3-Allyltrisulfanyl-alanine Formation during the Preparation of Aged Garlic Extract.

The formation of 3-allyltrisulfanyl-alanine (ATrSA) was investigated during the aging process to prepare aged garlic extract (AGE). In raw garlic, ATrSA and its possible precursor, S-allylmercaptocysteine (SAMC), were barely detectable. However, the ATrSA content in AGE increased steadily during the 22 month of aging, while the SAMC level increased to a maximum at 4 months and then gradually decreased. In a model reaction mimicking the AGE preparation process, ATrSA production was decreased when the formation of SAMC was blocked by a γ-glutamyl-transpeptidase inhibitor but its decrease was reversed by the addition of SAMC. We also found that ATrSA was formed by the incubation of SAMC with allylsulfides such as diallyldisulfide and diallyltrisulfide. These findings suggest that ATrSA is formed via the reaction involving SAMC during the aging process. In addition, we found that ATrSA inhibits the secretion of interleukin-6 induced by lipopolysaccharide in mouse splenic lymphocytes in culture.

Chemistry of aged garlic: Diversity of constituents in aged garlic extract and their production mechanisms via the combination of chemical and enzymatic reactions.

Raw garlic contains characteristic compounds, such as S-alk(en)ylcysteine sulfoxides, γ-glutamyl-S-alk(en)-ylcysteines and polysaccharides. These compounds undergo various transformation processes during the aging process. Among these compounds, the change of sulfur-containing molecules is diverse and time-dependent. Previously, by means of the liquid chromatography (LC)/LC-mass spectrometry (MS) method, a number of unidentified peaks corresponding to candidates of sulfur-containing molecules were detected in the chromatogram of aged garlic extract (AGE), and identified using MS and nuclear magnetic resonance (NMR). The production mechanisms of these compounds were then examined by model reactions and laboratory experiments mimicking the aging process. Three γ-glutamyl tripeptides [γ-glutamyl-γ-glutamyl-S-methylcysteine, γ-glutamyl-γ-gluta-myl-S-allylcysteine (GGSAC), γ-glutamyl-γ-glutamyl-S-1-propenylcysteine], γ-glutamyl-S-allylmercaptocysteine (GSAMC) and cis-S-1-propenylcysteine (cis-S1PC) were isolated and identified. GGSAC was produced from GSAC through the enzymatic reaction catalyzed by γ-glutamyltranspeptidase (GGT), and two other tripeptides could be produced in similar reactions. GSAMC was produced by the reaction between γ-glutamyl dipeptides and allicin. Furthermore, GSAMC was a precursor compound of S-allyl-mercaptocysteine (SAMC), and thus it was produced from GSAMC by GGT. cis-S1PC was produced from trans-S1PC by the isomerization reaction. A number of other compounds were also identified, including Maillard reaction products; however, their production mechanisms have not been elucidated. In this review, we present the changes in characteristic constituents in raw garlic and garlic extract during the aging process and discuss their production mechanisms involving the various chemical and enzymatic reactions.

Melanogenesis-Inhibitory and Cytotoxic Activities of Triterpene Glycoside Constituents from the Bark of Albizia procera.

Five oleanane-type triterpene glycosides including three new ones, proceraosides E-G (1-3), were isolated from a MeOH-soluble extract of Albizia procera bark. The structures of 1-3 were determined by use of NMR spectra, HRESIMS, and chemical methods. Compounds 1-5 exhibited inhibitory activities against the proliferation of the A549, SKBR3, AZ521, and HL60 human cancer cell lines (IC50 0.28-1.8 µM). Additionally, the apoptosis-inducing activity of compound 2 was evaluated by Hoechst 33342 staining and flow cytometry, while the effects of 2 on the activation of caspases-9, -8, and -3 in HL60 cells were revealed by Western blot analysis.

Differences in apoptotic signaling and toxicity between dimethylmonothioarsinic acid (DMMTAV) and its active metabolite, dimethylarsinous acid (DMAIII), in HepaRG cells: Possibility of apoptosis cascade based on diversity of active metabolites of DMMTAV.

Dimethylmonothioarsinical acid (DMMTAV), a metabolite of arsenosugars (AsSug) and arsenolipids (AsLP), which are major organoarsenicals contained in seafoods, has been a focus of our attention due to its toxicity. It has been reported that the toxicity of DMMTAV differs according to the host cell type and that dimethylarsinous acid (DMAIII), which is a higher active metabolite of inorganic and organo arsenic compounds, may be the ultimate substance. To further elucidate the details of the mechanisms of DMMTAV, we carried out toxicological characterization by comparing DMMTAV and DMAIII using HepaRG cells, which are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line that retains many characteristics, e.g, primary human hepatocytes including the morphology and expression of key metabolic enzymes (P450 s and GSTs, etc.) and complete expression of all nuclear receptors. HepaRG cells were induced to undergo differentiation by DMSO, which result red in increased levels of metabolic enzymes such as P450 and GST, in non-differentiated cells the cellular toxicities of DMMTAV and DMAIII were reduced and the induction of toxicity by DMMTAV was increased by GSH but not by DMAIII. Both DMAIII and DMMTAV induce apoptosis and increase caspase 3/7 activity. DMAIII exposure increased the activity of caspase-9. On the contrary, DMMTAV exposure resulted in markedly elevated activity of caspase-8 as well as caspase-9. These results suggest there are differences between the signaling pathways of apoptosis in DMAIII and DMMTAV and that between their active metabolites. Consequently, the ultimate metabolic substance of toxicity induction of DMMTAV may not only be DMAIII, but may also be partly due to other metabolic substances produced through the activation mechanism by GSH.

Antitumor Effect of Burchellin Derivatives Against Neuroblastoma.

BACKGROUND: Neuroblastoma is one of the most commonly encountered malignant solid tumors in the pediatric age group. We examined the antitumor effects of five burchellin derivatives against human neuroblastoma cell lines. MATERIALS AND METHODS: We evaluated cytotoxicity by the MTT assay for four human neuroblastoma and two normal cell lines. We also performed analysis of the apoptotic induction effect by flow cytometry, and examined the expression levels of apoptosis- and cell growth-related proteins by western blot analysis. RESULTS: We found that one of the burchellin derivatives (compound 4) exerted cytotoxicity against the neuroblastoma cell lines. Compound 4 induced caspase-dependent apoptosis via a mitochondrial pathway. The apoptosis mechanisms induced by compound 4 involved caspase-3, -7 and -9 activation and poly (ADP-ribose) polymerase cleavage. In addition, compound 4 induced cell death through inhibition of the cell growth pathway (via extracellular signal-regulated kinase 1 and 2, AKT8 virus oncogene cellular homolog, and signal transducer and activator of transcription 3). CONCLUSION: Compound 4 exerted cellular cytotoxicity against neuroblastoma cells via induction of caspase-dependent apoptosis, and may offer promise for further development as a useful drug for the treatment of advanced neuroblastoma.

Induction of cell death in pancreatic ductal adenocarcinoma by indirubin 3'-oxime and 5-methoxyindirubin 3'-oxime in vitro and in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a poor prognosis. To identify potential effective therapeutic drugs for PDAC, we established a screening system based on spheroid formation using 170#3 mouse PDAC cells with or without fibroblasts. We found that indirubin 3'-oxime (Indox) and 5-methoxyindirubin 3'-oxime (5MeOIndox) inhibited PDAC cell proliferation. Furthermore, PDAC xenograft growth was also inhibited in BALB/c nu/nu mice after administration of Indox and 5MeOIndox. Both phosphorylated CDK1 and cyclin B1 levels in 170#3 cells were significantly reduced by treatment with Indox and 5MeOIndox in vitro and in vivo. Cell cycle analysis revealed that 5MeOIndox, but not Indox, induced G2/M arrest. Annexin V-propidium iodide double-staining analysis demonstrated that Indox induced abundant non-apoptotic cell death of 170#3 cells, while 5MeOIndox predominantly induced early apoptosis, indicating that the cytotoxicity of 5MeOIndox is lower than that of Indox. These results suggest that one mechanism of 5MeOIndox is to induce G2/M arrest of PDAC cells via inhibition of CDK1/cyclin B1 levels, thereby leading to apoptosis. Our findings suggest 5MeOIndox as a potential useful anticancer agent in PDAC.

Triterpenoid glycosides from Ladenbergia hexandra Klotzsch.

From the bark of Ladenbergia hexandra Klotzsch, ten triterpenoid glycosides were isolated along with five known compounds, and their structures were determined based on extensive NMR and mass spectroscopic, GC and HPLC analyses. Some triterpenoid glycosides contained 6-deoxy-D-allose or D-allose as a sugar moiety. The absolute stereochemical assignment of the sugars was determined by comparison with synthetic samples, as well as by GC and HPLC analysis.

Silver nanoparticle functionalized glass fibers for combined surface-enhanced Raman scattering spectroscopy (SERS)/surface-assisted laser desorption/ionization (SALDI) mass spectrometry via plasmonic/thermal hot spots.

We presented the fabrication of a silver nanoparticle (Ag NP) functionalized glass fiber (Ag-GF) substrate for combined surface-enhanced Raman scattering spectroscopy (SERS)/surface-assisted laser desorption/ionization (SALDI) mass spectrometry. Ag NPs were immobilized onto the surface of glass fibers through a simple sputter deposition process. The SERS and SALDI activities strongly depended on the nanostructures of the deposited Ag NPs on the GFs. The closely-packed Ag NPs with a size of 20-50 nm and an inter-particle nanoscale gap of less than 10 nm were effective for the simultaneously enhanced SERS/SALDI substrate via plasmonic/thermal "hot spots", while the interconnected continuous Ag film reduced both the SERS/SALDI activities. The SERS enhanced factor (EFSERS) and SALDI enhanced factor (EFSALDI) were newly proposed. Finally, the concentration-dependent signal intensities of SERS and SALD-MS of sulfur compounds using an identical Ag NP-GF substrate were examined, and the linear dependence relationship in the log-log plot was demonstrated for the combined quantitative SERS/SALDI-MS analysis.

Biological Activities of Triterpenoids and Phenolic Compounds from Myrica cerifera Bark.

Seven triterpenoids, 1 - 7, two diarylheptanoids, 8 and 9, four phenolic compounds, 10 - 13, and three other compounds, 14 - 16, were isolated from the hexane and MeOH extracts of the bark of Myrica cerifera L. (Myricaceae). Among these compounds, betulin (1), ursolic acid (3), and myricanol (8) exhibited cytotoxic activities against HL60 (leukemia), A549 (lung), and SK-BR-3 (breast) human cancer cell lines (IC50 3.1 - 24.2 µm). Compound 8 induced apoptotic cell death in HL60 cells (IC50 5.3 µm) upon evaluation of the apoptosis-inducing activity by flow cytometric analysis and by Hoechst 33342 staining method. Western blot analysis on HL60 cells revealed that 8 activated caspases-3, -8, and -9 suggesting that 8 induced apoptosis via both mitochondrial and death receptor pathways in HL60. Upon evaluation of the melanogenesis-inhibitory activity in B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), erythrodiol (7), 4-hydroxy-2-methoxyphenyl ß-d-glucopyranoside (13), and butyl quinate (15) exhibited inhibitory effects (65.4 - 86.0% melanin content) with no, or almost no, toxicity to the cells (85.9 - 107.4% cell viability) at 100 µm concentration. In addition, 8, myricanone (9), myricitrin (10), protocatechuic acid (11), and gallic acid (12) revealed potent DPPH radical-scavenging activities (IC50 6.9 - 20.5 µm).

Cytotoxic and Apoptosis-inducing Activities of Taraxastane-type Triterpenoid Derivatives in Human Cancer Cell Lines.

Twenty-eight taraxastane-type triterpenoid derivatives 4 - 31 were prepared from the naturally occurring triterpenoids faradiol (1) and heliantriol C (3). The cytotoxic activities of these compounds and arnidiol (2) were evaluated in leukemia (HL60), lung (A549), duodenal (AZ521), and breast (SK-BR-3) cancer cell lines. 21-Oxoarnidiol (18) and faradiol 3,16-di-O-l-alaninate (31) exhibited potent cytotoxicity, with 50% inhibitory concentrations of 0.5 - 2.7 µm. In particular, flow cytometric analysis indicated that compound 31 induced typical apoptotic cell death in HL60 cells. These results suggested that taraxastane-type triterpenoid derivatives might provide useful antitumor agents with apoptosis-inducing activity.

Indirubin 3'-Epoxide Induces Caspase-Independent Cell Death in Human Neuroblastoma.

Indirubin inhibits cyclin-dependent kinases by binding to their ATP-binding site, thereby exerting potent cytotoxicity on some tumor cells. We examined the anti-tumor effect of indirubin 3'-epoxide on human neuroblastoma cell lines (IMR-32, SK-N-SH, and NB-39). The results revealed potent cytotoxicity of indirubin 3'-epoxide against the IMR-32 (IC50: 0.16 µM) and SK-N-SH (IC50: 0.07 µM) cells. Furthermore, it also induced an increase of the sub-G1 population in the IMR-32 cells. Examination by Hoechst 33342 staining revealed apoptosis characterized by cell shrinkage, nuclear condensation and nuclear fragmentation in a concentration-dependent manner. Furthermore, annexin V-propidium iodide (PI) double-staining revealed an increase in the percentage of early apoptotic cells following treatment of the cells with indirubin 3'-epoxide without activation of caspases. In addition, significant decreases in the protein level of survivin and poly(ADP-ribose)polymerase (PARP), and increase in that of apoptosis-inducing factor (AIF) were found in the nuclei of the cells. These results suggest that indirubin 3'-epoxide induced caspase-independent apoptosis through mechanisms involving DNA fragmentation and inhibition of DNA repair.

Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells.

Neuroblastoma is among the most fatal of solid tumors in the pediatric age group, even when treated aggressively. Therefore, a new effective therapeutic drug(s) for neuroblastoma is urgently needed. To clarify the anticancer effects of the sesquiterpene lactones dehydrocostus lactone and costunolide, derived from Saussurea lappa, we examined the cytotoxic and migration/invasion-inhibitory effects of these compounds against neuroblastoma cell lines. Both the compounds exerted significant cytotoxicity against the neuroblastoma cell lines IMR-32, NB-39, SK-N-SH, and LA-N-1. Evidence of cellular apoptosis, such as nuclear condensation and membrane inversion, were observed after treatment with these compounds. Both compounds induced caspase-7 activation and PARP cleavage as confirmed by Western blotting. Furthermore, the sesquiterpene lactones also suppressed invasion and migration of the neuroblastoma cells. These results suggest that dehydrocostus lactone and costunolide are promising candidates for being developed into novel anticancer drugs effective against neuroblastoma.

Melanogenesis-inhibitory activity and cancer chemopreventive effect of glucosylcucurbic acid from shea (Vitellaria paradoxa) kernels.

Two jasmonate derivatives, glucosylcucurbic acid (1) and methyl glucosylcucurbate (2), were isolated from the MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels. These and their deglucosylated derivatives, cucurbic acid (3) and methyl cucurbate (4), were evaluated for their melanogenesis-inhibitory and cancer chemopreventive potencies. Compounds 1, 3, and 4 exhibited potent melanogenesis-inhibitory activities in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells. Western-blot analysis revealed that compounds 1 and 3 reduced the protein levels of MITF (=microphthalmia-associated transcription factor), tyrosinase, TRP-1 (=tyrosine-related protein 1), and TRP-2 mostly in a concentration-dependent manner. In addition, compound 1 exhibited inhibitory effects against Epstein-Barr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, against TPA-induced inflammation in mice, and against skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter.

Triterpene glycosides and other polar constituents of shea (Vitellaria paradoxa) kernels and their bioactivities.

The MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels was investigated for its constituents, and fifteen oleanane-type triterpene acids and glycosides, two steroid glucosides, two pentane-2,4-diol glucosides, seven phenolic compounds, and three sugars, were isolated. The structures of five triterpene glycosides were elucidated on the basis of spectroscopic and chemical methods. Upon evaluation of the bioactivity of the isolated compounds, it was found that some or most of the compounds have potent or moderate inhibitory activities against the following: melanogenesis in B16 melanoma cells induced by α-melanocyte-stimulating hormone (α-MSH); generation of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-teradecanoylphorbol 13-acetate (TPA) in Raji cells; t TPA-induced inflammation in mice, and proliferation of one or more of HL-60, A549, AZ521, and SK-BR-3 human cancer cell lines, respectively. Western blot analysis established that paradoxoside E inhibits melanogenesis by regulation of expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1) and TRP-2. In addition, tieghemelin A was demonstrated to exhibit cytotoxic activity against A549 cells (IC50 13.5 µM) mainly due to induction of apoptosis by flow cytometry. The extract of defatted shea kernels and its constituents may be, therefore, valuable as potential antioxidant, anti-inflammatory, skin-whitening, chemopreventive, and anticancer agents.

Gold-decorated titania nanotube arrays as dual-functional platform for surface-enhanced Raman spectroscopy and surface-assisted laser desorption/ionization mass spectrometry.

In this report, we demonstrate gold-decorated titania nanotube arrays (Au-TNA substrate) as a dual-functional platform for surface-enhanced Raman spectroscopy (SERS) and surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). The Au nanoparticles are grown on the substrate using vapor deposition of Au. The resulting substrates perform better than Au colloids in terms of the reproducibility of the SERS measurements, long-term stability of the fabricated structures, and clean surface of the Au. The nanostructure of the Au-TNA substrate was designed to optimize the SALDI-MS and SERS performance. Excellent reproducibility of the SERS measurements using the Au-TNA substrate was obtained, with a standard error less than 6 %. SALDI activity was also demonstrated for the same Au-TNA substrates. Finally, the Au-TNA substrate was used for combined SERS and SALDI-MS analysis (i) to discriminate the structural isomers of pyridine compounds (para-, meta-, and ortho-pyridinecarboxylic acid) and (ii) to detect polycarbamate, a dithiocarbamate fungicide. These results are difficult to obtain using either approach alone.

Computer-aided parachute guiding system for closed reduction of diaphyseal fractures.

BACKGROUND: Closed reduction is of great benefit for fracture healing. However, achieving this without sacrificing the reduction accuracy and exposing the surgeon and patient to excessive radiation is difficult. METHODS: A novel parachute guiding system (ParaEx System) was developed for closed reduction of fractures based on computed tomography data. The system included two counter guides with stainless tubular markers that could be attached to the unilateral external fixator. Comminuted tibial diaphyseal fracture models were used to validate the ParaEx System. RESULTS: The mean errors (and standard deviations) of residual rotational and translational deformity were 0.67° ± 0.45°, 0.92° ± 1.00°, and 0.64° ± 0.50° in rotation and 1.30 ± 1.10 mm, 1.13 ± 0.70 mm, and 0.94 ± 0.92 mm in translation about the X, Y, and Z axes of the local coordinate axes, respectively. CONCLUSIONS: The ParaEx System was useful for accurate closed reduction of fractures at low cost.

Cytotoxic activities of amino acid-conjugate derivatives of abietane-type diterpenoids against human cancer cell lines.

Nine amino acid conjugate derivatives, each 2-10 and 12-20, were prepared from abietic acid (1) and dehydroabietic acid (11), respectively, and they were evaluated for their cytotoxicities against four human cancer cell lines, i.e., leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3). All compounds showed cytotoxicities against HL60 with IC50 values in the range of 2.3-37.3 µM. In addition, most of the derivatives exhibited moderate cytotoxicities against the other cancer cell lines. Among the derivatives, methyl N-[18-oxoabieta-8,11,13-trien-18-yl]-L-tyrosinate (19) exhibited potent cytotoxic activities against four cancer cell lines with IC50 values of 2.3 (HL60), 7.1 (A549), 3.9 (AZ521), and 8.1 µM (SK-BR-3). Furthermore, all derivatives were shown to possess high selective cytotoxic activities for leukemia cells, since they exhibited only weak cytotoxicities against normal lymphocyte cell line RPMI1788.

In vivo kinematics of high-flex mobile-bearing total knee arthroplasty, with a new post-cam design, in deep knee bending motion.

PURPOSE: The objective of this study was to evaluate the in vivo knee kinematics to assess the available functional motion of the characteristic mobile-bearing prosthesis design and to examine whether the artificial joint would work in vivo according to its design concept. METHODS: We studied 14 knees (11 patients) implanted with the Vanguard RP Hi-Flex prosthesis. This prosthesis has a highly original form of post-cam called a PS saddle design with high compatibility, and with a rotating plate mobile-bearing mechanism. The cylinder-type post-cam is designed to enable contact in early flexion ranges, and to prevent paradoxical anterior femoral component movement. Each patient performed weight-bearing deep knee bending under fluoroscopic surveillance. Motion between each component including the polyethylene insert was analyzed using the 2D/3D registration technique. RESULTS: The mean range of motion was 122.0°. The mean femoral component rotation for the tibial tray was 5.0°. No paradoxical anterior movement of the nearest point was confirmed between the femoral component and the tibial tray in the early flexion ranges. Initial contact of the post-cam was confirmed at a knee flexion angle of 33.8°. Subsequently, the wide contact of the post-cam was maintained until flexion reached 120° in all knees, but disengagement of the post-cam was observed in two knees when flexion was ≥130°. CONCLUSIONS: The results of this study demonstrated that the prosthesis design generally works in vivo as intended by its design concept. The present kinematic data may provide useful information for improvement of high-flex type prostheses.