Yong Soo Kwon

The Cyt P450 Enzyme CYP716A47 Catalyzes the Formation of Protopanaxadiol from Dammarenediol-II During Ginsenoside Biosynthesis in Panax ginseng

Ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs and contains pharmacologically active components, ginsenosides, in its roots. Ginsenosides, a class of tetracyclic triterpene saponins, are thought to be synthesized from dammarenediol-II after hydroxylation by the Cyt P450 (CYP) enzyme and then glycosylation by glycosyltransferase (GT). However, no genes encoding the hydroxylation and glycosylation in ginsenoside biosynthesis have been identified. Here, we identify protopanaxadiol synthase, which is a CYP enzyme (CYP716A47), to be involved in the hydroxylation of dammarenediol-II at the C-12 position to yield protopanaxadiol. Nine putative full CYP sequences were isolated from the expressed sequence tags (ESTs) of methyl jasmonate (MeJA)-treated adventitious ginseng roots. The CYP716A47 gene product was selected as the putative protopanaxadiol synthase because this gene was transcriptionally activated not only by MeJA treatment but also in transgenic ginseng that overexpresses squalene synthase and overproduces ginsenosides. In vitro enzymatic activity assays revealed that CYP716A47 catalyzed the oxidation of dammarenediol-II to produce protopanaxadiol. Ectopic expression of CYP716A47 in recombinant WAT21 yeasts that were fed dammarenediol-II yielded protopanaxadiol. Furthermore, co-expression of the dammarenediol synthase gene (PgDDS) and CYP716A47 in yeast yielded protopanaxadiol without adding dammarenediol-II. The chemical structures of the protopanaxadiol products from dammarenediol-II were confirmed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Thus, CYP716A47 is a dammarenediol 12-hydroxylase that produces protopanaxadiol from dammarenediol-II.

Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng.

Squalene epoxidase catalyzes the first oxygenation step in phytosterol and triterpenoid saponin biosynthesis and is suggested to represent one of the rate-limiting enzymes in this pathway. Here, we investigated the roles of two squalene epoxidase genes (PgSQE1 and PgSQE2) in triterpene and phytosterol biosynthesis in Panax ginseng. PgSQE1 and PgSQE2 encoded deduced proteins of 537 and 545 amino acids, respectively. Amino acid sequences deduced from PgSQE1 and PgSQE2 share 83% homology, but the N-terminal regions (first 60 amino acids) are highly different. PgSQE1 mRNA abundantly accumulated in all organs. PgSQE2 was only weakly expressed and preferentially in petioles and flower buds. Methyl jasmonate (MeJA) treatment enhanced the accumulation of PgSQE1 mRNA in roots, but rather suppressed expression of PgSQE2. Precursor (squalene) treatment coordinately upregulated the expression of both PgSQE1 and PgSQE2. In situ hybridization analysis established that both PgSQE1 and PgSQE2 mRNAs accumulated preferentially in vascular bundle tissue and resin ducts of petioles. RNA interference of PgSQE1 in transgenic P. ginseng completely suppressed PgSQE1 transcription. Concomitantly, the interference of PgSQE1 resulted in reduction of ginsenoside production. Interestingly, silencing of PgSQE1 in RNAi roots strongly upregulated PgSQE2 and PNX (cycloartenol synthase) and resulted in enhanced phytosterol accumulation. These results indicate that expression of PgSQE1 and PgSQE2 were regulated in a different manner, and that PgSQE1 will regulate ginsenoside biosynthesis, but not that of phytosterols in P. ginseng.

Cytotoxic constituents from the bark of Salix hulteni.

Eight compounds were isolated from the bark of Salix hulteni. Based on spectral data, the isolated compounds were identified as 4-hydroxyacetophenone (1), naringenin (2), aromadendrin (3), catechin (4), picein (5), sachaliside 1 (6), 1-p-coumaroyl-β-D-glucoside (7), and dihydromyricetin (8). Their cytotoxic activities against brine shrimp and a human lung cancer cell line (H1299) were evaluated.

Antioxidative constituents fromPaeonia lactiflora

The ethanol extract of the peony root (Paeonia Lactiflora Pall, Paeoniaceae) as well as its major active components including gallic acid and methyl gallate were evaluated for their protective effects against free radical generation and lipid peroxidation. In addition, the protective effects against hydrogen peroxide-induced oxidative DNA damage in a mammalian cell line were examined. The ethanol extracts of the peony root (PREs) and its active constituents, galdiphenyl-2-picryl hydrazine (DPPH) radical generation and had an inhibitory effect on lipid peroxidation, as measured by the level of malondialdehyde (MDA) formation. The PREs did not have any pro-oxidant effect. They strongly inhibited the hydrogen peroxide-induced DNA damage from NIH/3T3 fibroblasts, as assessed by single cell gel electrophoresis. Furthermore, the oral administration of 50% PRE (50% ethanol extract of peony root), gallic acid and methyl gallate potently inhibited the formation of micronucleated reticulocytes (MNRET) in the mouse peripheral blood induced by a KBrOP3 treatmentin vivo. Therefore, PREs containing gallic acid and methyl gallate may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative DNA damage without exhibiting any pro-oxidant effect.

3,4,5-Trihydroxycinnamic acid inhibits lipopolysaccharide (LPS)-induced inflammation by Nrf2 activation in vitro and improves survival of mice in LPS-induced endotoxemia model in vivo

NF-E2-related factor 2 (Nrf2) has been demonstrated to be a key transcription factor regulating the anti-inflammatory genes including heme oxygenase-1 (HO-1) in experimental sepsis models. Based on the fact that 3,4,5-trihydorxycinnamic acid (THC) has been reported to possess anti-inflammatory properties in BV2 microglial cells, the possible effects of THC and its underlying mechanism was examined against lipopolysaccharide (LPS)-induced RAW 264.7 cell culture and septic mouse models. Pretreatment of RAW 264.7 cells with THC significantly attenuated LPS-induced NO, PGE2 production, and expression of iNOS and COX-2. THC also significantly suppressed LPS-induced release of pro-inflammatory cytokines and degradation of IκB-α. Increased phosphorylation of Nrf2 and nuclear translocation of Nrf2 were observed with THC treatment with consequent expression of HO-1. The data demonstrated that multiple signaling pathways including Akt, p38, and PKC are involved in the THC-induced activation of Nrf2/HO-1 pathway. Treatment of THC resulted in significantly increased survival of LPS-induced septic mice. THC also significantly ameliorated LPS-induced septic features such as hypothermia and increased vascular leakage. In accordance with the data from cell culture model, THC exhibited increased expression of HO-1 in kidney and decreased serum level of pro-inflammatory mediators such as TNF-α, IL-1β, and NO. Taken together, the present study for the first time demonstrates that THC inhibits inflammation in LPS-induced RAW264.7 cells by Nrf2 activation and improves survival of mice in LPS-induced endotoxemia model.

3,4,5-Trihydroxycinnamic Acid Inhibits LPS-Induced iNOS Expression by Suppressing NF-κB Activation in BV2 Microglial Cells

Although various derivatives of caffeic acid have been reported to possess a wide variety of biological activities such as neuronal protection against excitotoxicity and anti-inflammatory property, the biological activity of 3,4,5-trihydroxycinnamic acid (THC), a derivative of hydroxycinnamic acids, has not been clearly examined. The objective of the present study is to evaluate the anti-inflammatory effects of THC on lipopolysaccharide (LPS)-stimulated BV2 microglial cells. THC significantly suppressed LPS-induced excessive production of nitric oxide (NO) and expression of iNOS, which is responsible for the production of iNOS. THC also suppressed LPS-induced overproduction of pro-inflammatory cytokines such as IL-1β and TNF-α in BV2 microgilal cells. Furthermore, THC significantly suppressed LPS-induced degradation of IκB, which retains NF-κB in the cytoplasm. Therefore, THC attenuated nuclear translocation of NF-κB, a major pro-inflammatory transcription factor. Taken together, the present study for the first time demonstrates that THC exhibits anti-inflammatory activity through the suppression of NF-κB transcriptional activation in LPS-stimulated BV2 microglial cells.

Protection against kainate neurotoxicity by ginsenosides: Attenuation of convulsive behavior, mitochondrial dysfunction, and oxidative stress

We previously demonstrated that kainic acid (KA)‐mediated mitochondrial oxidative stress contributed to hippocampal degeneration and that ginsenosides attenuated KA‐induced neurotoxicity and neuronal degeneration. Here, we examined whether ginsenosides affected KA‐induced mitochondrial dysfunction and oxidative stress in the rat hippocampus. Treatment with ginsenosides attenuated KA‐induced convulsive behavior dose‐dependently. KA treatment increased lipid peroxidation and protein oxidation and decreased the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio to a greater degree in the mitochondrial fraction than in the hippocampal homogenate. KA treatment resulted in decreased Mn‐superoxide dismutase expression anddiminished the mitochondrial membrane potential. Furthermore, KA treatment increased intramitochondrial Ca2+ and promoted ultrastructural degeneration in hippocampal mitochondria. Treatment with ginsenosides dose‐dependently attenuated convulsive behavior and the KA‐induced mitochondrial effects. Protection appeared to be more evident in mitochondria than in tissue homogenates. Collectively, the results suggest that ginsenosides prevent KA‐induced neurotoxicity by attenuating mitochondrial oxidative stress and mitochondrial dysfunction.

Selegiline potentiates the effects of EGb 761 in response to ischemic brain injury

We evaluated whether combined treatment with selegiline, a selective MAO-B inhibitor, and EGb 761, a standard extract of Ginkgo biloba, has synergistic effects against ischemic reperfusion injury (IRI) in gerbils. Interestingly, we observed that pretreatment with EGb 761 significantly attenuated selegiline-induced hyperactivity. This finding paralleled striatal fos-related antigen immunoreactivity (FRA-IR) in mice. Four minutes of bilateral carotid artery occlusion caused substantial cell loss in the CA1 of the hippocampus 5 days post-ischemic insult. Pretreatment with EGb 761, with or without selegiline, significantly attenuated this neuronal loss. Combined treatment with EGb 761 plus selegiline was more efficacious in preventing this loss. Synaptosomal formations of protein carbonyl, lipid peroxidation (malondialdehyde (MDA) + 4-hydroxyalkenal (4-HDA)), and reactive oxygen species (ROS) in the hippocampus remained elevated 5 days post-ischemic insult. The antioxidant effects appeared to be most significant in the group treated with EGb 761 plus selegiline. This combined treatment produced more significant attenuation of IRI-induced alterations in intramitochondrial calcium accumulation, the mitochondrial transmembrane potential, and mitochondrial Mn-superoxide dismutase-like immunoreactivity (Mn-SOD-IR) than either treatment alone. Our results suggest that co-administration of EGb 761 and selegiline produces significant neuroprotective effects via suppression of oxidative stress and mitochondrial dysfunction without affecting neurological function.

Antiviral Activity of Hederasaponin B from Hedera helix against Enterovirus 71 Subgenotypes C3 and C4a.

Enterovirus 71 (EV71) is the predominant cause of hand, foot and mouth disease (HFMD). The antiviral activity of hederasaponin B from Hedera helix against EV71 subgenotypes C3 and C4a was evaluated in vero cells. In the current study, the antiviral activity of hederasaponin B against EV71 C3 and C4a was determined by cytopathic effect (CPE) reduction method and western blot assay. Our results demonstrated that hederasaponin B and 30% ethanol extract of Hedera helix containing hederasaponin B showed significant antiviral activity against EV71 subgenotypes C3 and C4a by reducing the formation of a visible CPE. Hederasaponin B also inhibited the viral VP2 protein expression, suggesting the inhibition of viral capsid protein synthesis.These results suggest that hederasaponin B and Hedera helix extract containing hederasaponin B can be novel drug candidates with broad-spectrum antiviral activity against various subgenotypes of EV71.

Aromadendrin Inhibits Lipopolysaccharide-Induced Nuclear Translocation of NF-κB and Phosphorylation of JNK in RAW 264.7 Macrophage Cells.

Aromadendrin, a flavonol, has been reported to possess a variety of pharmacological activities such as anti-inflammatory, antioxidant, and anti-diabetic properties. However, the underlying mechanism by which aromadendrin exerts its biological activity has not been extensively demonstrated. The objective of this study is to elucidate the anti-inflammatory mechanism of aromadedrin in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Aromadendrin significantly suppressed LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2. In accordance, aromadendrin attenuated LPSinduced overexpression iNOS and COX-2. In addition, aromadendrin significantly suppressed LPS-induced degradation of IκB, which sequesters NF-κB in cytoplasm, consequently inhibiting the nuclear translocation of pro-inflammatory transcription factor NF- κB. To elucidate the underlying signaling mechanism of anti-inflammatory activity of aromadendrin, MAPK signaling pathway was examined. Aromadendrin significantly attenuated LPS-induced activation of JNK, but not ERK and p38, in a concentration-dependent manner. Taken together, the present study clearly demonstrates that aromadendrin exhibits anti-inflammatory activity through the suppression of nuclear translocation of NF-κB and phosphorylation of JNK in LPS-stimulated RAW 264.7 macrophage cells.