax protein.

Terpenoid Glucosides from Gentiana macrophylla That Attenuate TNF-α Induced Pulmonary Inflammation in A549 Cells.

Four previously undescribed terpenoid glucosides, including one sesquiterpenoid di-glucoside (1), two new iridoid glucosides (2, 3), and a new triterpenoid tri-glucoside (4), were isolated from a 70% ethanol extract of the root of Gentiana macrophylla (Gentianaceae), along with eight known terpenoids. Their structures were determined by spectroscopic techniques, including 1D, 2D NMR, and HRMS (ESI), as well as chemical methods. The absolute configuration of compound 1 was determined by quantum chemical calculation of its theoretical electronic circular dichroism (ECD) spectrum. The sugar moieties of all the new compounds were confirmed to be D-glucose by GC analysis after acid hydrolysis and acetylation. Anti-pulmonary inflammation activity of the iridoids were evaluated on a TNF-α induced inflammation model in A549 cells. Compound 2 could significantly alleviate the release of proinflammatory cytokines IL-1ß and IL-8 and increase the expression of anti-inflammatory cytokine IL-10.

Preparation of a water soluble aminated ß-1,3-D-glucan for gene carrier: The in vitro study of the anti-inflammatory activity and transfection efficiency.

ß-1,3-D-glucan has been reported to have a series of bioactivities including antitumor, antimicrobial, anti-inflammatory and antioxidative effects; however, its insolubility in neutral aqueous solution significantly restricts the potential application in biological and medicine fields. Herein, a water-soluble aminated ß-1,3-D-glucan (AG) was synthesized and the anti-inflammatory effect, cytotoxicity and plasmid DNA (pDNA) binding capacity of AG, serum stability, the particle sizes and zeta potentials of AG/pDNA nanocomposites, and the transfection efficiency and mechanism of action were studied. AG shows no obvious cytotoxicity within the range of working concentration (1-64 µg/ml) and it exerts potent anti-inflammatory effect independent on Dectin-1 and TLR2. AG/pDNA nanocomposites prepared by electrostatic interaction possess an appropriate particle size ranged from 192.8 to 118.4 nm and zeta potentials ranged from 20.880 to 27.16 mV with the N/P ratios from 5 to 100. AG/pDNA nanocomposites at the N/P ratios of 10 and 20 were able to show superior transfection efficiencies in RAW 264.7 cells as a result of their suitable particle size, zeta potential, anti-inflammatory effect, and the specific interaction with pattern recognition receptors (Dectin-1 and TLR2). These results indicate that AG is a potential candidate for DNA delivery system due to its potent anti-inflammatory effect and high transfection efficiency.

Synergistic effect of dual targeting vaccine adjuvant with aminated ß-glucan and CpG-oligodeoxynucleotides for both humoral and cellular immune responses.

Presently, clinically approved adjuvants (such as aluminum salts) fail to induce cellular immune responses, which is crucial to defend against intracellular pathogens (including HIV, malaria, tuberculosis and Ebola) and cancer. However, Freund's complete adjuvant potently stimulates both humoral and cellular immune responses, accompanying by high toxicity and severe side reactions. Here in this work, a CpG-oligodeoxynucleotides (CpG-OND) crosslinked aminated ß-glucan-Ovalbumin dual targeting nanoparticle (CpG-OND-AG-OVA) is prepared through a simple and mild ionic complexation method. The aminated ß-glucan plays dual roles as antigen presenting cells (APCs) targeted carrier and immunopotentiator (targeting and activating dectin-1 on APCs). Meanwhile, CpG-OND also plays dual roles as ionic crosslinker and immunopotentiator (targeting and activating Toll-like receptor 9 in APCs). The adjuvant activity of the particles is evaluated through in vitro and in vivo experiments. The particles significantly enhance uptake and sustained proteolytic processing of antigens, and result in APCs maturation, inducing robust Th1 and Th2-type immune responses comparable to Freund's adjuvant without obvious toxicity. The potent adjuvant activity of the nanoparticles may originate from dual targeting synergistic effects between aminated ß-glucan and CpG-OND. Accordingly, the dual targeting nanoparticles may be a promising vaccine adjuvant for inducing robust humoral and cellular immune responses against infectious diseases and cancers. STATEMENT OF SIGNIFICANCE: An ideal adjuvant for subunit vaccine should act as both a carrier to enhance the uptake, sustained processing and cytosolic delivery of antigens, and an immunopotentiator to stimulate antigen presenting cells (APCs) for activation of naive T cells. Additionally, it should be easy to obtain and safe with negligible toxicity. Unfortunately, both synthetic and natural polymers that have been developed into antigen delivery system cannot completely fulfill the requirements. In the present study, the authors design nanoparticles with aminated ß-glucan and CpG-oligodeoxynucleotides (CpG-OND) through a simple and mild method. ß-Glucan (a dectin-1 and TLR2 targeted PAMP) and CpG-OND (a TLR9 targeted PAMP) are readily accessible. Aminated ß-glucan plays dual roles in the nanoparticle as APCs targeted carrier and immunopotentiator. Meanwhile, CpG-OND also plays dual roles as crosslinker and APCs targeted immunopotentiator. By making use of synergistic effect of the dual targeting vaccine adjuvant with aminated ß-glucan and CpG-OND, the nanoparticles induce robust antigen specific immune responses comparable to Freund's adjuvant without obvious toxicity.

Antigen uptake and immunoadjuvant activity of pathogen-mimetic hollow silica particles conjugated with ß-glucan.

The aim of vaccines is to imitate the immune responses induced by pathogen infection without causing disease. Therefore, strategies of designing vaccine delivery systems by mimicking key features of pathogens are often used. For this purpose, the present study prepares pathogen-mimicking ß-glucan-conjugated hollow silica particles by using polystyrene or bacteria particles as templates. The particles perfectly duplicate the structure and morphology of pathogens and possess excellent properties of hollow silica particles, including large opening pore channels, large interior cavities, high loading of OVA (ovalbumin) and controlled release capability, biocompatibility, tunability of surface functionality and immunopotentiating activity. In addition, the particles are antigen presenting cells (APCs) targeted by specific interaction with ß-glucan specific receptors on the surface of APCs, which can enhance the uptake and sustained proteolytic processing of antigens and induce APC maturation. Eventually, potent Th1 and Th2-type immune responses are aroused. The size and shape of the particles have a significant impact on the antigen uptake and immunoadjuvant activity. The degree of antigen uptake enhancement is ranked in the following order: PS HSP@glucan (nanoscale spherical) > E. coli HSP@glucan (micron-sized rod-like) > S. aureus HSP@glucan (micron-sized spherical). The PS HSP@glucan is more apt to induce a Th1-type immune response, while the E. coli HSP@glucan is more apt to induce a Th2-type immune response. The particles may thus provide a promising strategy for development of novel vaccine delivery systems for inducing robust humoral and cellular immune responses against infectious diseases and cancers.