Exclusion Column Research Articles

Antimalarial evaluation of selected medicinal plants traditionally used for treatment of fever, by inhibition test of heme polymerization

World Health Organization (WHO) has reported 249 million cases infected by malaria worldwide and 608 thousands deaths in 2022. Investigations for new antimalarial drugs from traditional medicine have proven to be more effective and less expensive. The medicinal plants that have been used for treatment of malaria, generally known as types of fevers in traditional medicine, can be suitable candidates for evaluating antimalarial effects. The aim of the present study was to evaluate the in-vitro mechanism of antimalarial action of selected medicinal plants by inhibition test of heme detoxification (ITHD). The methanol extract and fractions were prepared through maceration of the dry powdered plants. The ITHD method was carried in 96-wells plate and the percentage of heme polymerization inhibition was determined. The cytotoxicity of the effective plants were examined on MDBK cell line by MTT assay. Bioassay guided fractionation was performed exposing to size exclusion column chromatography and liquid-liquid fractionation and was assayed by the ITHD method for the sample with the least IC50 value and lowest cytotoxic effect. The methanol fraction of Viola odorata whole plant showed the most considerable results among the tested plants with IC50 171.8 μg.mL-1 beside the lowest cytotoxic effects. This fraction by the bioassay guided fractionation led to fraction SB2 and this fraction demonstrated the most effective result with lowest IC50 = 14.8 ± 3µg.mL-1 in ITHD assay. Regarding the results of the present study and the traditional use of V. odorata for overcoming fever in Iranian traditional medicine, the final fraction of the plant could be proper candidate for further phytochemical and antimalarial studies.

A magnetic separation method for isolating and characterizing the biomolecular corona of lipid nanoparticles

Lipid nanoparticle (LNP) formulations are a proven method for the delivery of nucleic acids for gene therapy as exemplified by the worldwide rollout of LNP-based RNAi therapeutics and mRNA vaccines. However, targeting specific tissues or cells is still a major challenge. After LNP administration, LNPs interact with biological fluids (i.e., blood), components of which adsorb onto the LNP surface forming a layer of biomolecules termed the "biomolecular corona (BMC)" which affects LNP stability, biodistribution, and tissue tropism. The mechanisms by which the BMC influences tissue- and cell-specific targeting remains largely unknown, due to the technical challenges in isolating LNPs and their corona from complex biological media. In this study, we present a new technique that utilizes magnetic LNPs to isolate LNP-corona complexes from unbound proteins present in human serum. First, we developed a magnetic LNP formulation, containing >40 superparamagnetic iron oxide nanoparticles (IONPs)/LNP, the resulting LNPs containing iron oxide nanoparticles (IOLNPs) displayed a similar particle size and morphology as LNPs loaded with nucleic acids. We further demonstrated the isolation of the IOLNPs and their corresponding BMC from unbound proteins using a magnetic separation (MS) system. The BMC profile of LNP from the MS system was compared to size exclusion column chromatography and further analyzed via mass spectrometry, revealing differences in protein abundances. This new approach enabled a mild and versatile isolation of LNPs and its corona, while maintaining its structural integrity. The identification of the BMC associated with an intact LNP provides further insight into LNP interactions with biological fluids.

Polyethylene Glycol -Mediated Exosome Isolation: A Method for Exosomal RNA Analysis.

: Exosomal RNAs (ExoRNAs) offer valuable insights into their cellular origin. ExoRNA studies were faced with challenges in obtaining sufficient amounts of high-quality RNA. Herein, we aimed to compare three traditional exosome isolation methods to introduce an appropriate strategy to extract RNA from cancer-derived exosomes for further RNA analysis. Exosomes were isolated through ultracentrifugation, precipitation kit, and size exclusion column chromatography, and then characterized by dynamic light scattering and transmission electron microscopy, followed by extracting total RNA. The quality and quantity of the extracted RNAs were assessed by a NanoDrop and 2.5% agarose gel electrophoresis. Extracted exosomes displayed a similar range of size and morphology. We found that polyethylene glycol-precipitation method resulted in a higher RNA yield with a 260/280 ratio of 1.9. The obtained exoRNA appeared as a smear in the agarose gel, indicative of small exoRNAs. We provide researchers a suitable approach to isolate exosomes based on yield and purity of exoRNA.

Aspidopterys obcordata vine inulin fructan affects urolithiasis by modifying calcium oxalate crystallization

Aspidopterys obcordata vine is a Chinese Dai ethnic herb used to treat urolithiasis. However, the material basis and underlying mechanisms remain undefined. In this study, a 2.3 kD inulin-like A. obcordata fructan (AOFOS) was isolated by size exclusion column chromatography and characterized by ultrahigh-performance liquid chromatography-ion trap-time of flight mass spectrometry (UPLC-IT-TOF-MS), nuclear magnetic resonance (NMR) spectroscopy, gas chromatography mass spectrometry (GC–MS) and high-performance gel permeation chromatography (HGPC). In addition, AOFOS showed unique anti-urolithiasis activity in Drosophila kidney stone models. Mechanism study indicated that AOFOS reduced the size of calcium oxalate crystals by inhibiting the formation of large size crystals and the generation rate of calcium oxalate crystals as well as the crystal form conversion from calcium oxalate monohydrate (COM) to calcium oxalate dihydrate (COD).

Abstract 5326: Targeted extracellular vesicle to deliver combined chemotherapies to treat cancers

Abstract Extracellular vesicles (EV), facilitated with cancer-targeted antibody or peptide, have been demonstrated as an effective delivery vehicle of chemotherapy and gene therapy. This study aimed to develop and evaluate a targeted EV to deliver a newly identified natural cytotoxic marine compound verrucarin A (Ver-A) or combination with highly potent microtubule polymerization inhibitor mertansine (DM1). The tumor models, glioblastoma (GBM), triple-negative breast cancer (TNBC) and others, were used to validate the developed targeted therapy. First, the stirred-tank bioreactor-based EV biomanufacturing was optimized by developing a two-step purification procedure using size exclusion (300 kDa) and affinity (CD63) columns in liquid chromatography system. Second, both single and dual tumor-targeting monoclonal antibodies (mAbs), such as anti-SSTR2, CD276, EGFR and/or CD47 mAb either developed in house or used in clinics, were tagged on the surface of PEGylated EV to construct mAb-EV-drugs. The flow cytometry analysis, live-cell confocal images and IC50 assay revealed the effective surface binding, intracellular drug delivery and high anti-cancer cytotoxicity of mAb-EV-drugs in cancer cell lines, and the biodistribution study confirmed the tumor targeting in xenograft mouse models. Furthermore, the maximum tolerated dose study showed minimal systemic toxicity, as confirmed by hematoxylin and eosin staining of multiple important organs. Finally, the anti-tumor efficacy study demonstrated significant inhibition of tumor growth by mAb-EV-Ver-A or Ver-A/DM1 in cell line-derived xenograft models, patient-derived xenograft models, and/or immunocompetent xenograft models. This study suggested that the targeted EV is an efficient platform to deliver combined potent chemotherapies to treat cancer or tumor. Citation Format: Kai Chen, Yingnan Si, Seulhee Kim, Zhuoxin Zhou, Lufang Zhou, X. Margaret Liu. Targeted extracellular vesicle to deliver combined chemotherapies to treat cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5326.

Bioactive constituents isolated from the Sri Lankan endemic plant Artocarpus nobilis and their potential to use in novel cosmeceuticals

Two novel compounds, dicarbonyl ester (2) and artolankanin A (3) were isolated from the sequential ethyl acetate and ethanol extracts of stem bark of A. nobilis along with the known compounds, artonin E (1) and artobiloxanthone (4) by bioassay-guided fractionation using a combination of normal phase and size exclusion column chromatography. Their structures were elucidated by spectroscopic techniques, including NMR (1H, 13C, APT, 1H–1H COSY, HSQC, and HMBC), Mass, IR, and UV. The isolated compounds were evaluated for DPPH free radical scavenging activity, tyrosinase, elastase, hyaluronidase, and A5-lipoxygenase enzyme inhibitory activities lipopolysaccharide-induced nitric oxide production inhibitory activity in SIM-A9 microglial cells. Artonin E exhibited marked tyrosinase and A5-lipoxygenase enzyme inhibitory activities and NO inhibition activity in LPS-stimulated microglia cells having IC50 values of 60.92 ± 3.45, 56.38 ± 1.05, and 23.94 ± 1.08 µg/mL, respectively. Kinetic studies showed that the tyrosinase inhibitory activity of artonin E is via a noncompetitive inhibition mechanism. Further, artolankanin A and artobiloxanthone exhibited good A5-lipoxygenase enzyme inhibitory activity having IC50 values of 59.48 ± 1.06 and 62.45 ± 2.25 µg/mL, respectively. Insights from molecular docking and molecular dynamics simulations align with these experimental observations and propose plausible mechanisms of inhibition. Collectively these results suggest that these compounds have the potential to be used as depigmenting and anti-inflammatory agents.

Blockwise synthesis of polylactosamine fragments and keratan sulfate oligosaccharides comprised of dimeric Galβ(1 → 4)GlcNAc6Sβ

In this paper, the chemical synthesis of polylactosamine fragments up to docosasaccharide (22-mer) via the blockwise synthetic approach is reported. We used suitably protected tetrasaccharide and octasaccharide sequences as key building blocks. The use of such large building blocks as glycosyl donors and acceptors enabled the rapid construction of polysaccharide frameworks. Furthermore, the coupling reaction between these large building blocks facilitated the purification of glycosylated products, for which size exclusion column chromatography is highly effective. Then, we applied the building blocks to the synthesis of keratan sulfate glycan, which is partially sulfated poly-N-acetyllactosamine. Consequently, we achieved the synthesis of the octasaccharide of a keratan sulfate glycan comprised of a repeating Galβ(1 → 4)GlcNAc6Sβ disaccharide unit.

Purification and Characterization of Milk Clotting Enzyme from Edible Mushroom (Pleurotus florida)

Oyster mushroom (Pleurotus florida 14 MICC) is one of the most widely cultivated edible fungi in the world. Milk clotting enzyme (MCE) derived from the mold was developed as a calf rennet alternative; thus, it was purified and characterized, and its effect on different milk species was investigated. The highest MCE activity (75.49 SU/ml) was observed in mushroom fruit bodies dissolved in 0.2 M sodium acetate pH 5.0; as well as the highest total MCE activity (367.85 SU) was recorded at 20% of ammonium sulfate with a specific activity of 343.79 SU mg-1 protein while size exclusion column chromatography on Sephadex G-100 purified MCE 3.46-fold with 17.96% yield. Also, it could be capable of coagulating different milk species. Mushroom MCE exhibited their optimal activity at pH 5.0 for crude extract (CE) while at pH 6.0 for partial purified (PP) and purified (P) MCE fractions; as well as at 55 °C for CE and PP MCE fractions while 50 °C for P MCE. CaCl2 concentration (0.01%) recorded the maximal activity for CE while (0.04%) and (0.02%) for PP and P fractions, respectively. It could be concluded that MCE from Oyster mushroom may be a good candidate as a calf rennet substitute in cheese production.

Circulating Donor Lung-specific Exosome Profiles Enable Noninvasive Monitoring of Acute Rejection in a Rodent Orthotopic Lung Transplantation Model.

There is a critical need for development of biomarkers to noninvasively monitor for lung transplant rejection. We investigated the potential of circulating donor lung-specific exosome profiles for time-sensitive diagnosis of acute rejection in a rat orthotopic lung transplant model. Left lungs from Wistar transgenic rats expressing human CD63-GFP, an exosome marker, were transplanted into fully MHC-mismatched Lewis recipients or syngeneic controls. Recipient blood was collected between 4 h and 10 d after transplantation, and plasma was processed for exosome isolation by size exclusion column chromatography and ultracentrifugation. Circulating donor exosomes were profiled using antihuman CD63 antibody quantum dot on the nanoparticle detector and via GFP trigger on the nanoparticle flow cytometer. In syngeneic controls, steady-state levels of circulating donor exosomes were detected at all posttransplant time points. Allogeneic grafts lost perfusion by day 8, consistent with acute rejection. Levels of circulating donor exosomes peaked on day 1, decreased significantly by day 2, and then reached baseline levels by day 3. Notably, decrease in peripheral donor exosome levels occurred before grafts had histological evidence of acute rejection. Circulating donor lung-specific exosome profiles enable an early detection of acute rejection before histologic manifestation of injury to the pulmonary allograft. As acute rejection episodes are a major risk factor for the development of chronic lung allograft dysfunction, this biomarker may provide a novel noninvasive diagnostic platform that can translate into earlier therapeutic intervention for lung transplant patients.

In vitro evaluation of cytotoxic effects of di (2-ethylhexyl) phthalate (DEHP) produced by Bacillus velezensis strain RP137 isolated from Persian Gulf

Phthalates are widely used in polymer science and have potential toxicity related to their chemical structures. However, lots of evidence indicate that phthalate derivatives are undoubtedly produced as secondary metabolites by organisms, including plants, animals, and microorganisms. In the present study, Bacillus velezensis strain RP137 was cultured under optimized conditions. Its biomass was extracted with ethyl acetate with one fraction showing cytotoxic properties. A pure compound was isolated from the active fraction using combined silica gel and LH20 size exclusion column chromatography. Structural evaluation including FT-IR, 1H NMR, 13C NMR, HR-MS and CHN analysis identified the purified compound as di(2-ethylhexyl)phthalate (DEHP) with the formula C24H38O4 and the molecular weight of 389.29 Da. The microorganism-derived (stereospecific) DEHP was strongly reduced the proliferation and induced cytotoxic effects on various eukaryotic cell lines in compare to the synthetic racemic mixture of the compound when assessed by MTT assay. Furthermore, crystal violet assay and morphological changes confirmed the cytotoxic effect of DEHP. Interestingly, non-malignant SV40-immortalized fibroblast cells were less affected by the purified DEHP. Further evaluation on the antibacterial activity of DEHP documented no effect toward Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) pathogens even at a high concentration of 100 μM. In conclusion, existence of DEHP as byproduct of microorganism's metabolism can seriously be considered as a warning to human health.

Geldanamycin Alters Abundance and Composition of Low Molecular Weight Proteins and Multiprotein Complexes as Observed by Size Exclusion Column Chromatography

Size exclusion chromatography (SEC) is a commonly used and minimally disruptive liquid chromatography‐based technique that separates unbound cytosolic proteins and multiprotein complexes according to their perceived molecular weights. The purpose of this project was to develop a method using SEC to partially purify and isolate biomolecular complexes, including those containing the essential molecular chaperone protein hsp90, in the presence and absence of the hsp90 inhibitor geldanamycin. Using an automated SEC program and S‐200 column, unbound proteins and protein complexes of differing molecular weights were isolated from reticulocyte lysate incubated in the presence and absence of geldanamycin. In addition to hsp90 eluting in a complex of ~250–300 kDa, consistent with it being part of a multiprotein chaperone machinery, other predominant protein elution peaks corresponded to ~26 kDa and ~16 kDa. Interestingly, incubating cell lysate with geldanamycin depleted nearly all of the ~26 kDa peak, indicating an unexpected hsp90 effect. Continued examination of eluate fractions from + and − geldanamycin‐incubated lysates has allowed us to further characterize these geldanamycin‐sensitive low molecular weight proteins as we examine the multitude of intracellular hsp90 functional activities.Support or Funding InformationFunding was provided by the Seattle University College of Science & Engineering undergraduate research program.

Natural Toxins of Ageratum conyzoides from Hepatic Vein Occlusive Disease Affected Community in Ethiopia

Pyrrolizidine alkaloids (PAs) are phytotoxins produced by plants as a secondary metabolite against herbivores. PAs account for acute hepatotoxic, mutagenic, carcinogenic and teratogenic effects in humans and animals. In vivo PA intoxications of humans and animals are well documented all over the globe. The current study investigated the toxicological chemical make-up of Ageratum conyzoides L, collected from North Western Zones of Tigray region of Ethiopia, where episodic outbreak of hepatic veno-occlusive disease (HVOD) occurred. During the episodic outbreak multiple hundred deaths were reported due to PAs intoxications. PAs contamination occurred through food items such as millet in the farm fields. This study has led to take interventional measures against the causative agents to cease the morbidity and mortality. The identified PAs possesses the 1:2 double bond in the pyrrole ring necessary for in vivo activation and subsequent liver intoxications. The presence of Angeloyl-platynecine, Angeloyl-retronecine, and the macrocyclic, Seneciphylline alkaloids in A. conyzoides L. is not known previously. Isolation of the different PAs were realized by column chromatography (CC) with silica gel, sephadex-LH-20 for size exclusion CC and cationexchanger (CE). PA detection was performed by TLC with Ehrlich reaction method of Mattocks and their identification with gas-chromatography/mass-spectrometry (GC-MS) equipped with standard PAs library. PA containing plants and compounds should be under the watchlist to prevent similar outbreaks worldwide.

Construction of saturated odd- and even-numbered hyaluronan oligosaccharide building block library

Hyaluronic acid (HA) and its oligosaccharides (o-HAs) have been receiving much attention as medical treatments. Even-numbered o-HAs with two different reducing ends (HA2nAN and HA2nNA, n = 1, 2, 3…) can be prepared using bovine testicular hyaluronidase (BTH) and leech hyaluronidase (LHase) respectively, while preparation of odd-numbered o-HAs (HA2n+1AA and HA2n+1NN, n = 1, 2, 3…) is relatively difficult. A new enzymatic method was developed to prepare odd-numbered o-HAs in this study, taking advantage of differences in the degradation of glycosidic linkages by BTH and LHase. Through quantitative analysis of the yield of even-numbered o-HAs, a dynamic model for LHase hydrolysis was established, providing optimal preparation conditions for HA2nNA (n = 1–5). Then HA2nNA (n = 2–5) was further degraded by BTH to produce odd-numbered o-HAs. The above o-HAs were separated and purified by combining high-performance Q-Sepharose ion-exchange (Q HP) and size exclusion column chromatography steps.

An Application of pET SUMO Protein Expression System in Escherichia coli: Cloning, Expression, Purification, and Characterisation of Native Kras4BG12V Oncoprotein.

Being an important regulator of cell growth and survival, a point mutation at glycine-12 residue of Kras4B to valine (V), renders Kras4BG12V oncogenic. Kras4B recombinant protein is used as a bait to fish its potential ligands in the attempt of drugging this oncoprotein and to validate its pharmacologically relevant ligand in protein-ligand interaction studies. Nevertheless, synthesis of Kras4B recombinant protein is challenging as it was reported being susceptible to aggregation into inclusion bodies in the bacterial host, resulting in a poor yield of recombinant protein. Here, we describe a novel method to produce native Kras4BG12V protein by using pET SUMO protein expression system as a solution to the formation of inclusion bodies. Kras4BG12V oncogene was cloned into pET SUMO vector, followed by a 12 h chemically induced protein expression in Escherichia coli at 20°C. Native Kras4BG12V protein was produced in a series of protein purification steps involving immobilised nickel ion-affinity column chromatography, SUMO fusion protein and polyhistidine tag removal, and size exclusion column chromatography. The identity of the purified Kras4BG12V protein was validated by immunoblot analysis. The purified protein exhibited self-dimerising, indicating that the purified protein structurally resembles Kras4B. Its physical interaction with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a known binder of Kras4B, confirms the identity of the purified protein as Kras4BG12V. The native Kras4BG12V protein was successfully purified in a substantial amount by using the pET SUMO protein expression system.

Construction of a Macrophage-Targeting Bio-nanocapsule-Based Nanocarrier.

The construction protocol of bio-nanocapsule (BNC)-based nanocarriers, named GL-BNC and GL-virosome, for targeted drug delivery to macrophages is described here. First, genes encoding the Streptococcus sp. protein G-derived C2 domain (binds to IgG Fc) and Finegoldia magna protein L-derived B1 domain (binds to Igκ light chain) are prepared by PCR amplification. Subsequently, the genes encoding hepatic cell-specific binding domain of hepatitis B virus envelope L protein are replaced by these PCR products. The expression plasmid for this fused gene (encoding GL-fused L protein) can be used to transform Saccharomyces cerevisiae AH22R- cells. To obtain GL-BNC, the transformed yeast cells are disrupted with glass beads, treated with heat, and then subjected to IgG affinity column chromatography followed by size exclusion column chromatography. In addition, GL-BNCs can be fused with liposomes to form GL-virosome. The targeted delivery of GL-BNC and GL-virosome to macrophages can be confirmed by in vitro phagocytosis assays using the murine macrophage cell line RAW264.7.

Hemostatically potent small molecular weight serine protease from Maclura spinosa (Roxb. ex Willd.) accelerates healing of subcutaneous dermal wounds in Swiss albino mice

Latex of Maclura spinosa has been used by Kollamalayali tribal community of Western Ghats for curdling of milk. In our previous study we have reported the presence of multiple proteases in the latex, among which the serine protease possesses potent role in hemostasis. With a view to further characterize these proteases the current study was taken up. Maclura spinosa latex contains a serine protease which is resolved using molecular size exclusion column chromatography and an anion exchanger resin in a consecutive manner. The specific activity of the enzyme Maclura spinosa latex protease (MSLP) was found to be 56.15 units/mg and recovery to be 2.68% with a fold purity of 0.41. Being a typical serine protease, MSLP is significantly inhibited by PMSF up to 72.97%. The optimum temperature and pH for the enzyme were found to be 50 °C and 8 respectively. Excision wound healing assay in Swiss albino mice using MSLP, showed accelerated wound closure up to 89.35 ± 1.209% compared to 91.938 ± 1.649% shown by positive control. Further PMSF treated MSLP sample did not show considerable wound healing which confirms exclusive involvement of serine proteases. Examination of biochemical markers viz, hydroxy proline content in healing tissue and catalase activity of fatty tissue also ascertain the potential of MSLP in wound healing. Histopathological studies of healing tissue provide confirmatory evidence with dense collagenation of tissue and fibroblast proliferation rendered by MSLP in the respective treated subjects.

193-OR: Glycemic Control via Glucagon Modulation by a Novel Brown Adipose Peptide Hormone

Subcutaneous transplants of embryonic brown adipose tissue (BAT) lead to euglycemia without insulin in mouse models of T1D [Diabetes 2012 61:674]. Euglycemia is accompanied by normalized plasma glucagon levels, restored healthy white adipose tissue, reduced inflammatory cytokines, and increased anti-inflammatory adipokines. Elevated IGF-1 was shown to play a role in successful transplants, putatively by direct stimulation of the insulin receptor [Am. J. Physiol. Endocrinol. Metab. 2015 308:E1043]. However, the mechanism underlying this effect remained unknown. We hypothesized that a secreted product from the transplanted BAT communicates with the recipient allowing a new metabolic equilibrium in the absence of insulin. We tested this using buffer conditioned by cultured immortalized BAT cells. We injected this buffer into diabetic NOD mice once daily for 7 consecutive days. In 5 of the 9 animals studied, blood glucose levels dropped back to the normal range (∼150 mg/dl) for at least 2 months with one animal remaining euglycemic for 5 months with no further intervention. As in embryonic BAT transplants, conditioned buffer injections led to a rapid decrease in plasma glucagon. We found that applying this conditioned buffer to isolated mouse and human islets lowers glucagon secretion directly (without significant changes in insulin or somatostatin). To purify and identify the active BAT-secreted compound, we established a multi-step fractionation protocol using size exclusion, ion exchange, and hydrophobicity columns to concentrate the active compound ∼10 million-fold over the initial BAT-conditioned buffer. Protease, denaturation, and spectroscopy experiments revealed a modified peptide of ∼1600 Da. Initial screening of GPCR targets [Nat Struct Mol Biol. 2015 22:362] revealed binding to the μ-opioid receptor, which is expressed preferentially in α-cells within the islet of Langerhans. Thus, these data suggest a novel path towards clinical antagonism of glucagon secretion. Disclosure D.W. Piston: Consultant; Self; Pfizer Inc. A. Ustione: None. S.C. Gunawardana: None. J. Hughes: None. Funding National Institutes of Health (DK098659); The Leona M. and Harry B. Helmsley Charitable Trust

Structure of a serine-type glutathione S-transferase of Ceriporiopsis subvermispora and identification of the enzymatically important non-canonical residues by functional mutagenesis

Ceriporiopsis subvermispora (C. subvermispora), one of the white-rot fungi, is known as a selective lignin degrader of the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that are capable of catalyzing the reactions involved in detoxification and metabolic pathways. In this study, a GST of C. subvermispora, named CsGST63524, was overexpressed in E. coli, and then purified by affinity, anion exchange, and size exclusion column chromatography. The crystal structures of the CsGST63524 in ligand-free and complex with GSH were refined at 2.45 and 2.50 Å resolutions, respectively. The sulfur atom of glutathione forms a hydrogen bond with Ser21 of CsGST63524, indicating it is a serine-type GST. Mutagenesis of Ser21 unexpectedly indicated that this serine residue is not essential for the enzymatic activity of CsGST63524. Comparative sequence and structural analyses, together with functional mutagenesis, newly identified the enzymatically important non-canonical amino acid residues, Asn23 and Tyr45, other than the serine residue.

A Bioactive Resveratrol Trimer from the Stem Bark of the Sri Lankan Endemic Plant Vateria copallifera.

A new resveratrol trimer, vateriferol (1), having four cis-oriented methine protons and constituting four contiguous stereocenters, was isolated from the bark extract of Vateria copallifera by bioassay-guided fractionation using a combination of normal, reversed phase, and size exclusion column chromatography. The structure was established based on its spectroscopic data. Vateriferol (1) was evaluated in vitro for its antioxidant capacity, enzyme inhibitory activity, growth inhibitory activity on a number of cancer cell lines, neuroprotective activity, and anti-inflammatory activity. Vateriferol (1) exhibited AChE inhibitory activity (IC50 8.4 ± 0.2 μM), ORAC activity (2079 ± 0.20 TE/g), and neuroprotective activity at 1.5 μM using PC12 cells deprived of oxygen and glucose and lowered NO levels in lipopolysaccharide-stimulated SIM-A9 microglial cells at 14.7 and 73.6 μM. Vateriferol (1) exhibited weak cytotoxic potency (<50% growth inhibition) against the tested cell lines at 147.2 μM.

Large-scale preparation of sulfated polysaccharides with anti-angionenic and anti-inflammatory properties from Antrodia cinnamomia

Sulfated polysaccharides (SPSs) were isolated from 0.5mM potassium-sulfate fed Antrodia cinnamomea. We investigated the chemical properties and bio-activities of the five different fractions (SPS-K1, SPS-K2, SPS-K3, SPS-K4, and SPS-K5) with molecular weights ranging from 0.51 to 523.48kDa. SPS-K3 was consisted mainly of glucose, galactose and sulfate in a molar ratio of 15:1:30 with Mn value of 6.82kDa. It showed maximal inhibition of the tumor necrosis factor (TNF-α), interleukin-1beta (IL-1β) and interleukin-6 (IL-6) on bacterial LPS-induced inflammation in RAW264.7 macrophage and the production was recorded as 26.19 and 51.06%, respectively. SPS-K2 showed inhibition of endothelial cell tube formation in an in vitro assay of angiogenesis, and IC50 was determined to be 160.92μg/ml. Large-scale preparation of SPS was performed in the 3-L fermentation of A. cinnamomea and the yield of the SPS was 5.38%. The area percentage of high-molecular-weight SPSs (1470–1590kDa) covered almost half of the SPSs mixture characterized by size exclusion column chromatography. The SPSs from fermented A. cinnamomea had significant inhibition on TNF-α, IL-1β and IL-6 production. This study is the first report to large-scale produce SPSs and demonstrates sulfated galactoglucan with strong anti-inflammatory activity.