Sun-Hye Choi

Gintonin, Newly Identified Compounds from Ginseng, Is Novel Lysophosphatidic Acids-Protein Complexes and Activates G Protein-Coupled Lysophosphatidic Acid Receptors with High Affinity

Recently, we isolated a subset of glycolipoproteins from Panax ginseng, that we designated gintonin, and demonstrated that it induced [Ca2+]i transients in cells via G-protein-coupled receptor (GPCR) signaling pathway(s). However, active components responsible for Ca2+ mobilization and the corresponding receptor(s) were unknown. Active component(s) for [Ca2+]i transients of gintonin were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry and ion-mobility mass spectrometry, respectively. The corresponding receptor(s) were investigated through gene expression assays. We found that gintonin contains LPA C18:2 and other LPAs. Proteomic analysis showed that ginseng major latex-like protein and ribonuclease-like storage proteins are protein components of gintonin. Gintonin induced [Ca2+]i transients in B103 rat neuroblastoma cells transfected with human LPA receptors with high affinity in order of LPA2 > LPA5 > LPA1 > LPA3 > LPA4. The LPA1/LPA3 receptor antagonist Ki16425 blocked gintonin action in cells expressing LPA1 or LPA3. Mutations of binding sites in the LPA3 receptor attenuated gintonin action. Gintonin acted via pertussis toxin (PTX)-sensitive and -insensitive G protein-phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3)-Ca2+ pathways. However, gintonin had no effects on other receptors examined. In human umbilical vein endothelial cells (HUVECs) gintonin stimulated cell proliferation and migration. Gintonin stimulated ERK1/2 phosphorylation. PTX blocked gintonin-mediated migration and ERK1/2 phosphorylation. In PC12 cells gintonin induced morphological changes, which were blocked by Rho kinase inhibitor Y-27632. Gintonin contains GPCR ligand LPAs in complexes with ginseng proteins and could be useful in the development of drugs targeting LPA receptors.

Gintonin, a Ginseng-Derived Lysophosphatidic Acid Receptor Ligand, Attenuates Alzheimer's Disease-Related Neuropathies: Involvement of Non-Amyloidogenic Processing

Ginseng extracts show cognition-enhancing effects in Alzheimers disease (AD) patients. However, little is known about the active components and molecular mechanisms of how ginseng exerts its effects. Recently, we isolated a novel lysophosphatidic acid (LPA) receptor-activating ligand from ginseng, gintonin. AD is caused by amyloid-β protein (Aβ) accumulation. Aβ is derived from amyloid-β protein precursors (AβPPs) through the amyloidogenic pathway. In contrast, non-amyloidogenic pathways produce beneficial, soluble AβPPα (sAβPPα). Here, we describe our investigations of the effect of gintonin on sAβPPα release, Aβ formation, Swedish-AβPP transfection-mediated neurotoxicity in SH-SY5Y neuroblastoma cells, and Aβ-induced neuropathy in mice. Gintonin promoted sAβPPα release in a concentration- and time-dependent manner. Gintonin action was also blocked by the Ca2+ chelator BAPTA, α-secretase inhibitor TAPI-2, and protein-trafficking inhibitor brefeldin. Gintonin decreased Aβ1-42 release and attenuated Aβ1-40-induced cytotoxicity in SH-SY5Y cells. Gintonin also rescued Aβ1-40-induced cognitive dysfunction in mice. Moreover, in a transgenic mouse AD model, long-term oral administration of gintonin attenuated amyloid plaque deposition as well as short- and long-term memory impairment. In the present study, we demonstrated that gintonin mediated the promotion of non-amyloidogenic processing to stimulate sAβPPα release to restore brain function in mice with AD. Gintonin could be a useful agent for AD prevention or therapy.

Identification of ginsenoside interaction sites in 5-HT3A receptors.

We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.

Ginsenoside Rg3 Inhibits Human Kv1.4 Channel Currents by Interacting with the Lys531 Residue

We have demonstrated previously that the 20(S) but not the 20(R) form of ginsenoside Rg3 inhibited K+ currents flowing through Kv1.4 (hKv1.4) channels expressed in Xenopus laevis oocytes, pointing to the presence of specific interaction site(s) for Rg3 in the hKv1.4 channel. In the current study, we sought to identify this site(s). To this end, we first assessed how point mutations of various amino acid residues of the hKv1.4 channel affected inhibition by 20(S)-ginsenoside Rg3 (Rg3). Lys531 residue is known to be a key site for K+ activation and to be part of the extracellular tetraethylammonium (TEA) binding site; the mutation K531Y abolished the Rg3 effect and made the Kv1.4 channel sensitive to TEA applied to the extracellular side of the membrane. Mutations of many other residues, including the pH sensitive-site (H507Q), were without any significant effect. We next examined whether K+ and TEA could alter the effect of Rg3 and vice versa. We found that 1) raising [K+]o reduced the inhibitory effect of Rg3 on hKv1.4 channel currents, whereas Rg3 shifted the K+ activation curve to the right, and 2) TEA caused a rightward shift of the Rg3 concentration-response curve of wild-type hKv1.4 channel currents, whereas Rg3 caused a rightward shift of the TEA concentration-response curve of K531Y mutant channel currents. The docked modeling revealed that Lys531 plays a key role in forming hydrogen bonds between Rg3 and hKv1.4 channels. These results indicate that Rg3 inhibits the hKv1.4 channel current by interacting with residue Lys531.

Effects of Korean red ginseng extract on cisplatin-induced nausea and vomiting

Ginseng, the root ofPanax ginseng C. A. Meyer, is well known as a tonic medicine for restoring and enhancing human health. In traditional medicine, ginseng is utilized for the alleviation of emesis, which includes nausea and vomiting. However, it has not yet been demonstrated whether ginseng exhibitsin vivo anti-nausea and anti-vomiting properties. In this study, we examined the anti-emetic effect of Korean red ginseng total extract (KRGE) on cisplatin-induced nausea and vomiting using ferrets. Intraperitoneal administration (i.p.) of cisplatin (7.5 mg/kg) induced both nausea and vomiting with one-hour latency. The episodes of nausea and vomiting reached a peak after 1.5 h and persisted for 3 h. Treatment with KRGEvia oral route significantly reduced the cisplatin-induced nausea and vomiting in a dose-dependent manner. The anti-emetic effect was 12.7±8.6, 31.8±6.9, and 67.6±4.0% with doses of 0.3, 1.0, and 3.0 g/kg of KRGE, respectively. Pretreatment with KRGEvia oral route 1 and 2 h before cisplatin administration also significantly attenuated the cisplatin-induced nausea and vomiting. However this did not occur with a pretreatment 4 h before cisplatin administration. These results are supportive of KRGE being utilized as an anti-emetic agent against nausea and vomiting caused by chemotherapy (i.e. cisplatin).

A simple method for the preparation of crude gintonin from ginseng root, stem, and leaf.

Ginseng has been used as a general tonic agent to invigorate the human body as an adaptogenic agent. In a previous report, we have shown that ginseng contains a novel glycolipoprotein called gintonin. The main function of gintonin is to transiently enhance intracellular free Ca2+ [Ca2+]i levels in animal cells. The previous method for gintonin isolation included multiple steps using organic solvents. In the present report, we developed a simple method for the preparation of crude gintonin from ginseng root as well as stem and leaf, which produced a higher yield of gintonin than the previous one. The yield of gintonin was 0.20%, 0.29%, and 0.81% from ginseng root, stem, and leaf, respectively. The apparent molecular weight of gintonin isolated from stem and leaf through sodium dodecyl sulfate polyacrylamide gel electrophoresis was almost same as that from root but the compositions of amino acids, carbohydrates or lipids differed slightly between them. We also examined the effects of crude gintonin from ginseng root, stem, and leaf on endogenous Ca2+-activated Cl- channel (CaCC) activity of Xenopus oocytes through mobilization of [Ca2+]i. We found that the order of potency for the activation of CaCC was ginseng root > stem > leaf. The ED50 was 1.4±1.4, 4.5±5.9, and 3.9±1.1 μg/mL for root, stem and leaf, respectively. In the present study, we demonstrated for the first time that in addition to ginseng root, ginseng stem and leaf also contain gintonin. Gintonin can be prepared from a simple method with higher yield of gintonin from ginseng root, stem, and leaf. Finally, these results demonstrate the possibility that ginseng stem and leaf could also be utilized for ginstonin preparation after a simple procedure, rather than being discarded.

Effects of Minor Ginsenosides, Ginsenoside Metabolites, and Ginsenoside Epimers on the Growth of Caenorhabditis elegans

In the previous report, we have demonstrated that ginsenoside Rc, one of major ginsenosides, is a major component for the restoration for normal growth of worms in cholesterol-deprived medium. In the present study, we further investigated the roles of minor ginsenosides, such as ginsenoside Rh1 and Rh2, ginsenoside metabolites such as compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT) and ginsenoside epimers such as 20(R)- and 20(S)-ginsenoside Rg3 in cholesterol-deprived medium. We found that ginsenoside Rh1 almost restored normal growth of worms in cholesterol-deprived medium in F1 generation. However, supplement of ginsenoside Rh2 caused a suppression of worm growths in cholesterol-deprived medium. In addition, CK and PPD also slightly restored normal growth of worms in cholesterol-deprived medium but PPT not. In experiments using ginsenoside epimers, supplement of 20(S)- but not 20(R)-ginsenoside Rg3 in cholesterol-deprived medium also almost restored worm growth. These results indicate that the absence or presence of carbohydrate component at backbone of ginsenoside, the number of carbohydrate attached at carbon-3, and the position of hydroxyl group at carbon-20 of ginsenoside might plays important roles in restoration of worm growth in cholesterol-deprived medium.

Ginsenoside Rg3 activates human KCNQ1 K+ channel currents through interacting with the K318 and V319 residues: a role of KCNE1 subunit.

The slowly activating delayed rectifier K(+) channels (I(Ks)) are one of the main pharmacological targets for development of drugs against cardiovascular diseases. Cardiac I(Ks) consists of KCNQ1 plus KCNE1 subunits. Ginsenoside, one of the active ingredient of Panax ginseng, enhances cardiac I(Ks) currents. However, little is known about the molecular mechanisms of how ginsenoside interacts with channel proteins to enhance cardiac I(Ks). In the present study, we investigated ginsenoside Rg(3) (Rg(3)) effects on human I(Ks) by co-expressing human KCNQ1 plus KCNE1 subunits in Xenopus oocytes. Rg(3) enhanced I(Ks) currents in concentration- and voltage-dependent manners. The EC(50) was 15.2+/-8.7 microM. However, in oocytes expressing KCNQ1 alone, Rg(3) inhibited the currents with concentration- and voltage-dependent manners. The IC(50) was 4.8+/-0.6 microM. Since Rg(3) acts opposite ways in oocytes expressing KCNQ1 alone or KCNQ1 plus KCNE1 subunits, we examined Rg(3) effects after co-expression of different ratios of KCNE1 and KCNQ1. The increase of KCNE1/KCNQ1 ratio converted I(Ks) inhibition to I(Ks) activations. One to ten ratio of KCNE1 and KCNQ1 subunit is required for Rg(3) activation of I(Ks). Mutations of K318 and V319 into K318Y and V319Y of KCNQ1 channel abolished Rg(3) effects on KCNQ1 or KCNQ1 plus KCNE1 channel currents. The docked modeling revealed that K318 residue plays a key role in stabilization between Rg(3) and KCNQ1 plus KCNE1 or KCNQ1 subunit. These results indicate that Rg(3)-induced activation of I(Ks) requires co-assembly of KCNQ1 and KCNE1 subunits and achieves this through interaction with residues K318 and V319 of KCNQ1 subunit.

Ginsenoside Rg3 decelerates hERG K+ channel deactivation through Ser631 residue interaction

The human ether-a-go-go-related gene (hERG) cardiac K(+) channels are one of the representative pharmacological targets for development of drugs against cardiovascular diseases such as arrhythmia. Panax ginseng has been known to have cardio-protective effects. However, little is known about the molecular mechanisms of how ginsenosides, the active ingredients in Panax ginseng, interact with hERG K(+) channel proteins. In the present study, we first examined the effects of various ginsenosides on hERG K(+) channel activity by expressing human α subunits in Xenopus oocytes. Among them ginsenoside Rg(3) (Rg(3)) most potently enhanced outward I(hERG) and peak I(tail). Rg(3) induced a large persistent deactivating-tail current (I(deactivating-tail)) and profoundly decelerated deactivating current decay in both concentration- and voltage-dependent manners. The EC(50) for steady-state I(hERG), peak I(tail), and persistent I(deactivating-tail) was 0.41±0.05, 0.61±0.11, and 0.36±0.04μM, respectively. Rg(3) actions were blocked by bepridil, a hERG K(+) channel antagonist. Site-directed mutation of S631, which is located at the channel pore entryway, to S631C in hERG K(+) channel abolished Rg(3) actions on hERG K(+) channels. These results indicate that S631 residue of hERG K(+) channel plays an important role in Rg(3)-mediated induction of a persistent I(deactivating-tail) and in a deceleration of hERG K(+) channel deactivation.

Effects of quercetin on α9α10 nicotinic acetylcholine receptor-mediated ion currents

Quercetin, one of the flavonoids, is a low molecular weight substance found in fruits and vegetables. Quercetin, like other flavonoids, has a wide range of neuropharmacological actions and antioxidant effects. The α9α10 nicotinic acetylcholine receptor is one of the numerous nicotinic acetylcholine receptors that exist as a heteropentameric form between efferent olivocochlear fibers and hair cells of the cochlea. In this study, we report the effects of quercetin on rat α9α10 nicotinic acetylcholine receptor-mediated ion currents using the two-electrode voltage-clamp technique. Treatment with acetylcholine evoked inward currents (I(ACh)) in oocytes heterologously expressing the α9α10 nicotinic acetylcholine receptor. Quercetin blocked I(ACh) in concentration-dependent and reversible manners, and the blocking effect on I(ACh) was stronger with pre-application than co-application of quercetin. The half maximal inhibitory concentration (IC(50)) of quercetin was 45.4±10.1μM. Quercetin-mediated I(ACh) inhibition was not affected by acetylcholine concentration and was independent of membrane-holding potential. Although the inhibitory effect of quercetin was significantly attenuated in the absence of extracellular Ca(2+), the action of quercetin was independent of extracellular Ca(2+) concentration, indicating that the presence of extracellular Ca(2+) might be needed for quercetin-related effects and might play an important role in quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor. These results indicate that quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor could provide a molecular basis for quercetin actions at the cellular level.