Elicitation of induced resistance against Pectobacterium carotovorum and Pseudomonas syringae by specific individual compounds derived from native Korean plant species.

Plants have developed general and specific defense mechanisms for protection against various enemies. Among the general defenses, induced resistance has distinct characteristics, such as broad-spectrum resistance and long-lasting effectiveness. This study evaluated over 500 specific chemical compounds derived from native Korean plant species to determine whether they triggered induced resistance against Pectobacterium carotovorum supsp. carotovorum (Pcc) in tobacco (Nicotiana tabacum) and Pseudomonas syringae pv. tomato (Pst) in Arabidopsis thaliana. To select target compound(s) with direct and indirect (volatile) effects, a new Petri-dish-based in vitro disease assay system with four compartments was developed. The screening assay showed that capsaicin, fisetin hydrate, jaceosidin, and farnesiferol A reduced the disease severity significantly in tobacco. Of these four compounds, capsaicin and jaceosidin induced resistance against Pcc and Pst, which depended on both salicylic acid (SA) and jasmonic acid (JA) signaling, using Arabidopsis transgenic and mutant lines, including npr1 and NahG for SA signaling and jar1 for JA signaling. The upregulation of the PR2 and PDF1.2 genes after Pst challenge with capsaicin pre-treatment indicated that SA and JA signaling were primed. These results demonstrate that capsaicin and jaceosidin can be effective triggers of strong induced resistance against both necrotrophic and biotrophic plant pathogens.

Anti-inflammatory effects of the Zingiber officinale roscoe constituent 12-dehydrogingerdione in lipopolysaccharide-stimulated Raw 264.7 cells.

Ginger has long been used worldwide as a spice, seasoning, and wine and is also used as a traditional medicine. There have been no previous studies of the potential beneficial effects of the ginger constituent 12-dehydrogingerdione (12-DHGD). We investigated the anti-inflammatory effect of 12-DHGD on lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. The cytotoxicity of 12-DHGD was measured using the MTT assay, and production of prostaglandin E2 (PGE2 ) and the inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α was measured by ELISA. Production of nitric oxide (NO) was measured using Griess reagent and expression of cyclooxygenase-2 (COX-2) and inducible NO (iNOS) enzymes was assessed by reverse transcriptase-polymerase chain reaction. Treatment of Raw 264.7 cells with 12-DHGD significantly inhibited LPS-stimulated production of NO (at 12-DHGD concentrations of 150 and 200 ng/ml), IL-6 (at 50, 100, 150, and 200 ng/ml), and PGE2 (at 200 ng/ml). Consistent with the effects on NO and PGE2 production, 12-DHGD treatment also inhibited the LPS-stimulated increase in iNOS and COX-2 mRNA levels. However, 12-DHGD did not affect production of IL-1ß or TNF-α in response to LPS. 12-DHGD, a constituent of ginger, is a potent inhibitor of proinflammatory mediator production in Raw 264.7 macrophage cells.

Comparison of neuroprotective effects of five major lipophilic diterpenoids from Danshen extract against experimentally induced transient cerebral ischemic damage.

We observed neuroprotective effects of five major lipophilic diterpenes derived from Danshen (Radix Salvia miltiorrhiza) extract, such as cryptotanshinone (CTs), dihydrotanshinone I (DTsI), tanshinone I (TsI), tanshinone IIA (TsIIA) and tanshinone IIB (TsIIB), in the hippocampal CA1 region (CA1) against transient ischemic damage in gerbils. These diterpenes were administered 30min before ischemia-reperfusion and the animals were sacrificed 4days after ischemia-reperfusion. In the vehicle-treated-group, cresyl violet positive (CV(+)) cells and neuronal nuclei (NeuN)(+) neurons were significantly decreased in the CA1. However, in the TsI- and CTs-treated-ischemia-groups, CV(+) and NeuN(+) neurons were abundant in the CA1. In the other groups, the number of CV(+) and NeuN(+) neurons was less than the TsI- and CTs-treated-ischemia-groups. In addition, gliosis induced by ischemic damage was apparently blocked in the TsI- and CTs-treated-ischemia-groups. These results suggest that TsI and CTs among five major lipophilic diterpenes have strong potentials for neuroprotection against ischemic damage.

Decursin, an active compound isolated from Angelica gigas, inhibits fat accumulation, reduces adipocytokine secretion and improves glucose tolerance in mice fed a high-fat diet.

Decursin (De), an active component of Angelica gigas, is known to exert anticancer and neuroprotective effects. However, its antiobesity and antidiabetic potential has not yet been investigated. This study evaluated the antiobesity effect of decursin, particularly focusing on its ability to inhibit adipocyte differentiation in 3T3-L1 cells. Decursin treatment resulted in the inhibition of adipocyte differentiation and the expression of fatty acid synthase. The study further investigated these antiobesity effects using mice fed a normal diet (ND), a high-fat diet (HFD) and a HFD plus decursin 200 mg/kg diet (HFD + De) for 7 weeks. Mice administered HFD plus decursin showed a drastic decrease in weight gain, triglyceride content, total cholesterol content and fat size compared with those that received the HFD alone; this was observed despite similar quantities of total food intake. Furthermore, decursin improved glucose tolerance in mice fed a HFD. Finally, administration of decursin along with the HFD significantly reduced the secretion of HFD-induced adipocytokines such as leptin, resistin, IL-6 and MCP-1. These results suggest that decursin might be useful for the treatment of obesity and diabetes.

Hepatoprotective effect of Platycodon grandiflorum against chronic ethanol-induced oxidative stress in C57BL/6 mice.

AIMS: This study was carried out to evaluate the hepatoprotective effect of Platycodon grandiflorum (PG) in ethanol (EtOH)-induced liver damage. METHODS AND RESULTS: PG treatment (both the total extract and saponin fraction) significantly blocked EtOH-induced oxidative stress through the preservation of activities of antioxidant enzymes in HepG2 cells. Furthermore, while the administration of EtOH to C57BL/6 mice for 6 weeks induced liver damage, along with a significant increase in plasma glutamic oxalacetic transaminase, glutamic pyruvic transaminase, hepatic triglyceride and thiobarbituric acid reactive substance levels, PG treatment significantly decreased glutamic oxalacetic transaminase, glutamic pyruvic transaminase, hepatic triglyceride and thiobarbituric acid reactive substance levels compared with the EtOH-treated control group (p < 0.05). Histological observation by hematoxylin-eosin and oil red O staining in the liver showed more effective inhibition of lipid accumulation in PG-treated groups, as compared to the EtOH-treated control group. Additionally, PG treatments appeared to enhance the activities of superoxide dismutase and catalase in the liver (p < 0.05). CONCLUSION: These results suggest that PG has a protective effect against EtOH-induced oxidative damage, possibly by inhibition of lipid accumulation and peroxidation through the enhancement of the antioxidant defense system. PG might be useful as a therapeutically potent natural ingredient for the prevention of chronic EtOH-induced oxidative stress and liver damage.

Zizyphus enhances cell proliferation and neuroblast differentiation in the subgranular zone of the dentate gyrus in middle-aged mice.

Zizyphus jujuba is considered to have various physiological functions in the brain. We obtained a Z. jujuba methanol extract (ZJE) and observed its effects on neurogenesis in middle-aged mice. Twelve-month-old mice received repeated oral administrations of ZJE for 30 days. The administration of ZJE significantly increased the number of Ki67 (a marker for cell proliferation)-positive cells in the subgranular zone of the dentate gyrus of middle-aged mice. Furthermore, ZJE significantly increased doublecortin (a marker for neuroblast differentiation)-immunoreactive neuroblasts with tertiary dendrites, but not those without tertiary dendrites, in the dentate gyrus. In addition, doublecortin protein levels in the ZJE-treated groups tended to increase dose-dependently. These results suggest that the repeated supplement of ZJE may increase the hippocampal plasticity in middle-aged mice.

Galbanic acid, a cytotoxic sesquiterpene from the gum resin of Ferula asafoetida, blocks protein farnesyltransferase.

Farnesylation of the activated RAS oncogene product by protein farnesyltransferase (FTase) is a critical step for its oncogenic function. Bioassay-guided purification of Ferula asafoetida (Umbelliferae) extract led to the isolation of the coumarin-derived sesquiterpene galbanic acid (1) as an active principal for FTase inhibitory activity, together with the four structurally related sesquiterpenes karatavicinol (2), umbelliprenin (3), farnesiferol B (4), and farnesiferol C (5). The 50 % inhibitory concentration (IC (50)) of 1 against FTase in an enzyme-based assay was calculated as 2.5 µM. Compound 1 also demonstrated potent inhibition of the proliferation of oncogenic RAS-transformed NIH3T3/Hras-F in a dose-dependent manner. The IC (50) value of 1 on the proliferation of oncogenic RAS-transformed NIH3T3/Hras-F cells was calculated as 16.2 µM, whereas its IC (50) value on control vector-transfected normal RAS-containing NIH3T3/ZIPneo cells was 58.5 µM.

New guaiane sesquiterpene lactones from Ixeris dentata.

Three new guaiane-type sesquiterpene lactones (1-3), together with nine related sesquiterpenes (4-12), were isolated from the whole extract of Ixeris dentata (Asteraceae). The chemical structures of isolates 1-12 were established by spectroscopic analyses as 3 ß,8 ß-dihydroxy-guaia-10(14)-en-1 α,4 α,5 α,6 ß,7 α,11 ßH-12,6 α-olide (1), ixerin N 6'- O-acetate (2), ixerisoside A 6'- O-acetate (3), ixerin N ( 4), ixerisoside A (5), ixerin M (6), tectroside (7), 8-epidesacylcynaropicrin glucoside (8), 8-epiisolipidiol (9), 11 ßH-11,13-dihydrointegrifolin (10) 8 ß-hydroxy-4 ß,15-dihydrozaluzanin C (11), and integrifolin (12). Compounds 1-12 were evaluated for their inhibitory effect on the proliferation of the cultured human tumor cell lines MES-SA, MES-SA/DX5, HCT-15, and HCT15/CL02 in vitro.

In vitro BACE-1 inhibitory activity of resveratrol oligomers from the seed extract of Paeonia lactiflora.

A new resveratrol oligomer (1) together with eight related components (2- 9) were isolated from the seed extract of Paeonia lactiflora (Paeoniaceae) as active principles responsible for the inhibition of beta-site APP-cleaving enzyme 1 (BACE-1) in vitro. The chemical structure of 1 was established as (-)-7a,8a- CIS- ε-viniferin with the aid of spectroscopic analyses including NOESY experiments. All isolated resveratrol oligomers (1- 9) demonstrated significant inhibition on baculovirus-expressed BACE-1 in a dose-dependent manner, which was assessed by the FRET assay using Rh-EVNLDAEFK as a substrate in vitro.

Euonymus alatus extract attenuates LPS-induced NF-κB activation via IKKß inhibition in RAW 264.7 cells.

AIM OF THE STUDY: The present study was performed to investigate the underlying mechanisms of anti-inflammatory effects with the extract of Euonymus alatus (EEA), and specially focused on nuclear factor κB (NF-κB) signaling pathway by targeting the IκB kinase ß (IKKß). MATERIALS AND METHODS: The effect of EEA for IKKß activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The effect of EEA on lipopolysaccharide (LPS)-induced NF-κB activation in murine macrophage RAW 264.7 cells with western blotting and immunofluorescent staining was evaluated. RESULTS: IKKß studies based on IMAP-TR-FRET showed that EEA possesses a potent IKKß inhibitory activity with IC(50) value of 11.83µg/ml. EEA (10, 30µg/ml) also attenuated the LPS-induced IκBα phosphorylation/degradation, NF-κB translocation and subsequent NO synthesis in RAW 264.7 cells. CONCLUSIONS: These results suggest that EEA abrogates LPS-induced NF-κB signaling pathway by targeting the IKKß in RAW 264.7 cells and these properties may provide a molecular basis for understanding the inhibitory effects of EEA on LPS-mediated inflammation.

Yeast α-glucosidase inhibition by isoflavones from plants of Leguminosae as an in vitro alternative to acarbose.

In the course of searching for new classes of α-glucosidase inhibitors originated from natural resources, 11 kinds of isoflavones, i.e., medicarpin (1), formononetin (2), mucronulatol (3), (3R)-calussequinone (5), (3R)-5'-methoxyvestitol (6), tectorigenin (7), biochanin A (8), tuberosin (9), calycosin (10), daidzein (11), and genistein (12), as well as a flavone, liquritigenin (4), were isolated as active principles responsible for the yeast α-glucosidase inhibitory activity from two leguminous plant extracts, i.e., the heartwood extract of Dalbergia odorifera and the roots extract of Pueraria thunbergiana. Each components (1-12) demonstrated a significantly potent inhibition on yeast α-glucosidase in a dose dependent manner when the p-nitrophenyl-α-D-glucopyranoside was used as a substrate in vitro. The concentration required for 50% enzyme inhibition (IC50) were calculated as 2.93 mM (1), 0.51 mM (2), 3.52 mM (7) 0.35 mM (8), 3.52 mM (9), 0.85 mM (11), and 0.15 mM (12) when that of reference drug acarbose was evaluated as 9.11 mM, in vitro. However, isoflavone glycosides, i.e., puerarin (13), daidzin (14), formononetin-7-O-ß-glucopyranoside (15), and genistin (16), exhibited a relatively poor inhibitory activity on yeast α-glucosidase as compared with the corresponding isoflavone (2, 11, 12), respectively.

Zizyphus attenuates ischemic damage in the gerbil hippocampus via its antioxidant effect.

The fruit of Zizyphus jujuba has been used as a traditional Chinese medicinal herb and considered for thousands of years to affect various physiological functions in the body. We obtained a Z. jujuba extract (ZJE) and observed the neuroprotective effects of ZJE against ischemic damage in gerbils that had received repeated oral administrations of ZJE for 10 days. In the ZJE-treated ischemia group, neuronal nuclei (a marker for neurons)-immunoreactive neurons were abundant (58.4% vs. sham group) in the hippocampal CA1 region 4 days after ischemia/reperfusion compared to those in the vehicle-treated ischemia group (11.3%). In addition, ZJE treatment significantly decreased the reactive gliosis of astrocytes and microglia in the CA1 region compared to that in the vehicle-treated group 4 days after ischemia/reperfusion. Immunoreactivities of Cu,Zn-superoxide dismutase (SOD1) and brain-derived neurotrophic factor in the ZJE-treated ischemia group were higher those in the vehicle-treated ischemia group 4 days after ischemia/reperfusion. In addition, in the ZJE-treated ischemia group, levels of hydroxynonenal, an indicator of lipid peroxidation, were much lower than those in the vehicle-treated ischemia group after ischemia/reperfusion. These results suggest that the repeated supplements of ZJE can protect neurons from ischemic damage via up-regulation of SOD1 and reduction of lipid peroxidation in the ischemic hippocampal CA1 region.

Preventative effects of Platycodon grandiflorum treatment on hepatic steatosis in high fat diet-fed C57BL/6 mice.

Platycodon grandiflorum (PG) (Korean name, Doraji; Chinese name, Jiegeng; and Japanese name, Kikyo) is a perennial plant in the Campanulaceae family that contains triterpenoid saponins, carbohydrates, and fibers. This study was carried out to investigate effects of root of PG on fatty liver inhibition in high fat diet (HFD)-fed C57BL/6 mice. C57BL/6 mice were divided into control, total extract of PG (T-PG, 500 mg/kg) and saponin fraction (S-PG, 50 mg/kg)-treated groups. Significant decreases in body weight, associated with fat mass reduction, were observed in PG-treated groups (p<0.05). Hepatic lipid content and score index calculated from morphometric observations on fatty liver were significantly decreased in the PG-treated groups (p<0.05). Moreover, activities of fatty acid synthase (FAS) and carnitine palmitoyl-transferase (CPT) were significantly suppressed and increased as compared with the control group, respectively (p<0.05). mRNA expressions of the sterol regulatory element binding protein (SREBP1c) and stearoyl-CoA desaturase (SCD1) gene were suppressed in the T-PG and S-PG groups (p<0.05). From these findings, we speculate that fatty liver inhibition effects of PG extract and its saponins appear to be conferred by hepatic lipogenesis and acceleration of energy expenditure, along with modulation of liver FAS and CPT activities in HFD-fed C57BL/6 mice.

Stimulatory effect of undecylenic acid on mouse osteoblast differentiation.

Natural compounds with bone-forming (or anabolic) activity have been recently focused on in bone research. The present study investigated the effect of undecylenic acid (UA) on osteoblast differentiation in mouse osteoblastic MC3T3-E1 subclone 4 cells and primary mouse calvarial cells. Low concentrations of UA (up to 5 microM) exhibited no cytotoxicity and significantly increased the expression and activity of alkaline phosphatase (early differentiation marker of osteoblast) and calcium deposition with the induction of expression of the osteocalcin gene in both cells. Interestingly, at low concentration of UA, the induction of NF-kappaB p65 translocation into nucleus and the up-regulation of AP-1 and NFATc1 transcript levels were also observed, suggesting that the stimulatory effect of UA on osteoblast differentiation could be mediated through the activation of transcription factors. Additionally, although the patterns of UA-induced activation of MAP kinases (JNK and p38) were not completely consistent with the increase of both ALP activity and calcium deposition by UA, MAP kinases might be partially involved in the biological function of UA during the early and late stages of osteoblast differentiation.

Melanin-concentrating hormone-1 receptor binding activity of pheophorbides isolated from Morus alba leaves.

The time-resolved fluorescence technique based on melanin-concentrating hormone (MCH) receptor subtype-1 (MCH-1 receptor) binding assay was adopted to carry out a bioassay-guided fractionation of the methanol extract of Morus alba leaves. This fractionation and purification led to the isolation of two compounds identified as pheophorbide a methyl ester and 13(2)(S)-hydroxypheophorbide a methyl ester. These active pheophorbides exhibited potent inhibitory activity in binding of europium-labeled MCH to the human recombinant MCH-1 receptor (IC(50) value; 4.03 and 0.33 microM, respectively). Besides binding activity, the pheophorbides inhibited MCH-mediated extracellular signal-regulated kinase (ERK) phosphorylation in Chinese hamster ovary cells expressing human MCH-1 receptor. These results suggest that pheophorbide a methyl ester and 13(2)(S)-hydroxypheophorbide a methyl ester act as modulators of MCH-1 receptor and MCH-mediated ERK signaling.

Platycodin D and 2''-O-acetyl-polygalacin D2 isolated from Platycodon grandiflorum protect ischemia/reperfusion injury in the gerbil hippocampus.

Platycodi radix is used as a folk remedy for several conditions. In this study, we investigated the neuroprotective effects of five major extracts; deapioplatycoside E (DPE), platycoside E (PE), platyconic acid A (PA), platycodin D (PD) and 2''-o-acetyl-polygalacin D2 (PD2) isolated from the P.radix in the hippocampal CA1 region (CA1) 4 or 10 days after ischemia/reperfusion (I/R). Each extract was administered into gerbils with intraperitoneal injection (5 mg/kg/day) 10 days before ischemic surgery and the gerbils were sacrificed 4 or 10 days after I/R. Fluoro-Jade B (F-J B, a marker for neurodegeneration) positive ((+)) neurons increased significantly in the stratum pyramidale of the CA1 region in the vehicle-treated group after I/R. A similar pattern was observed in the DPE-, PE- and PA-treated groups; however, in the PD- and PD2-treated groups, F-J B(+) neurons were small in number. We also observed that activations of astrocytes and microglia in the CA1 region after I/R were blocked by the PD- and PD2 treatments. In addition, we found that Cu,Zn-superoxide dismutase (SOD1) immunoreactivity in the pyramidal layer of the PD- and PD2-treated groups was similar to that of the sham group and COX-2(+) and NF-kappaB(+) cells were significantly lower in the PD- and PD2-treated group than those in the vehicle-treated group after I/R. These results suggest that PD and PD2 rescue neurons in the CA1 region from an ischemic damage.

Melanin-concentrating hormone-1 receptor antagonism and anti-obesity effects of ethanolic extract from Morus alba leaves in diet-induced obese mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In Korea, Morus alba leaves have been traditionally administered as natural therapeutic agent for the alleviating dropsy and diabetes. AIM OF THE STUDY: The present study was performed to evaluate melanin-concentrating hormone receptor subtype 1 (MCH1) antagonism of the ethanol extract of Morus alba leaves (EMA) and its anti-obesity effect in diet-induced obese (DIO) mice. MATERIALS AND METHODS: The binding affinity of EMA for the MCH1 receptor with europium-labeled MCH (Eu-MCH), the function of recombinant MCH1 receptors expressed in CHO cells, and the anti-obesity effects in DIO mice were evaluated. RESULTS: MCH1 receptor binding studies showed, EMA exhibited a potent inhibitory activity with IC50 value of 2.3+/-1.0 microg/ml. EMA (10-100 microg/ml) also inhibited the intracellular calcium mobilization with the recombinant MCH1 receptors expressed in CHO cells. In an anti-obesity study with DIO mice, longterm oral administrations of EMA for 32 consecutive days produced a dose-dependent decrease in body weight and hepatic lipid accumulation. CONCLUSIONS: These results suggest that chronic treatment with EMA exerts an anti-obesity effect in DIO mice, and its direct MCH1 receptor antagonism may contribute to decrease body weight.

A new specific BACE-1 inhibitor from the stembark extract of Vitis vinifera.

A new resveratrol dimer, (+)-vitisinol E (1) which demonstrated inhibitory activity on BACE-1 (beta-site APP-cleaving enzyme 1) in vitro, was isolated from the stembark extract of Vitis vinifera (Vitaceae) together with four known resveratrol oligomers, (+)-epsilon-viniferin (2), (+)-ampelopsin A (3), (+)-vitisin A (4) and (-)-vitisin B (5). The chemical structure of 1 was established by MR spectroscopic analyses, including HMBC. All isolated resveratrol derivatives (1-5) demonstrated significant inhibition on baculovirus-expressed BACE-1 in a dose-dependent manner, which was assessed with the FRET assay using Rh-EVNLDAEFK as a substrate in vitro.

Germinated Buckwheat extract decreases blood pressure and nitrotyrosine immunoreactivity in aortic endothelial cells in spontaneously hypertensive rats.

Thee present study analysed the quantification of rutin in raw buckwheat extract (RBE) and germinated buckwheat extract (GBE) by high performance liquid chromatography (HPLC), and examined changes in body weight, systolic blood pressure (SBP) and nitrotyrosine (a marker for peroxynitrite formation) immunoreactivity in aortic endothelial cells in spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats after treatment with RBE and GBE for 5 weeks. In the HPLC study, RBE and GBE contained a mean content of rutin of 1.52 +/- 0.21 and 2.92 +/- 0.88 mg/g, respectively. In the 600 mg/kg GBE-treated group, SBP was lower than that in the 600 mg/kg RBE-treated group. The treatment with RBE and/or GBE significantly reduced oxidative damage in aortic endothelial cells by lowering nitrotyrosine immunoreactivity. These results suggest that GBE has an antihypertensive effect and may protect arterial endothelial cells from oxidative stress.

Modulation of macrophage functions by compounds isolated from Zingiber officinale.

Bioactivity-guided fractionation of Zingiber Officinale (zingiberaceae) led us to isolate 14 compounds, -gingerol ( 1), -gingerol ( 2), -gingerol ( 3), -gingerol ( 4), -paradol ( 5), -shogaol ( 6), -shogaol ( 7), 1-dehydro- -gingerdione ( 8), -gingerdione ( 9), hexahydrocurcumin ( 10), tetrahydrocurcumin ( 11), gingerenone A ( 12), 1,7-bis-(4' hydroxyl-3' methoxyphenyl)-5-methoxyhepthan-3-one ( 13), and methoxy- -gingerol ( 14). Using the RAW 264.7 cell line, the inhibitory effects on nitric oxide production induced by lipopolysaccharide and the stimulatory effects on phagocytosis of these compounds were evaluated. Compounds 7, 8, and 9 significantly decreased lipopolysaccharide-induced nitric oxide production, and compounds 7 and 8 significantly reduced inducible nitric oxide synthase expression. Among them, compound 8 also showed significant stimulatory effects on phagocytosis.