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The cytotoxic activities of several diterpenoid constituents of the medicinal plant, “hempedu bumi,” or Andrographis paniculata Nees (Acanthaceae), were evaluated. The seven diterpenoid compounds used were andrographolide, 14-deoxyandrographolide, andrographiside, deoxyandrographiside, 14-deoxy-12-methoxyandrographolide, neoandrographolide, and 14-deoxy-11,12-didehydroandrographolide. The activities of these compounds were evaluated with various human tumor cell lines such as Caov-3 (human ovarian carcinoma cell line), T-47D (human breast carcinoma cell line), Hs-578T (human breast carcinoma cell line), Hep G2 (human hepatocarcinoma cell line), and NCI–H23 (human lung adenocarcinoma cell line). Cell survival was measured using the MTS assay after 72 h of incubation. Andrographolide, neoandrographolide, andrographiside, deoxyandrographiside, and 14-deoxy-12-methoxyandrographolide appeared to be noncytotoxic against all the cell lines. Only 14-deoxyandrographolide and 14-deoxy-11,12-didehydroandrographolide exhibited cytotoxic activities (based on EC 50 values), but this was limited to the T-47D cell line (EC 50 values of 2.8 µg/ml and 1.5 µg/ml, respectively). As one of the principle diterpenoids of Andrographis paniculata , 14-deoxy-11,12-didehydroandrographolide appeared to be the most potent when compared with the rest of the compounds examined. The effects of 14-deoxy-11,12-didehydroandrographolide on T-47D cells were further confirmed to be nonapoptotic, non-necrotic, but programmed in nature, as demonstrated using a DNA fragmentation detection assay, Trypan blue exclusion assay, and annexin V–propidium iodide staining. [ABSTRACT FROM AUTHOR]

In order to evaluate the state of activation in a cyclotron facility used for the radioisotope production of PET diagnostics, we measured the neutron flux by using gold foils and TLDs. Then, the spatial distribution of neutrons and induced activity inside the cyclotron vault were simulated with the Monte Calro calculation code for neutron transport and DCHAIN-SP for activation calculation. The calculated results are in good agreement with measured values within factor 3. Therefore, the adaption of the advanced evaluation procedure for activation level is proved to be important for the planning of decommissioning of these facilities.

Background: The effect of velamentous cord insertion (VCI) on perinatal outcomes in twin pregnancies is unclear due to conflicting findings. This retrospective study aimed to examine VCI prevalence and related risk factors in twin pregnancies and its association with adverse perinatal outcomes., Methods: Women with twin pregnancies who delivered between January 2012 and December 2021 in a single tertiary hospital were included. The participants were divided into dichorionic (DC) and monochorionic diamniotic (MCDA) groups, and their maternal and fetal characteristics and VCI rates were compared. Logistic regression models were used to identify risk factors for VCI and VCI-related perinatal outcomes., Results: Among the 694 twin pregnancies included in this study, the VCI rate was significantly higher in MCDA than in DC twins. Body mass index and MCDA twins were significant risk factors for VCI, whereas assisted reproductive technology pregnancy was a significant protective factor against VCI. In DC twins, VCI did not affect perinatal outcomes. In MCDA twins, VCI was a significant risk factor for fetal growth restriction, twin-to-twin transfusion syndrome, and preterm birth at <36 weeks., Conclusions: VCI was a prominent risk factor for adverse perinatal outcomes only in MCDA twins. Antenatal sonographic assessment of the umbilical cord insertion site would be beneficial.

Rapid radiation associated with phenotypic divergence and convergence provides an opportunity to study the genetic mechanisms of evolution. Here we investigate the genus Takifugu that has undergone explosive radiation relatively recently and contains a subset of closely-related species with a scale-loss phenotype. By using observations during development and genetic mapping approaches, we show that the scale-loss phenotype of two Takifugu species, T. pardalis Temminck & Schlegel and T. snyderi Abe, is largely controlled by an overlapping genomic segment (QTL). A search for candidate genes underlying the scale-loss phenotype revealed that the QTL region contains no known genes responsible for the evolution of scale-loss phenotype in other fishes. These results suggest that the genes used for the scale-loss phenotypes in the two Takifugu are likely the same, but the genes used for the similar phenotype in Takifugu and distantly related fishes are not the same. Meanwhile, Fgfrl1 , a gene predicted to function in a pathway known to regulate bone/scale development was identified in the QTL region. Since Fgfr1a1 , another memebr of the Fgf signaling pathway, has been implicated in scale loss/scale shape in fish distantly related to Takifugu , our results suggest that the convergence of the scale-loss phenotype may be constrained by signaling modules with conserved roles in scale development.

A methanol extract of the leaves of Cephalotaxus harringtonia var. nana and its ethyl acetate (EtOAc)-soluble fraction demonstrated strong antitumor activity against A549 and HT-29 cell lines. The EtOAc-soluble fraction was purified by column chromatography and high-performance liquid chromatography (HPLC) using a reverse-phase column to yield three novel acyl flavonoids and a biflavonoid, along with 15 other known compounds that included flavonoids, biflavonoids, and other phenolics. The structures of the new compounds were elucidated using spectral data from HR-MS and NMR, including two-dimensional NMR studies, as (2R,3R)-3-O-eicosanoyltaxifolin (1), (2R,3R)-3-O-docosanoyltaxifolin (2), (2R,3R)-3-O-tetracosanoyltaxifolin (3), and 6-methyl-4',7,7″-tri-O-methylamentoflavone (4). The isolated compounds, including the known compounds, were tested for possible antitumor activity; some of the biflavones were found to be active. The potent antitumor activity of the extract was attributed to Cephalotaxus alkaloids, such as homoharringtonine (20).

Transannular cyclizations of germacrone-4,5-epoxide under acidic and thermal conditions have been reported in our previous study. However, this process gave the different and interesting results under basic conditions. (4S,5S)-Germacrone-4,5-epoxide (1) was treated under basic conditions to yield four products (2-5). Compound 2 was an isomer of 1--(4S,5S,9Z)-4,5-epoxygermacra-7(11),9-dien-8-one--and the remaining three compounds (3-5) were eudesmane-type derivatives. Compounds 4 and 5 are new compounds. The structures of the new compounds were determined using high resolution (HR)-MS, one dimensional (1D)-NMR, 2D-NMR and circular dichroism (CD) spectroscopic data. Products 3-5 had the same carbon skeleton as that of eudesmane-type compounds; however, these compounds showed different arrangement of isoprene units to the natural eudesmane-type sesquiterpenes.

This study aimed to develop a novel wound dressing composed of hyaluronic acid (HA) spongy sheet containing bioactive components. The wound dressing prepared by the freeze-drying method has a two-layered structure: an upper layer composed of cross-linked high-molecular-weight HA (HMW-HA) and a lower layer composed of low-molecular-weight HA (LMW-HA) containing arginine (Arg), magnesium ascorbyl phosphate (vitamin C derivative: VC), and epidermal growth factor (EGF) (referred to as EGF-dressing). A wound dressing containing only Arg and VC was prepared in a similar manner (referred to as EGF-free-dressing). The potential of each wound dressing was evaluated in animal tests using Sprague Dawley (SD) rats and diabetic mice. In the first experiment, each wound dressing was applied to a full-thickness skin defect in the abdominal region of SD rats. Wound conditions after 1 week and 2 weeks of treatment were evaluated based on macroscopic and histological appearance. A commercially available non-woven alginate wound dressing (Alg-dressing) was used in a control group. Both EGF-free-dressing and EGF-dressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with Alg-dressing. In particular, EGF-dressing promoted re-epithelialization. In the second experiment, each wound dressing was applied to a full-thickness skin defect in the dorsal region of diabetic mice. Wound conditions after 1 week and 2 weeks of treatment were evaluated based on macroscopic and histological appearance. A commercially available Alg-dressing was used in a control group. Both EGF-free-dressing and EGF-dressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with Alg-dressing. These findings indicate that EGF-free-dressing and EGF-dressing have the potential for more effective wound healing when compared with Alg-dressing. In particular, EGF-dressing has a higher potential for wound healing when compared with EGF-free-dressing.

The present study aimed to develop a two-layered cultured dermal substitute (CDS). The upper layer is a hyaluronic acid (HA) and collagen (Col) spongy sheet with or without epidermal growth factor (EGF). The lower layer is a HA spongy sheet and Col gel containing fibroblasts. The CDS is prepared in serum-free medium, followed by placing on the wound surface. Corresponding to clinical application, CDS was incubated in serum-free medium for a period of 1, 3 or 5 days, followed by placing onto the air and culture medium interface (wound surface model), and culture for 6 days using conventional culture medium supplemented with serum. Metabolic activity and cytokine production were considerably higher in EGF-incorporating CDS, as compared with EGF-free CDS. Metabolic activity of EGF-incorporating CDS was maintained for a period of 3 days, but decreased slightly after 5 days. EGF-incorporating CDS is able to effectively stimulate fibroblasts within CDS to release increased amounts of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing. CDS is promising for wound therapy, because there is no risk of cellular damage caused by cryopreservation, thawing and rinsing processes. The critical issue is how to reduce the cellular damage during a prolonged period of incubation in serum-free medium. EGF-incorporating CDS can be used after a period of 3-5 days incubation in serum-free medium. This period is sufficient for transport of CDS from manufacturing facilities to hospitals.

Anti-adhesive products need to be designed while considering the concept of wound healing. Two main events must proceed simultaneously: facilitating wound healing in surgically excised tissue, as well as preventing injured tissue from adhering to the surrounding tissue. The present study aimed to develop an anti-adhesive spongy sheet composed of hyaluronic acid and collagen (Col) containing epidermal growth factor, and to investigate the potential of this spongy sheet using an in vitro wound surface model (placing a spongy sheet on a fibroblast-incorporating Col gel sheet) and an in vitro inter-tissue model (placing a spongy sheet between two fibroblast-incorporating Col gel sheets). These in vitro experiments demonstrated that this spongy sheet effectively stimulates fibroblasts to release an increased amount of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing to proceed succesfully. In addition, anti-adhesive performance of this spongy sheet was evaluated in animal experiments using Sprague Dawley rats. Under anesthesia, a 1 cm × 2 cm segment of peritoneum was superficially excised from walls, and the cecum was then abraded by scraping with a scalpel blade over a 1 cm × 2 cm area. A piece of spongy sheet was placed on the peritoneal defect. Both defects were placed in contact, and the incision was closed by suturing. Peritoneal condition was evaluated after one week. This spongy sheet was capable of facilitating the wound healing of surgically excised tissue and preventing surgically excised tissue from adhering to surrounding tissues.

Background: In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported., Results: In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries., Conclusions: These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

1. When benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) was incubated with liver microsomes of untreated rats in the presence of NADPH, the 5-hydroxylated metabolite, 2,5-dihydroxy-4-methoxybenzophenone (5-OH-BP-3), was formed as a major novel metabolite of BP-3. The 4-desmethylated metabolite, 2,4-dihydroxybenzophenone (2,4-diOH-BP), previously reported as the major in vivo metabolite of BP-3, was also detected. However, the amount of 5-OH-BP-3 formed in vitro was about the same as that of 2,4-diOH-BP. 2. The oxidase activity affording 5-OH-BP-3 was inhibited by SKF 525-A and ketoconazole, and partly by quinidine and sulfaphenazole. The oxidase activity affording 2,4-diOH-BP was inhibited by SKF 525-A, ketoconazole and α-naphthoflavone, and partly by sulfaphenazole. 3. The oxidase activity affording 5-OH-BP-3 was enhanced in liver microsomes of dexamethasone-, phenobarbital- and 3-methylcholanthrene-treated rats. The activity affording 2,4-diOH-BP was enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats. 4. When examined recombinant rat cytochrome P450 isoforms catalyzing the metabolism of BP-3, 5-hydroxylation was catalyzed by P450 3A2, 1A1, 2B1, 2C6 and 2D1, while 4-desmethylation was catalyzed by P450 2C6 and 1A1.

Germacrone (1) and (4S,5S)-germacrone-4,5-epoxide (2) were isolated, along with guaiane and secoguaiane-type sesquiterpenes, from Curcuma aromatica plants. Compound 2 was derived from 1 and cyclized through transannular (T-A) reactions into various guaiane and secoguaiane-type sesquiterpenes in C. aromatica. The cyclization reaction of 2 was initiated by protonation at an epoxide oxygen atom, followed by cleavage of the epoxide ring and the formation of a C-C bond between C-1 and C-5 to give guaiane-type derivatives. Acidic and thermal treatments of 2 produced twelve sesquiterpenes having guaiane and secoguaiane skeletons. The structures of these products were elucidated by spectral methods, including 2D-NMR spectroscopy. Most were identified as sesquiterpenes isolated from C. aromatica as natural products. The T-A cyclization of 2 occurred via two transition states, a cross conformation and a parallel conformation. The mechanism of the T-A cyclization reaction of 2 is discussed.

14-Deoxy-11,12-didehydroandrographolide is one of the principle compounds of the medicinal plant, Andrographis paniculata Nees. This study explored the mechanisms of 14-deoxy-11,12-didehydroandrographolide-induced toxicity and non-apoptotic cell death in T-47D breast carcinoma cells. Gene expression analysis revealed that 14-deoxy-11,12-didehydroandrographolide exerted its cytotoxic effects by regulating genes that inhibit the cell cycle or promote cell cycle arrest. This compound regulated genes that are known to reduce/inhibit cell proliferation, induce growth arrest and suppress cell growth. The growth suppression activities of this compound were demonstrated by a downregulation of several genes normally found to be over-expressed in cancers. Microscopic analysis revealed positive monodansylcadaverine (MDC) staining at 8h, indicating possible autophagosomes. TEM analysis revealed that the treated cells were highly vacuolated, thereby suggesting that 14-deoxy-11,12-didehydroandrographolide may cause autophagic morphology in these cells. This morphology may be correlated with the concurrent expression of genes known to affect lysosomal activity, ion transport, protein degradation and vesicle transport. Interestingly, some apoptotic-like bodies were found, and these bodies contained multiple large vacuoles, suggesting that this compound is capable of eliciting a combination of apoptotic and autophagic-like morphological characteristics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In the course of screening for leishmanicidal constituents from Asian and South American medicinal plants, a Pakistani medicinal plant, Withania coagulans, showed activity. We therefore studied the active components of the methanol extract of aerial parts of W. coagulans. From the ethyl acetate soluble fraction of the extract, four new withanolides (1-4) were isolated along with seven known withanolides (5-11). The new compounds were elucidated to be (14R,15R,17S,20S,22R)-14,15,17,20-tetrahydroxy-1-oxowitha-2,5,24-trienolide (1), (14R,15R,17S,20S,22R)-14,15,17,20-tetrahydroxy-1-oxowitha-3,5,24-trienolide (2), (14S,17R,20S,22R)-14,17,20-trihydroxy-1-oxowitha-2,5,24-trienolide (3), and (14S,17R,20S,22R)-14,17,20-trihydroxy-1-oxowitha-3,5,24-trienolide (4), from 1H-NMR, 13C-NMR, 2D-NMR and high resolution (HR)-MS data. Some of these compounds having the partial structure 1-oxo-2,5-diene showed strong leishmanicidal activity against Leishmania major.

Aims: Cytochrome P450 (CYP) enzymes have been implicated in a large number of preventable drug-herb interactions. Andrographis paniculata Nees, a tropical herb widely used for various health conditions contains two major diterpenoids, andrographolide and 14-Deoxy-11, 12-Didehydroandrographolide. These compounds were evaluated systematically for their effects on CYP1A2, CYP2D6 and CYP3A4 expressions in HepG2 cells., Main Methods: Quantitative RT-PCR and Western blot analysis were used to assess the mRNA and protein expression of the three CYPs. CYP3A4 enzyme activity was evaluated using P450-Glo™ Assays. The LanthaScreen® TR-FRET PXR (SXR) Competitive Binding Assay was used to determine if the compounds are potential PXR-ligands., Key Findings: Both diterpenoids inhibited the mRNA and protein expressions of CYP1A2, CYP2D6, and CYP3A4. Interestingly, the lowest concentration of both diterpenoids produced a more than 50% reduction in the mRNA and protein expression of CYP3A4 and this reduction was consistent with the enzyme activity. Further experiments revealed that both diterpenoids were also capable of attenuating the ability of dexamethasone to induce CYP3A4 expression, and 14-Deoxy-11, 12-Didehydroandrographolide tended to bind to the PXR-LBD site in a concentration-dependent manner., Significance: These diterpenoids are potential CYP3A4 inhibitors and the effects on CYP3A4 may be clinically significant. 14-Deoxy-11, 12-Didehydroandrographolide inhibits CYP3A4 by binding and antagonizing PXR function., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The production of sesquiterpene-type phytoalexins with a vetispyradiene skeleton by Hyoscyamus albus hairy roots induced by methyl jasmonate (MeJA) was reported in a previous paper. The production pattern on co-treatment with cupper sulfate and MeJA (CuSO(4)-MeJA) showed a TLC profile differing from that on treatment with MeJA. Thus, we studied the production of phytoalexins on hairy root culture involving co-treatment with CuSO(4)-MeJA. In the experiment, many sesquiterpene-type phytoalexins with a vetispyradiene skeleton were isolated, most of which were different from the products reported in the previous paper. Here, we isolated four new phytoalexins (1-4) along with known compounds 5-10 from the culture medium of H. albus hairy roots co-treated with MeJA-CuSO(4). The structures of the new compounds (1-4) were determined as: (3R,4S,5R,7S,9R)-3-acetoxy-9-(2-methylpropionyloxy)solavetivone (1), (3R,4S,5R,7S,9R)-3-hydroxy-9-(3-methylbutanoyloxy)solavetivone (2), (3R,4S,5R,7S,9R)-3-acetoxy-9-(3-methyl-butanoyloxy)solavetivone (3), and (3R,4S,5R,7S,9R)-3-acetoxy-9-(3-methyl-2-butenoyloxy)-solavetivone (4) based on MS and NMR including 2D-NMR data. These findings indicated that the production of phytoalexins in H. albus hairy roots yielded different products based on treatment with different chemicals (CuSO(4), MeJA, and MeJA-CuSO(4)).

Lancemaside A, a triterpenoid saponin isolated from the roots of Codonopsis lanceolata, has been reported to ameliorate the reduction of blood testosterone levels induced by immobilization stress in mice. In the present study, we investigated the metabolism and absorption of lancemaside A in mice. After oral administration of lancemaside A at 100 mg/kg body weight, the unmetabolized compound appeared rapidly in plasma (t (max) = 0.5 h). Lancemaside A has a low bioavailability (1.1%) because of its metabolism by intestinal bacteria and its poor absorption in the gastrointestinal tract. Furthermore, we identified four metabolites from the cecum of mice after oral administration of lancemaside A: codonolaside II, echinocystic acid, echinocystic acid 28-O-beta-D: -xylopyranosyl-(1 --> 4)-alpha-L: -rhamnopyranosyl-(1 --> 2)-alpha-L: -arabinopyranosyl ester, and echinocystic acid 28-O-alpha-L: -rhamnopyranosyl-(1 --> 2)-alpha-L: -arabinopyranosyl ester. Among these metabolites, codonolaside II and echinocystic acid were detected in plasma, and their t (max) values were 4 and 8 h, respectively. These findings should be helpful for understanding the mechanism of the biological effect of lancemaside A.

Hyperici erecti herba (Hypericum erectum THUNB.) showed a suppressive effect on generation of isovaleric acid by Corynebacterium xerosis. An ethyl acetate (AcOEt) soluble fraction of methanol extract of H. erectum showed the activity. The AcOEt fraction was separated by various successive choromatographical methods to give seven new compounds 1-7 along with some known compounds. The structures of the new compounds were elucidated to be polyprenylated benzoylphloroglucinol derivatives by means of HR-MS and NMR spectra including 2D-NMR. Some of these compounds had novel cage structures having benzoyltricyclo[3,3,1,1(3,7)]decane and benzoyltricyclo[4,3,1,1(3,8)]undecane skeletons arising from a polyprenylated phloroglucinol precursor by a transannular cyclization reaction. The isolated compounds were tested for suppressive activity, but they showed only weak activity.

In the course of screening leishmanicidal active compounds from Asian and South American medicinal plants, a Nepalese medicinal plant, Tulsi (Ocimum sanctum L.), showed strong activity. We therefore studied the isolation and structural elucidation of the active constituents from O. sanctum L. From the ethyl acetate soluble fraction of the plant, seven new novel neolignan derivatives were isolated along with 16 known compounds. The structures of the new compounds (1-7) were elucidated as 6-allyl-3',8-dimethoxy-flavan-3,4'-diol (1), 6-allyl-3-(4-allyl-2-methoxyphenoxy)-3',8-dimethoxyflavan-4'-ol (2), 5-allyl-3-(4-allyl-2-methoxyphenoxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran (3), 1,2-bis(4-allyl-2-methoxyphenoxy)-3-(4-hydroxy-3-methoxyphenyl)-3-methoxypropane (4), 1-(4-hydroxy-3-methoxyphenyl)-1,2,3-tris(4-allyl-2-methoxyphenoxy)propane (5), 1-allyl-4-(5-allyl-2-hydroxy-3-methoxyphenoxy)-3-(4-allyl-2-methoxyphenoxy)-5-methoxybenzene (6), and 3-(5-allyl-2-hydroxy-3-methoxyphenyl)-1-(4-hydroxy-3-methoxyphenoxy)-prop-1-ene (7) by means of (1)H-NMR, (13)C-NMR, and 2D-NMR spectral data. Some of these compounds showed leishmanicidal activity.

We developed a rapid and simple analytical method for the simultaneous determination of seven 3,28-bidesmosidic triterpenoid saponins in the roots of Codonopsis lanceolata. The saponins are lancemaside A, lancemaside B, lancemaside C, lancemaside E, lancemaside G, foetidissimoside A, and aster saponin Hb. Root samples were extracted with 50% methanol and prepared for analysis. Saponins were detected by reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry, and ginsenoside Rb(1) was used as an internal standard. The overall recoveries of all saponins were 92-116%, and the relative standard deviation values of intra- and inter-day precision were lower than 3.7 and 7.7%, respectively. Eight root samples collected from Korea and Japan were analyzed using the developed method. Lancemaside A was the most abundant saponin in the root samples from Korea, ranging from 2.65 to 3.64 mg/g dry root. However, the maximum content of lancemaside A among Japanese samples was 0.101 mg/g dry root.

On the basis of monitoring the prevention of accumulation of lipid droplets in mouse 3T3-L1 preadipocyte cells and inhibition of the proliferation of human colon cancer HT-29 cells, effective anti-corpulence and anticancer compounds were isolated from the peel of Citrus fruits. These bioactive components were identified as polymethoxyflavones and coumarin derivatives by spectroscopic analyses. 5-Hydroxy-6,7,8,3',4'-pentamethoxyflavone had the greatest anti-corpulence effects and 3,5,6,7,8,3',5'-heptamethoxyflavone had the greatest anticancer effects. Furthermore, distributions of those bioactive components in the peel of 10 species of Citrus fruits were demonstrated by HPLC analyses.

Further chemical study led to two new A-seco-rearranged lanostane triterpenoid derivatives (1-2) and one known compound Abiesanolide C (3) from the ethyl acetate (EtOAc) soluble fraction of the MeOH extract of Abies sachalinensis needles. The new compounds were identified as 3,4-seco-8-(14-->13R)abeo-17,13-friedo-9beta-lanosta-4(28), 7, 14, 24-tetraen-26,23-olide-23-hydroxy-3-oic acid and methyl-3,4-seco-8-(14-->13R) abeo-17,13-friedo-9beta-lanosta-4(28), 7, 14, 24-tetraen-26,23-olide-23-hydroxy-3-oate, respectively. Structural determination of these compounds were carried out by the spectral studies especially by the two digital (2D)-NMR and high-resolution (HR)-MS experiences.

Liquid chromatography coupled with sequential mass spectrometry (LC-MS(n)) has been used to identify 3,28-bidesmosidic triterpenoid saponins, lancemaside A (1), foetidissimoside A (2), aster saponin Hb (3), lancemaside E (4), lancemaside B (5), lancemaside F (6), lancemaside G (7), lancemaside C (8), and lancemaside D (9) in the roots of Codonopsis lanceolata. Structural information about both the aglycone and the sugar moiety at the C-3 position of saponins was obtained in the negative-ion mode. On the other hand, positive-ion spectra mainly provide structural information about the sugar chains of saponins, especially the oligosaccharide moiety at the C-28 position. During subsequent fragmentation of the product ions derived from the oligosaccharide moiety at the C-28 position, fragments produced by sequential loss of a monosaccharide unit were observed. Furthermore, the structural features of two unknown saponins in the roots of C. lanceolata were assigned on the basis of the fragmentation patterns of the known saponins. These studies demonstrate that LC-MS(n) analysis has great potential for the identification and characterization of triterpenoid saponins in plant extracts.

To isolate more rearranged lanostane-type triterpenes from Abies sachalinensis, continuous chemical investigation of the ethyl acetate soluble fraction of the methanol extract of A. sachalinensis afforded two new rearranged lanostane-type triterpenes (1, 2). Their structures were elucidated to be 3,4-seco-4(28),7,12,24-mariesatetraen-26,23-olide-23-hydroxy-3-oic acid (1) and ethyl 3,4-seco-8(14-->13R)abeo-17,13-friedo-4(28),7,14,24-lanostatetraen-26,23-olide-23-hydroxy-3-oate (2), respectively. The structure of these compounds was determined by spectral studies, especially by two-dimensional (2D)-NMR and high-resolution (HR)-MS. Compounds 1 and 2 have a tautomeric lactone structure in the side chain.

Introduction: Lancemaside A is a saponin that inhibits decreases in blood testosterone level and thus prevents or ameliorates symptoms associated with male climacteric disorder. Our initial attempt to preparative isolation of lancemaside A from the saponin fraction of Codonopsis lanceolata roots by a preparative HPLC did not give a clear result., Objective: To develop a simple and efficient method for the preparative isolation of lancemaside A from the hot water extract of C. lanceolata roots using centrifugal partition chromatography (CPC)., Methodology: The saponin fraction obtained from the hot water extract of C. lanceolata roots was used as the sample for preparative-scale separation of lancemasides by CPC using n-hexane:n-butanol:methanol:0.1% aqueous formic acid (3:4:1:6, v/v) as the two-phase solvent system. The upper phase (organic phase) of the two-phase solvent system was used as the mobile phase, and 0.5 g of saponin fraction was applied for separation by CPC. Each fraction that was separated by CPC was analysed by HPLC, and the fractions containing each of the separated compounds were pooled together, and then were purified by simple preparative HPLC., Results: The demonstrated separation sequence, hot water extraction, DIAION HP-20 column chromatography, CPC and preparative HPLC, yielded lancemaside A, foetidissimoside A and astersaponin Hb in their pure forms., Conclusion: The simple and efficient method for the preparative isolation of lancemaside A along with two other saponins, foetidissimoside A and astersaponin Hb, from the saponin fraction of C. lanceolata was established using CPC.

In the course of studies on biological active constituents from woody plants, we previously reported the isolation of many lignan derivatives as neurite outgrowth-promoting compounds from an ethyl acetate soluble fraction of Japanese Cypress (Chamaecyparis obtusa). Further chemical investigation on the residual parts of the ethyl acetate soluble fraction of a methanol extract of Japanese Cypress resulted in the isolation of four new beyerene type derivatives and a novel sesquiterpene dimer formed between cryptomeridiol and hinokiic acid. Their structures were elucidated as 18-O-(Z)-p-coumaroylbeyer-15-ene-18-ol (1), 18-O-(E)-p-coumaroylbeyer-15-ene-18-ol (2), 18-O-(E)-p-coumaroylbeyer-15-ene-11beta,18-diol (3), 18-O-(Z)-p-coumaroylbeyer-15-ene-11beta,18-diol (4) and ent-cryptomeridiol-4-yl-hinokiiate (5) by (1)H-NMR, (13)C-NMR, 2D-NMR, and HR-MS spectral analysis.

In the previous work we reported five A-seco-rearranged lanostane triterpenoids as antibacterial constituents from the ethyl acetate soluble fraction of Abies sachalinensei leaves. In further study on the isolation of constituents from the ethyl acetate soluble fraction, two new rearranged lanostane and lanostane-type triterpenoids (3, 4) and three reported compound (1, 2, 5) were isolated. The structures of new compound 3 and 4 were determined to be 3,4-seco-4(28),6,8(14),24-mariesatetraen-26,23-olide-23-hydroxy-3-oic acid and 3,4-seco-4(28),7,24-lanostatrien-26,23-olide-23-hydroxy-3-oic acid, respectively, by spectral studies on HR-MS, (1)H-NMR, (13)C-NMR, and 2D-NMR spectra. Compound 1 was identified with pindrolactone and its structure was revised as 7,14,22Z,24-mariesatetraen-26,23-olide-3alpha-ol. Structures of 2 and 5 were determined as 7,14,24-mariesatrien-26,23-olide-3alpha,23-diol and 3alpha-hydroxy-7,14,24E-mariesatrien-23-oxo-26-oic acid. Of these compounds, 2, 3 and 4 were obtained as lactol tautomer mixtures at gamma-lactone structures of side chains.

In the course of the development of new designer foods using the roots of Codonopsis lanceolata, we found that hot-water extracts of C. lanceolata recovered decreased testosterone levels in the blood and accelerated the restoration of reproductive dysfunction induced by hyperthermic treatment in male mice. Thus we studied the constituents of the polar fraction of the roots of C. lanceolata and identified six new triterpene saponins, lancemasides B (2), C (3), D (4), E (5), F (6), and G (7), along with the known saponin lancemasaide A (1) and phenylpropanoid glycosides 8-10. The structures of the new compounds 2-7 were determined by means of spectral data including 2D-NMR studies and chemical reactions to be oleanan-type bisdesmoside with sugars at C-3 and C-28. Compounds 2-6 have echinocystic acid as an aglycone, and compound 7 has asterogenic acid as an aglycone. Identification of the sugars and determination of their D,L-chiralities were carried out by application of the exciton chirality method to the per-O-p-bromobenzoylmethyl sugar derived from saponins.

Grapefruit seed extract (GSE), derived from the seeds of grapefruit (Citrus paradisi MCAF.), is listed as a natural food additive in Japan. Products containing GSE are used as disinfectants made from only natural sources, especially after Japanese researchers found that GSE prevents the growth of norovirus. On the other hand, recent overseas studies indicated that synthetic disinfectants, such as benzalkonium and benzethonium chlorides, were present in some commercial GSE products. To confirm the quality of commercial GSE products available in Japanese markets, we carried out comprehensive research to identify the major constituents of commercial GSE products which are used as food additives (13 products from 6 manufacturers), dietary supplements (5 products from 4 manufacturers), cosmetic materials (16 products from 10 manufacturers) and disinfectant or deodorant sprays (7 products from 7 manufacturers). By means of NMR and LC/MS analysis, synthetic disinfectants such as benzethonium or benzalkonium salts were detected in most of the commercial GSE products.

In the screening of biologically active constituents from woody plants, the methanol extract of leaves of Chamaecyparis obtusa showed potent neurite outgrowth-promoting activity in neuronal PC12 cells. The ethyl acetate-soluble fraction of the methanol extract showed potent activity and was separated by means of various chromatographic methods to give the two new compounds 1 and 2, as well as 11 known lignan and sesquiterpene derivatives. The structures of the new compounds were determined to be 9-O-acetyldihydrosesamin (1) and 9-O-(11-hydroxyeudesman-4-yl)dihydrosesamin (2), respectively, in NMR studies including 2D-NMR experiments. Of the 13 compounds, the known compound hinokinin (5) and the new compound 2 showed potent neurite outgrowth-promoting activity in PC 12 cells.

Molasses of tangerine orange (Citrus unshiu Markovich) is obtained as a waste product in the course of tangerine orange juice production. This molasses is expected to be a useful source of organic compounds such as flavonoids and limonoids. To elucidate a use for this molasses waste, we isolated and identified its organic constituents. Two new flavanonol glycosides were isolated from tangerine orange molasses, along with several flavonoids such as hesperidine, narirutin, eriodictyol, 3',4',5,6,7,8-hexamethoxy-3-O-beta-D-glucopyranosyloxyflavone, and 3',4',5,6,7,8-hexamethoxy- 3-beta-D-[4-O-(3-hydroxy-3-methylglutaloyl)]-glucopyranosyloxyflavone, and limonoids such as limonin, nomilin, and cyclic peptide, citrusin III. The structures of the new flavanonol glycosides were determined as (2R,3R)-7-O-(6-O-alpha-L-rahmnopyranosyl-beta-D-glucopyranosyl)-aromadendrin and 7-O-(6-O-alpha-L-rahmnopyranosyl-beta-D-glucopyranosyl)-3,3',5,7-tetrahydroxy-4'-methoxyflavanone by means of spectral analyses using (1)H-NMR, (13)C-NMR, and 2D-NMR. Of these compounds, flavanone glycoside, hesperidin and narirutin were isolated as the main constituents. Thus, molasses is a promising source of flavonoid glycosides.

Isodonis Herba is used as a Japanese dietary supplement and folk medicine. The extract of the herb (Isodonis extract) is also used as a food additive whose major compound is enmein (1). Here we compared internal transcribed spacer sequences of nuclear ribosomal DNA from Isodonis Herba available on the Japanese and Chinese crude drug markets, and found that the former derived from Isodon japonicus and Isodon trichocarpus, while the latter derived from distinct species such as Isodon eriocalyx. The liquid chromatography/mass spectrometry profiles of Isodonis Herba were classified into four chemotypes (A to D) according to the ratio of the major constituents. Types B and C contained 1 and oridonin (2) as major components, respectively. An intermediate (or mixed) form of types B and C in various ratios was designed type A. Type D contained eriocalyxin B (3) as its major component. Japanese herba were types A-C, while Chinese herba were types C and D. The commercial Isodonis extract products tested were classified as type D, suggesting that they originated from Chinese Herba. Understanding the relationship between extract constituents and DNA profiles is important for the official specification of dietary supplements and food additives of plant origin.

In the process of decommissioning a tritium-handling laboratory, direct-immersion and water-extraction methods for measuring tritium contamination in concrete were compared to a heating-cooling method. For this evaluation, concrete pieces chipped from contaminated concrete walls and floors were used. The direct-immersion method was about 2.5 times less sensitive at determining the level of contamination compared to the heating-cooling method. The water-extraction method was comparable with the heating-cooling method if the water extraction time was adequate.

Sandarac resin, a natural gum base, is described as "a substance composed mainly of sandaracopimaric acid obtained from the secretion of sandarac trees" in the List of Existing Food Additives in Japan. To evaluate its quality as a food additive, the main constituents in a sandarac resin product were investigated. Three constituents were isolated and identified as sandaracopimaric acid, sandaracopimarinol and 4-epidehydroabietic acid by MS and 2D-NMR. Quantification of the main constituent, sandaracopimaric acid, was performed by HPLC and its content in the product was determined to be 11.6%.

In the course of research on the bioactive constituents of woody plants in the Cyugoku area of Japan, a methanol extract of the leaves of Alnus japonica were found to have strong antioxidative activity. Ethyl acetate soluble and n-buthanol soluble fractions of the methanol extract had a potent antioxidative effect. Both fractions were purified by silica gel column chromatography and HPLC using an ODS column to give four new diarylheptanoids along with known diarylheptanoids and flavonoids. These new compounds were elucidated to be 7-(3,4-dihydroxyphenyl)-5-hydroxy-1-(4-hydroxyphenyl)-3-heptanone-5-O-beta-D-xylopyranoside (1), 1-(3,4-dihydroxyphenyl)-5-hydroxy-7-(4-hydroxyphenyl)-3-heptanone-5-O-beta-D-xylopyranoside (2), 1,7-bis-(3,4-dihydroxyphenyl)-5-hydroxy-3-heptanone-5-O-[2-(2-methylbutenoyl)]-beta-D-xylopyranoside (3) and 1,7-bis-(3,4-dihydroxyphenyl)-5-methoxy-3-heptanone (4) using spectral methods and especially 1H-, 13C-NMR and 2D-NMR measurements. The isolated compounds including their main constituent, oregonin (5), were tested for antioxidative activity. Some of these compounds having two catechol structures showed potent antioxidative activity. Compounds having one catechol structure showed moderate antioxidative activity, but a peracetate of 5 having no catechol structure exhibited no antioxidative activity. Thus the catechol structure of the diarylheptanoids is indispensable for antioxidative activity.

Some compatible pathogens secrete toxins to induce host cell death and promote their growth. The toxin-induced cell death is a pathogen strategy for infection. To clarify the executioner of the toxin-induced cell death, we examined a fungal toxin (fumonisin B1 (FB1))-induced cell death of Arabidopsis plants. FB1-induced cell death was accompanied with disruption of vacuolar membrane followed by lesion formation. The features of FB1-induced cell death were completely abolished in the Arabidopsis vacuolar processing enzyme (VPE)-null mutant, which lacks all four VPE genes of the genome. Interestingly, an inhibitor of caspase-1 abolished FB1-induced lesion formation, as did a VPE inhibitor. The VPE-null mutant had no detectable activities of caspase-1 or VPE in the FB1-treated leaves, although wild-type leaves had the caspase-1 and VPE activities, both of which were inhibited by a caspase-1 inhibitor. gammaVPE is the most essential among the four VPE homologues for FB1-induced cell death in Arabidopsis leaves. Recombinant gammaVPE recognized a VPE substrate with Km = 30.3 microm and a caspase-1 substrate with Km = 44.2 microm, which is comparable with the values for mammalian caspase-1. The gammaVPE precursor was self-catalytically converted into the mature form exhibiting caspase-1 activity. These in vivo and in vitro analyses demonstrate that gammaVPE is the proteinase that exhibits a caspase-1 activity. We show that VPE exhibiting a caspase-1 activity is a key molecule in toxin-induced cell death. Our findings suggest that a susceptible response of toxin-induced cell death is caused by the VPE-mediated vacuolar mechanism similar to a resistance response of hypersensitive cell death (Hatsugai, N., Kuroyanagi, M., Yamada, K., Meshi, T., Tsuda, S., Kondo, M., Nishimura, M., and Hara-Nishimura, I. (2004) Science 305, 855-858).

Apoptotic cell death in animals is regulated by cysteine proteinases called caspases. Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE deficiency prevents cell death during hypersensitive response and cell death of limited cell layers at the early stage of embryogenesis. Because plants do not have macrophages, dying cells must degrade their materials by themselves. VPE plays an essential role in the regulation of the lytic system of plants during the processes of defense and development. VPE is localized in the vacuoles, unlike animal caspases, which are localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.

The glucosylation of some coumarin and flavone derivatives on incubation with the hairy roots of morning glory (Pharbitis nil) was previously reported. We further studied the biotransformation of benzaldehyde- and acetophenone-type derivatives. Vanillin and isovanillin were reduced to alcoholic derivatives and glucosylated at the phenolic and the alcoholic hydroxyl groups. In the case of 3,4-dihydroxybenzaldehyde, the formyl group was reduced and the 3-hydroxyl or 4-hydroxyl groups were glucosylated to give monoglucosides. The 3-hydroxyl group was predominantly glucosylated to the 4-hydroxyl group. 4-beta-D-Glucopyranosyloxy-3-methoxybenzylalcohol was obtained in low yield. In time-course experiments with vanillin, it was found that the high-level reduction of the formyl group and glucosylation of the phenolic hydroxyl group occurred, and finally 4-O-beta-D-glucopyranosylvanillylalcohol was obtained as the main product. In the case of 3,4-dimethoxybenzaldehyde, 3,4,5-trimethoxybenzaldehyde, and salicylaldehyde, the formyl groups were reduced, and then the hydroxyl groups at the benyl position were glucosylated to give alcoholic glucosides in relatively high yields. In 4-hydroxy-3-methoxyacetophenone, the 4-hydroxyl group was glucosylated and two dimerized glucosides, biphenyl and biphenylether types, were obtained in low yields. In acetophenone, 1-beta-D-glucopyranosyloxy-1-phenylethane and 2-beta-D-glucopyranosyloxyacetophenone were obtained. As mentioned above P. nil hairy roots showed various biotransformative activities including glucosylation of phenolic and benzylic hydroxyl groups, reduction of the formyl group near the benzene ring, and phenol oxidation dimerization. The glucosylation reaction was especially interesting for the production of valuable glucosides.

Hairy roots of medicinal morning glory (Pharbitis nil) showed potent glucosylation activity against umbelliferone and aesculetin, so the glucosylation activity against several phenolic compounds was tested. Some coumarin derivatives and flavone derivatives having phenolic hydroxyl groups were incubated with the hairy roots. The coumarin derivatives and flavone derivatives almost disappeared from the culture medium in half a day. In the case of the coumarin derivatives, a 7-hydroxyl group was easily glucosylated. A methyl group at C-8 somewhat decreased the glucosylation to a hydroxyl group at C-7 of the coumarin skeleton. The 4-hydroxy coumarin derivatives were changed to acetophenone-type glucosides by incubation with the hairy roots through decarboxylation. Several flavonol derivatives were tested for glucosylation by the hairy roots. 3-Hydroxy flavone, 3.6-dihydroxyflavone and 3,7-dihydroxyflavone were glucosylated to give 3-glucosylated derivatives. Of these, 3,6-dihydroxyflavone was highly glucosylated, but not 3-hydroxyflavone or 3,7-dihydroxyflavone to the same degree. In the case of the flavones, a 3-hydroxy group could be predominantly glucosylated, and hydroxyl groups on the A and B ring of the flavones affected glucosylation by the hairy roots.

Hairy root cultures of Pharbitis nil treated with CuSO4 and methyl jasmonate (MeJA) produced umbelliferone (1) and scopoletin (2) in the culture medium, and skimmin (3), a beta-D-glucopyranoside of 1, was isolated from the hairy roots. While 1 in the medium increased and reached a maximal level 16 h after the treatment with CuSO4, the amount of 3 in the hairy roots decreased, reaching a minimal level after 8 h, before recovering to a level higher than the basal level after 24 h and then continuously increasing. These observations suggest that 1 was released by the hydrolysis of 3. Umbelliferone (1) inhibited hairy root growth, while skimmin (3) did not. This result suggests that, after the release of 1 as a phytoalexin, the hairy roots glycosylated 1 for the detoxification and re-use of 3 as a source of phytoalexin.