Chin Hsu

Isolation and Identification of Cucurbitane-Type Triterpenoids with Partial Agonist/Antagonist Potential for Estrogen Receptors from Momordica charantia

This study aims at investigating the estrogenic activity and active cucurbitane-type triterpenoid compounds of bitter gourd (Momordica charantia, MC) using a transactivation assay for estrogen receptors (ER) α and β. The lyophilized fruits of MC were exhaustively extracted with ethyl acetate (EA) and 95% ethanol (EtOH), sequentially. The nonsaponifiable fraction (NS) of the EA extract as well as the acid hydrolyzed EtOH extract (AH) was fractionated and isolated by repeated column chromatography and further purified by preparative HPLC or RP-HPLC. One known compound, 5β,19-epoxycucurbita-6,24-diene-3β,23ξ-diol (6), was isolated from the NS, and five new compounds (1-5) were isolated from AH and identified as cucurbita-6,22(E),24-trien-3β-ol-19,5β-olide (1), 5β,19-epoxycucurbita-6,22(E),24-triene-3β,19-diol (2), 3β-hydroxycucurbita-5(10),6,22(E),24-tetraen-19-al (3), 19-dimethoxycucurbita-5(10),6,22(E),24-tetraen-3β-ol (4), and 19-nor-cucurbita-5(10),6,8,22(E),24-pentaen-3β-ol (5). In the noncytotoxic concentration range, compounds 1, 2, 5 and 6 showed weak agonistic activity via ER α and β. Compounds 1, 2, 3 and 6 significantly antagonized the transactvation of 17β-estradiol (E(2)) via both ER α and β. In conclusion, this study demonstrates, for the first time as far as we know, the partial agonist/antagonist activity via ER of four new and one known cucurbitane-type triterpenoids from MC. Further studies are worthy to explore the selective estrogen receptor modulator (SERM) activity of MC.

Phytoestrogenic Compounds in Alfalfa Sprout (Medicago sativa) beyond Coumestrol

Coumestrol has long been known as the phytoestrogenic compound in alfalfa. However, it has been demonstrated that the ethyl acetate extract of alfalfa sprout (AEA) attenuated the disease severity and increased survival and life span of autoimmune-prone MRL-lpr/lpr mice. Coumestrol, on the contrary, decreased the survival. This study thus aimed to isolate and identify phytoestrogenic compounds other than coumestrol in AEA. AEA was fractionated and separated by successive silica gel chromatography and preparative HPLC. The activity of collected fractions was tracked by a transactivation assay for ERα and ERβ, respectively. In addition to coumestrol, liquiritigenin, isoliquiritigenin, loliolide, and (4S,6S)- and (4R,6S)-4-hydroxy-6-pentadecyltetrahydropyr-2-one were isolated and chemically identified. Except for loliolide, these compounds showed higher transactivation via ERβ than via ERα. The maximal activation via ERα of coumestrol reached 80% that of 1 nM 17β-estradiol (E(2)), whereas the activations of the remaining five compounds as well as AEA ranged from 8 to 49%. In addition, isoliquiritigenin, loliolide, and (4S,6S)- and (4R,6S)-4-hydroxy-6-pentadecyltetrahydropyr-2-one, but not coumestrol, preferentially inhibited 1 nM E(2) induced ERα activation, compared to that ERβ activation. The selectivity of these phytoestrogens might account for the difference between the effects of AEA and coumestrol in autoimmune-prone MRL-lpr/lpr mice observed previously.

A novel mechanism of cytokine release in phagocytes induced by aggretin, a snake venom C-type lectin protein, through CLEC-2 ligation

Summary  Background: Macrophages are major immune cells and play an important role in modulating homeostasis and the immune defense mechanism. In inflammatory responses to the infection of pathogens, macrophages are activated, producing various inflammatory mediators. Snake venom C‐type lectin proteins (snaclecs) have diverse targets, including platelet GPVI, GPIb, integrin α2β1 or CLEC‐2 expressed in platelets, endothelial cells or myeloid cells. Methods: In this study, murine macrophages (RAW 264.7 cells) and human monocytes (THP‐1) were treated with different snaclecs, including aggretin, gramicetin, trowaglerix and convulxin, in the absence or presence of LPS for 24 h. Results: The production of cytokines, such as tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6), in supernatants was measured by ELISA. Aggretin increased the production of TNF‐α and IL‐6 in both RAW264.7 and THP‐1 cells; however, the other snaclecs did not. Aggretin induced extracellular signal‐regulated kinase 1/2 (ERK1/2) and c‐Jun N‐terminal kinase (JNK) tyrosine phosphorylation of RAW264.7 cells. Pretreatments with inhibitor of ERK, JNK, p38 or NF‐κB abolished cytokine release caused by aggretin. Aggretin bound to THP‐1 cells in a concentration‐dependent manner and it displaced the CLEC‐2 mAb binding to THP‐1 cells and the immobilized aggretin selectively bound to CLEC‐2 of both platelets and THP‐1 cell lysates. Furthermore, aggretin elevated the plasma level of IL‐6 in ICR mice as it was administered intramuscularly. Conclusion: These results indicate that aggretin may induce cytokine TNF‐α/IL‐6 release via interacting with CLEC‐2 receptor and the subsequent MAPK and NF‐κB activation in monocytes/macrophages.

Metabolite identification for mass spectrometry-based metabolomics using multiple types of correlated ion information.

Metabolite identification remains a bottleneck in mass spectrometry (MS)-based metabolomics. Currently, this process relies heavily on tandem mass spectrometry (MS/MS) spectra generated separately for peaks of interest identified from previous MS runs. Such a delayed and labor-intensive procedure creates a barrier to automation. Further, information embedded in MS data has not been used to its full extent for metabolite identification. Multimers, adducts, multiply charged ions, and fragments of given metabolites occupy a substantial proportion (40-80%) of the peaks of a quantitation result. However, extensive information on these derivatives, especially fragments, may facilitate metabolite identification. We propose a procedure with automation capability to group and annotate peaks associated with the same metabolite in the quantitation results of opposite modes and to integrate this information for metabolite identification. In addition to the conventional mass and isotope ratio matches, we would match annotated fragments with low-energy MS/MS spectra in public databases. For identification of metabolites without accessible MS/MS spectra, we have developed characteristic fragment and common substructure matches. The accuracy and effectiveness of the procedure were evaluated using one public and two in-house liquid chromatography-mass spectrometry (LC-MS) data sets. The procedure accurately identified 89% of 28 standard metabolites with derivative ions in the data sets. With respect to effectiveness, the procedure confidently identified the correct chemical formula of at least 42% of metabolites with derivative ions via MS/MS spectrum, characteristic fragment, and common substructure matches. The confidence level was determined according to the fulfilled identification criteria of various matches and relative retention time.

The highly specific platelet glycoprotein (GP) VI agonist trowaglerix impaired collagen-induced platelet aggregation ex vivo through matrix metalloproteinase-dependent GPVI shedding

Summary.  Background: C‐type lectin proteins (CLPs) have diverse targets including platelet GPIb, GPVI and integrin α2β1, and affect platelet function in a various way. In this study, we characterized a huge, heterodimeric venom protein, trowaglerix, which belongs to the CLP family. Methods: We purified a potent platelet‐aggregation inducer, trowaglerix, from the crude venom of Tropidolaemus wagleri. Biotinylated trowaglerix was used for binding assays, and immunoblotting was used to investigate the signal transduction involved. Results: Two distinct subunits of trowaglerix with similar masses of around 16 kDa were eluted by high‐performance liquid chromatography after reduction and alkylation. Trowaglerix induced platelet aggregation of washed human platelets and platelet‐rich plasma (PRP) in a concentration‐dependent manner. Biotinylated trowaglerix specifically bound to platelet membrane GPVI, but not to GPIb or α2 integrin. Treatment with trowaglerix induced GPVI loss in human platelets in vitro and impaired the platelet aggregation of mouse PRP ex vivo in response to collagen but not in response to adenosine diphosphate (ADP). However, GM6001, a matrix metalloproteinase (MMP) inhibitor, inhibited trowaglerix‐induced GPVI cleavage and restored the platelet responsiveness of PRP to collagen. Conclusions: Trowaglerix activates platelets through specific binding to GPVI, leading to kinases‐dependent exposure of functional αIIbβ3 and platelet aggregation, and also induces MMP‐dependent GPVI shedding from platelets.

Improvements in endotoxemic syndromes using a disintegrin, rhodostomin, through integrin αvβ3‐dependent pathway

Summary.  Background and objectives: Septic shock is a major cause of morbidity and mortality in intensive care units, but there is still no effective therapy for the patients. We evaluated the effects of rhodostomin (Rn), an Arg‐Gly‐Asp‐containing snake venom disintegrin, on lipopolysaccharide (LPS)‐activated phagocytes in vitro and LPS‐induced endotoxemia in vivo. Methods and results: Rn inhibited adhesion, migration, cytokine production and mitogen‐activated protein kinase (MAPK) activation of macrophage induced by LPS. Flow cytometric analysis revealed that Rn specifically blocked anti‐αv mAb binding to RAW264.7. Besides inhibiting MAPK activation of THP‐1, Rn bound to LPS‐activated THP‐1 and specifically blocked anti‐αvβ3 mAb binding to THP‐1. Binding assays proved that integrin αvβ3 was the binding site for rhodostomin on phagocytes. Rn reversed the enhancement of fibronectin and vitronectin on LPS‐induced monocyte adhesion and cytokine release. Transfection of integrin αv siRNA also inhibited LPS‐induced activation of monocyte, and Rn exerted no further inhibitory effect. Furthermore, Rn significantly decreased the production of tumor necrosis factor‐α (TNF‐a), interleukin (IL)‐6, ‐1β and ‐10 and attenuated cardiovascular dysfunction, including blood pressure and heart pulse, and thrombocytopenia in LPS‐induced endotoxemic mice. Rn also protected against tissue inflammation as evidenced by histological examination. Conclusions: Rn may interact with αvβ3 integrin of monocytes/macrophages leading to interfere with the activation of phagocytes triggered by LPS. These results suggest that the protective function of Rn in LPS‐induced endotoxemia may be attributed to its anti‐inflammation activities in vivo.

Role of GLP-1 in the Hypoglycemic Effects of Wild Bitter Gourd

This study aimed to examine the role of GLP-1 in the hypoglycemic activity of wild bitter gourd (Momordica charantia L., BG). In vitro, the GLP-1 secretion in STC-1, a murine enteroendocrine cell line, was dose dependently stimulated by water extract (WE), its fractions (WEL, >3 kD and WES, <3 kD), and a bitter compounds-rich fraction of BG. These stimulations were partially inhibited by probenecid, a bitter taste receptor inhibitor, and by U-73122, a phospholipase Cβ2 inhibitor. These results suggested that the stimulation might involve, at least in part, certain bitter taste receptors and/or PLCβ2-signaling pathway. Two cucurbitane triterpenoids isolated from BG, 19-nor-cucurbita-5(10),6,8,22-(E),24-pentaen-3β-ol, and 5β,19-epoxycucurbita-6,24-diene-3β,23ξ-diol (karavilagenine E,) showed relative high efficacy in the stimulation. In vivo, mice fed BG diet showed higher insulinogenic index in an oral glucose tolerance test. A single oral dose of WE or WES pretreatment significantly improved intraperitoneal glucose tolerance. A single oral dose of WES significantly decreased glucose and increased insulin and GLP-1 in serum after 30 min. This acute hypoglycemic effect of WES was abolished by pretreatment with exendin-9, a GLP-1 receptor antagonist. Our data provide evidence that BG stimulates GLP-1 secretion which contributes, at least in part, to the antidiabetic activity of BG through an incretin effect.

Inhibitory effects of polypeptides derived from a snake venom C‐type lectin, aggretin, on tumor cell‐induced platelet aggregation

Podoplanin, a transmembrane sialoglycoprotein, is expressed by lymphatic endothelial cells and many tumor cells, and is involved in tumor cell‐induced platelet aggregation and tumor metastasis. A recent study found that C‐type lectin‐like receptor 2 (CLEC‐2) is a physiologic receptor for podoplanin. Previous studies showed that aggretin, a snake venom‐derived protein, activates platelets by targeting platelet CLEC‐2. We hypothesized that the C‐terminal fragment of aggretin may bind to platelet CLEC‐2 and displace podoplanin, in turn exerting antitumor metastatic effects.

Synthesis of nanoparticles using an atmospheric pressure plasma jet

Summary form only given. Nanocrystalline metal oxide particles are synthesized by an atmospheric pressure plasma jet (APPJ). The APPJ is sustained using a repetitive pulse source with O2 and N2 as the plasma gas. Precursors of several types are used to synthesize simple and complex metal oxides via multiple approaches to assess the potential using such a process to fabricate nanoparticles. The first approach uses Zn(NO3)2 and NaNO3 solution to fabricate ZnO nanoparticles using the salt-assisted spray pyrolysis-like process. The ultrasonically nebulized precursor is carried into the downstream of the APPJ using a carrier gas. This process is able to fabricate nanocrystalline ZnO particles within a short contact time (a few ms) between the precursor and the plasma jet. When N2 plasma is used, N-doped ZnO is formed and the doping level can be controlled using the plasma gas flow rate. The second approach utilizes nebulized the LiNO3 and Titanium bis(ammonium lactate)-dehydroxide solution as the precursor to fabricate Li4Ti5O12 using O2 and N2 plasmas. The fabricated powders include Li4Ti5O12. TiO2, and Li2TiO3. The third approach uses titanium tetraisopropoxide as the precursor. The precursor is injected into the system using a heated bubbler. With this approach, nearly mono-dispersed N-doped TiO2 nanoparticles are synthesized using N2 plasmas. Preliminary studies show that the first approach is the most stable process while the approach using organic metal precursors exhibits the highest throughput. In this presentation, key features of different approaches and key factors that dominate the fabricated nanoparticle characteristics will be discussed.

Decomposition of cellulose by plasma in salt solutions

Plasmas in salt solution are able to generate active species and can be used to decompose organic compounds. In this study, plasmas driven by AC or DC power sources in various saline-solutions are used to decompose cellulose. The solutions used include NaCl, NaNO3, NaOH, Zn(NO3)2, ZnCl2, CaCl2, Ca(NO3)2, and Ca(OH)2. The electrode at which the plasma is ignited is a platinum wire 0.5 mm in diameter covered by a glass tube while the grounding electrode is a bare platinum wire of the same diameter. The plasma voltage and current waveforms are monitored using electrical probes. Optical emission spectrometer is used to monitor the time-averaged emission spectra emanating from the plasma. The conductivity and the pH of the solution in which the plasma is ignited are monitored using commercially available meters. The decomposed products are identified and quantified using high performance liquid chromatography. It is shown that the plasma is able to effectively decompose cellulose into smaller molecules, such as glucose, fructose, glycolic acid, and several unknown products. The decomposition efficiency increases with the salt concentration. When different salt solution is used, it shows significantly different decomposition efficiencies and decomposed products. This observation strongly suggests that the decomposition process using plasmas in salt solution is chemical in nature and obtaining reaction selectivity is highly possible. Preliminary studies show that Ca(OH)2 and ZnCl2 solutions can most effectively decompose cellulose under optimized conditions.