Preparation of Red Ginseng Marc-Derived Gintonin and Its Application as a Skin Nutrient.

Ginseng is one of the traditional herbal medicines for tonic. Gintonin is a new material derived from white/red ginseng and its lysophosphatidic acids (LPAs) play as a ligand for G protein-coupled LPA receptors. Korean red ginseng marc (KRGM) is a by-product after the KRG processes. We developed a low-cost/high-efficiency method for KRGM gintonin production. We further studied the KRGM gintonin-mediated anti-skin aging effects under UVB exposure using human dermal fibroblasts (HDFs). KRGM gintonin yield is about 8%. KRGM gintonin contains a high amount of LPA C18:2, lysophosphatidylcholine (LPC), and phosphatidylcholine (PC), which is similar to white ginseng gintonin. KRGM gintonin induced [Ca2+]i transient via LPA1/3 receptors and increased cell viability/proliferation under UVB exposure. The underlying mechanisms of these results are associated with the antioxidant action of KRGM gintonin. KRGM gintonin attenuated UVB-induced cell senescence by inhibiting cellular ß-galactosidase overexpression and facilitated wound healing. These results indicate that KRGM can be a novel bioresource of KRGM gintonin, which can be industrially utilized as new material for skin nutrition and/or skin healthcare.

Effect of the Gintonin-Enriched Fraction on Glucagon-Like-Protein-1 Release.

Ginseng-derived gintonin reportedly contains functional lysophosphatidic acids (LPAs) as LPA receptor ligands. The effect of the gintonin-enriched fraction (GEF) on in vitro and in vivo glucagon-like protein-1 (GLP-1) secretion, which is known to stimulate insulin secretion, via LPA receptor(s) remains unclear. Accordingly, we examined the effects of GEF on GLP-1 secretion using human enteroendocrine NCI-H716 cells. The expression of several of LPA receptor subtypes in NCI-H716 cells using qPCR and Western blotting was examined. LPA receptor subtype expression was in the following order: LPA6 > LPA2 > LPA4 > LPA5 > LPA1 (qPCR), and LPA6 > LPA4 > LPA2 > LPA1 > LPA3 > LPA5 (Western blotting). GEF-stimulated GLP-1 secretion occurred in a dose- and time-dependent manner, which was suppressed by cAMP-Rp, a cAMP antagonist, but not by U73122, a phospholipase C inhibitor. Furthermore, silencing the human LPA6 receptor attenuated GEF-mediated GLP-1 secretion. In mice, low-dose GEF (50 mg/kg, peroral) increased serum GLP-1 levels; this effect was not blocked by Ki16425 co-treatment. Our findings indicate that GEF-induced GLP-1 secretion could be achieved via LPA6 receptor activation through the cAMP pathway. Hence, GEF-induced GLP secretion via LPA6 receptor regulation might be responsible for its beneficial effects on human endocrine physiology.

Wound Healing Effect of Gintonin Involves Lysophosphatidic Acid Receptor/Vascular Endothelial Growth Factor Signaling Pathway in Keratinocytes.

Gintonin, a novel compound of ginseng, is a ligand of the lysophosphatidic acid (LPA) receptor. The in vitro and in vivo skin wound healing effects of gintonin remain unknown. Therefore, the objective of this study was to investigate the effects of gintonin on wound healing-linked responses, especially migration and proliferation, in skin keratinocytes HaCaT. In this study, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, Boyden chamber migration assay, scratch wound healing assay, and Western blot assay were performed. A tail wound mouse model was used for the in vivo test. Gintonin increased proliferation, migration, and scratch closure in HaCaT cells. It also increased the release of vascular endothelial growth factor (VEGF) in HaCaT cells. However, these increases, induced by gintonin, were markedly blocked by treatment with Ki16425, an LPA inhibitor, PD98059, an ERK inhibitor, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester), a calcium chelator, and U73122, a PLC inhibitor. The VEGF receptor inhibitor axitinib also attenuated gintonin-enhanced HaCaT cell proliferation. Gintonin increased the phosphorylation of AKT and ERK1/2 in HaCaT cells. In addition, gintonin improved tail wound healing in mice. These results indicate that gintonin may promote wound healing through LPA receptor activation and/or VEGF release-mediated downstream signaling pathways. Thus, gintonin could be a beneficial substance to facilitate skin wound healing.

Comparative associations between anticholinergic burden and emergency department visits for anticholinergic adverse events in older Korean adults: a nested case-control study using national claims data for validation of a novel country-specific scale.

BACKGROUND: Considering the limited generalizability of previous anticholinergic burden scales, the Korean Anticholinergic Burden Scale (KABS) as a scale specific to the Korean population was developed. We aimed to validate the KABS by detecting the associations between high anticholinergic burden, measured with the KABS, and emergency department (ED) visits compared to the pre-existing validated scales in older Korean adults. METHODS: A nested case-control study was conducted using national claims data. The cases included the first anticholinergic ED visits between July 1 and December 31, 2016. Anticholinergic ED visits were defined as ED visits with a primary diagnosis of constipation, delirium, dizziness, fall, fracture, or urinary retention. Propensity score-matched controls were identified. Average daily AB scores during 30 days before the index date were measured. Multivariate logistic regression analyses were performed. RESULTS: In total, 461,034 were included. The highest proportion of those with high AB was identified with KABS (5.0%). Compared with those who had a KABS score of 0, older adults with a score ≥ 3 were at higher risk for overall anticholinergic ED visits (aOR, 1.62, 95% CI, 1.53-1.72), as well as visits for falls/fractures (aOR: 1.54, 95% CI: 1.40-1.69), dizziness (aOR: 1.44, 95% CI: 1.30-1.59), delirium (aOR: 2.96, 95% CI: 2.28-3.83), constipation (aOR: 1.84, 95% CI: 1.68-2.02), and urinary retention (aOR: 2.13, 95% CI: 1.79-2.55). High AB by KABS showed a stronger association with overall anticholinergic ED visits and visits due to delirium and urinary retention than those by other scales. CONCLUSIONS: In conclusion, KABS is superior to pre-existing scales in identifying patients with high AB and predicting high AB-related ED visits in older Korean adults.

Effects of gintonin-enriched fraction on hippocampal gene expressions.

BACKGROUND: Recently, gintonin and gintonin-enriched fraction (GEF) have been isolated from ginseng, a herbal medicine. Gintonin induces [Ca2+]i transition in cultured hippocampal neurons and stimulates acetylcholine release through LPA receptor activation. Oral administration of GEF is linked to hippocampus-dependent cognitive enhancement and other neuroprotective effects; however, effects of its long-term administration on hippocampal gene expression remains unknown. Here, we used next-generation sequence (NGS) analysis to examine changes in hippocampal gene expressions after long-term oral administration of GEF. METHODS: C57BL/6 mice were divided into three groups: control group, GEF50 (GEF 50 mg/kg, p.o.), and GEF100 (GEF 100 mg/kg, p.o.). After 22 days, total RNA was extracted from mouse hippocampal tissues. NGS was used for gene expression profiling; quantitative-real-time PCR and western blot were performed to quantify the changes in specific genes and to confirm the protein expression levels in treatment groups. RESULTS: NGS analysis screened a total of 23,282 genes, analyzing 11-related categories. We focused on the neurogenesis category, which includes four genes for candidate markers: choline acetyltransferase (ChAT) gene, ß3-adrenergic receptor (Adrb3) gene, and corticotrophin-releasing hormone (Crh) gene, and tryptophan 2,3-dioxygenase (Tdo2) gene. Real-time PCR showed a marked overexpression of ChAT, Adrb3, and Crh genes, while reduced expression of Tdo2. Western blot analysis also confirmed increased ChAT and decreased Tdo2 protein levels. CONCLUSION: We found that GEF affects mouse hippocampal gene expressions, associated with memory, cognitive, anti-stress and anti-anxiety functions, and neurodegeneration at differential degree, that might explain the genetic bases of GEF-mediated neuroprotective effects.

Ginseng Gintonin Contains Ligands for GPR40 and GPR55.

Gintonin, a novel ginseng-derived glycolipoprotein complex, has an exogenous ligand for lysophosphatidic acid (LPA) receptors. However, recent lipid analysis of gintonin has shown that gintonin also contains other bioactive lipids besides LPAs, including linoleic acid and lysophosphatidylinositol (LPI). Linoleic acid, a free fatty acid, and LPI are known as ligands for the G-protein coupled receptors (GPCR), GPR40, and GPR55, respectively. We, herein, investigated whether gintonin could serve as a ligand for GPR40 and GPR55, using the insulin-secreting beta cell-derived cell line INS-1 and the human prostate cancer cell line PC-3, respectively. Gintonin dose-dependently enhanced insulin secretion from INS-1 cells. Gintonin-stimulated insulin secretion was partially inhibited by a GPR40 receptor antagonist but not an LPA1/3 receptor antagonist and was down-regulated by small interfering RNA (siRNA) against GPR40. Gintonin dose-dependently induced [Ca2+]i transients and Ca2+-dependent cell migration in PC-3 cells. Gintonin actions in PC-3 cells were attenuated by pretreatment with a GPR55 antagonist and an LPA1/3 receptor antagonist or by down-regulating GPR55 with siRNA. Taken together, these results demonstrated that gintonin-mediated insulin secretion by INS-1 cells and PC-3 cell migration were regulated by the respective activation of GPR40 and GPR55 receptors. These findings indicated that gintonin could function as a ligand for both receptors. Finally, we demonstrated that gintonin contained two more GPCR ligands, in addition to that for LPA receptors. Gintonin, with its multiple GPCR ligands, might provide the molecular basis for the multiple pharmacological actions of ginseng.

Effects of Gintonin-Enriched Fraction on Methylmercury-Induced Neurotoxicity and Organ Methylmercury Elimination.

Gintonin is a newly discovered ingredient of ginseng and plays an exogenous ligand for G protein-coupled lysophosphatidic acid receptors. We previously showed that gintonin exhibits diverse effects from neurotransmitter release to improvement of Alzheimer's disease-related cognitive dysfunctions. However, previous studies did not show whether gintonin has protective effects against environmental heavy metal. We investigated the effects of gintonin-enriched fraction (GEF) on methylmercury (MeHg)-induced neurotoxicity and learning and memory dysfunction and on organ MeHg elimination. Using hippocampal neural progenitor cells (hNPCs) and mice we examined the effects of GEF on MeHg-induced hippocampal NPC neurotoxicity, on formation of reactive oxygen species (ROS), and on in vivo learning and memory functions after acute MeHg exposure. Treatment of GEF to hNPCs attenuated MeHg-induced neurotoxicity with concentration- and time-dependent manner. GEF treatment inhibited MeHg- and ROS inducer-induced ROS formations. Long-term treatment of GEF also improved MeHg-induced learning and memory dysfunctions. Oral administration of GEF decreased the concentrations of MeHg in blood, brain, liver, and kidney. This is the first report that GEF attenuated MeHg-induced in vitro and in vivo neurotoxicities through LPA (lysophosphatidic acids) receptor-independent manner and increased organ MeHg elimination. GEF-mediated neuroprotection might achieve via inhibition of ROS formation and facilitation of MeHg elimination from body.

Bioactive lipids in gintonin-enriched fraction from ginseng.

BACKGROUND: Ginseng is a traditional herbal medicine for human health. Ginseng contains a bioactive ligand named gintonin. The active ingredient of gintonin is lysophosphatidic acid C18:2 (LPA C18:2). We previously developed a method for gintonin-enriched fraction (GEF) preparation to mass-produce gintonin from ginseng. However, previous studies did not show the presence of other bioactive lipids besides LPAs. The aim of this study was to quantify the fatty acids, lysophospholipids (LPLs), and phospholipids (PLs) besides LPAs in GEF. METHODS: We prepared GEF from white ginseng. We used gas chromatography-mass spectrometry for fatty acid analysis and liquid chromatography-tandem mass spectrometry for PL analysis, and quantified the fatty acids, LPLs, and PLs in GEF using respective standards. We examined the effect of GEF on insulin secretion in INS-1 cells. RESULTS: GEF contains about 7.5% linoleic (C18:2), 2.8% palmitic (C16:0), and 1.5% oleic acids (C18:1). GEF contains about 0.2% LPA C18:2, 0.06% LPA C16:0, and 0.02% LPA C18:1. GEF contains 0.08% lysophosphatidylcholine, 0.03% lysophosphatidylethanolamine, and 0.13% lysophosphatidylinositols. GEF also contains about 1% phosphatidic acid (PA) 16:0-18:2, 0.5% PA 18:2-18:2, and 0.2% PA 16:0-18:1. GEF-mediated insulin secretion was not blocked by LPA receptor antagonist. CONCLUSION: We determined four characteristics of GEF through lipid analysis and insulin secretion. First, GEF contains a large amount of linoleic acid (C18:2), PA 16:0-18:2, and LPA C18:2 compared with other lipids. Second, the main fatty acid component of LPLs and PLs is linoleic acid (C18:2). Third, GEF stimulates insulin secretion not through LPA receptors. Finally, GEF contains bioactive lipids besides LPAs.

Gintonin, a Ginseng-Derived Exogenous Lysophosphatidic Acid Receptor Ligand, Protects Astrocytes from Hypoxic and Re-oxygenation Stresses Through Stimulation of Astrocytic Glycogenolysis.

Astrocytes are a unique brain cell-storing glycogen and express lysophosphatidic acid (LPA) receptors. Gintonin is a ginseng-derived exogenous G protein-coupled LPA receptor ligand. Accumulating evidence shows that astrocytes serve as an energy supplier to neurons through astrocytic glycogenolysis under physiological and pathophysiological conditions. However, little is known about the relationships between LPA receptors and astrocytic glycogenolysis or about the roles of LPA receptors in hypoxia and re-oxygenation stresses. In the present study, we examined the functions of gintonin-mediated astrocytic glycogenolysis in adenosine triphosphate (ATP) production, glutamate uptake, and cell viability under normoxic, hypoxic, and re-oxygenation conditions. The application of gintonin or LPA to astrocytes induced glycogenolysis in concentration- and time-dependent manners. The stimulation of gintonin-mediated astrocytic glycogenolysis was achieved through the LPA receptor-Gαq/11 protein-phospholipase C-inositol 1,4,5-trisphosphate receptor-intracellular calcium ([Ca2+]i) transient pathway. Gintonin treatment to astrocytes increased the phosphorylation of brain phosphorylase kinase, with sensitive manner to K252a, an inhibitor of phosphorylase kinase. Gintonin-mediated astrocytic glycogenolysis was blocked by isofagomine, a glycogen phosphorylase inhibitor. Gintonin additionally increased astrocytic glycogenolysis under hypoxic and re-oxygenation conditions. Moreover, gintonin increased ATP production, glutamate uptake, and cell viability under the hypoxic and re-oxygenation conditions. Collectively, we found that the gintonin-mediated [Ca2+]i transients regulated by LPA receptors were coupled to astrocytic glycogenolysis and that stimulation of gintonin-mediated astrocytic glycogenolysis was coupled to ATP production and glutamate uptake under hypoxic and re-oxygenation conditions, ultimately protecting astrocytes. Hence, the gintonin-mediated astrocytic energy that is modulated via LPA receptors helps to protect astrocytes under hypoxia and re-oxygenation stresses.

Effects of Gintonin-Enriched Fraction in an Atopic Dermatitis Animal Model: Involvement of Autotaxin Regulation.

Ginseng extract has been used for prevention of atopic dermatitis (AD) in experimental animal models. However, little is known about its active ingredients and the molecular mechanisms underlying its anti-AD effects. Recently, we isolated a unique lysophosphatidic acid (LPA) receptor ligand, gintonin, from ginseng. Gintonin, the glycolipoprotein fraction of ginseng, contains LPAs, mainly LPA C18 : 2 with other minor lysophospholipid components. A line of evidence showed that serum autotaxin (ATX) activity and level are significantly elevated in human AD patients compared to those in normal controls, which indicates that ATX may be involved in human AD. In a previous study, we demonstrated that gintonin exerted anti-inflammatory effects via inhibition of microglial activation and proinflammatory cytokine production by immune cells and that it strongly inhibited ATX activity. In this study, we investigated whether oral administration of the gintonin-enriched fraction (GEF) could ameliorate the symptoms of 2,4-dinitrofluorobenzene (DNFB)-induced AD in NC/Nga mice. We found that oral administration of GEF to DNFB-induced AD mice for 2 weeks reduced ear swelling and AD skin index. In addition, oral administration of GEF reduced the serum levels of immunoglobulin E, histamine, interleukin-4, and interferon-γ. Histological examination showed that oral administration of GEF attenuated skin inflammation and significantly reduced eosinophil and mast cell infiltration into the skin. Moreover, oral administration of GEF not only decreased serum ATX level but also reduced serum ATX activity. The present study shows that the anti-AD effects of ginseng might be attributed to GEF-induced anti-inflammatory activity and ATX regulation.

Gintonin attenuates depressive-like behaviors associated with alcohol withdrawal in mice.

BACKGROUND: Panax ginseng Meyer extracts have been used to improve mood and alleviate symptoms of depression. However, little is known about the extracts' active ingredients and the molecular mechanisms underlying their reported anti-depressive effects. METHODS: Gintonin is an exogenous lysophosphatidic acid (LPA) receptor ligand isolated from P. ginseng. BON cells, an enterochromaffin cell line, and C57BL/6 mice were used to investigate whether gintonin stimulates serotonin release. Furthermore, the effects of gintonin on depressive-like behaviors following alcohol withdrawal were evaluated using the forced swim and tail suspension tests. RESULTS: Treatment of BON cells with gintonin induced a transient increase in the intracellular calcium concentration and serotonin release in a concentration- and time-dependent manner via the LPA receptor signaling pathway. Oral administration of the gintonin-enriched fraction (GEF) induced an increase in the plasma serotonin concentration in the mice. Oral administration of the GEF in mice with alcohol withdrawal decreased the immobility time in two depression-like behavioral tests and restored the alcohol withdrawal-induced serotonin decrease in plasma levels. LIMITATIONS: We cannot exclude the possibility that the gintonin-mediated regulation of adrenal catecholamine release in the peripheral system, and acetylcholine and glutamate release in the central nervous system, could also contribute to the alleviation of depressive-like behaviors. CONCLUSION: The GEF-mediated attenuation of depressive-like behavior induced by alcohol withdrawal may be mediated by serotonin release from intestinal enterochromaffin cells. Therefore, the GEF might be responsible for the ginseng extract-induced alleviation of depression-related symptoms.

Evidence-based guidelines for fall prevention in Korea.

Falls and fall-related injuries are common in older populations and have negative effects on quality of life and independence. Falling is also associated with increased morbidity, mortality, nursing home admission, and medical costs. Korea has experienced an extreme demographic shift with its population aging at the fastest pace among developed countries, so it is important to assess fall risks and develop interventions for high-risk populations. Guidelines for the prevention of falls were first developed by the Korean Association of Internal Medicine and the Korean Geriatrics Society. These guidelines were developed through an adaptation process as an evidence-based method; four guidelines were retrieved via systematic review and the Appraisal of Guidelines for Research and Evaluation II process, and seven recommendations were developed based on the Grades of Recommendation, Assessment, Development, and Evaluation framework. Because falls are the result of various factors, the guidelines include a multidimensional assessment and multimodal strategy. The guidelines were developed for primary physicians as well as patients and the general population. They provide detailed recommendations and concrete measures to assess risk and prevent falls among older people.

Effects of gintonin-enriched fraction on hippocampal cell proliferation in wild-type mice and an APPswe/PSEN-1 double Tg mouse model of Alzheimer's disease.

We previously showed that gintonin, an exogenous lysophosphatidic acid (LPA) receptor ligand, attenuated ß-amyloid plaque formation in the cortex and hippocampus, and restored ß-amyloid-induced memory dysfunction. Both endogenous LPA and LPA receptors play a key role in embryonic brain development. However, little is known about whether gintonin can induce hippocampal cell proliferation in adult wild-type mice and an APPswe/PSEN-1 double Tg mouse model of Alzheimer's disease (AD). In the present study, we examined the effects of gintonin on the proliferation of hippocampal neural progenitor cells (NPCs) in vitro and its effects on the hippocampal cell proliferation in wild-type mice and a transgenic AD mouse model. Gintonin treatment increased 5-bromo-2'-deoxyuridine (BrdU) incorporation in hippocampal NPCs in a dose- and time-dependent manner. Gintonin (0.3 µg/ml) increased the immunostaining of glial fibrillary acidic protein, NeuN, and LPA1 receptor in hippocampal NPCs. However, the gintonin-induced increase in BrdU incorporation and immunostaining of biomarkers was blocked by an LPA1/3 receptor antagonist and Ca2+ chelator. Oral administration of the gintonin-enriched fraction (50 and 100 mg/kg) increased hippocampal BrdU incorporation and LPA1/3 receptor expression in adult wild-type and transgenic AD mice. The present study showed that gintonin could increase the number of hippocampal neurons in adult wild-type mice and a transgenic AD mouse model. Our results indicate that gintonin-mediated hippocampal cell proliferation contributes to the gintonin-mediated restorative effect against ß-amyloid-induced hippocampal dysfunction. These results support the use of gintonin for the prevention or treatment of neurodegenerative diseases such as AD via promotion of hippocampal neurogenesis.

Gintonin enhances performance of mice in rotarod test: Involvement of lysophosphatidic acid receptors and catecholamine release.

Ginseng has a long history of use as a tonic for restoration of vigor. One example of ginseng-derived tonic effect is that it can improve physical stamina under conditions of stress. However, the active ingredient and the underlying molecular mechanism responsible for the ergogenic effect are unknown. Recent studies show that ginseng contains a novel ingredient, gintonin, which consists of a unique class of herbal-medicine lysophosphatidic acids (LPAs). Gintonin activates G protein-coupled LPA receptors to produce a transient [Ca(2+)]i signal, which is coupled to diverse intra- and inter-cellular signal transduction pathways that stimulate hormone or neurotransmitter release. However, relatively little is known about how gintonin-mediated cellular modulation is linked to physical endurance. In the present study, systemic administration of gintonin, but not ginsenosides, in fasted mice increased blood glucose concentrations in a dose-dependent manner. Gintonin treatment elevated blood glucose to a maximum level after 30min. This elevation in blood glucose level could be abrogated by the LPA1/3 receptor antagonist, Ki16425, or the ß-adrenergic receptor antagonist, propranolol. Furthermore, gintonin-dependent enhanced performance of fasted mice in rotarod test was likewise abrogated by Ki16425. Gintonin also elevated plasma epinephrine and norepinephrine concentrations. The present study shows that gintonin mediates catecholamine release through activation of the LPA receptor and that activation of the ß-adrenergic receptor is coupled to liver glycogenolysis, thereby increasing the supply of glucose and enhancing performance in the rotarod test. Thus, gintonin acts via the LPA-catecholamine-glycogenolysis axis, representing a candidate mechanism that can explain how ginseng treatment enhances physical stamina.

Ginseng pharmacology: a new paradigm based on gintonin-lysophosphatidic acid receptor interactions.

Ginseng, the root of Panax ginseng, is used as a traditional medicine. Despite the long history of the use of ginseng, there is no specific scientific or clinical rationale for ginseng pharmacology besides its application as a general tonic. The ambiguous description of ginseng pharmacology might be due to the absence of a predominant active ingredient that represents ginseng pharmacology. Recent studies show that ginseng abundantly contains lysophosphatidic acids (LPAs), which are phospholipid-derived growth factor with diverse biological functions including those claimed to be exhibited by ginseng. LPAs in ginseng form a complex with ginseng proteins, which can bind and deliver LPA to its cognate receptors with a high affinity. As a first messenger, gintonin produces second messenger Ca(2+) via G protein-coupled LPA receptors. Ca(2+) is an intracellular mediator of gintonin and initiates a cascade of amplifications for further intercellular communications by activation of Ca(2+)-dependent kinases, receptors, gliotransmitter, and neurotransmitter release. Ginsenosides, which have been regarded as primary ingredients of ginseng, cannot elicit intracellular [Ca(2+)]i transients, since they lack specific cell surface receptor. However, ginsenosides exhibit non-specific ion channel and receptor regulations. This is the key characteristic that distinguishes gintonin from ginsenosides. Although the current discourse on ginseng pharmacology is focused on ginsenosides, gintonin can definitely provide a mode of action for ginseng pharmacology that ginsenosides cannot. This review article introduces a novel concept of ginseng ligand-LPA receptor interaction and proposes to establish a paradigm that shifts the focus from ginsenosides to gintonin as a major ingredient representing ginseng pharmacology.

Preparation of a Monoclonal Antibody against Gintonin and Its Use in an Enzyme Immunoassay.

Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an [Ca(2+)]i transient in animal cells via activation of LPA receptors. In vitro studies have shown that gintonin regulates various calcium-dependent ion channels and receptors. In in vivo studies, gintonin elicits anti-Alzheimer's disease activity through the activation of the non-amyloidogenic pathway and anti-metastatic effects through the inhibition of autotaxin. However, a method for gintonin quantitation in ginseng has not been developed. In the present study, we developed an enzyme immunoassay (EIA) to measure gintonin. A monoclonal antibody was raised in a mouse using gintonin as the immunogen, and an indirect competitive EIA was used to measure gintonin. The working range was 0.01-10 µg per assay. The anti-gintonin monoclonal antibody did not cross-react with the ginsenosides Ra, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and Rg3 or with LPAs such as LPA C16:0, LPA C18:0, LPA C18:1, and LPA C18:2. Using a standard curve, we measured the amount of gintonin in various ginseng extract fractions. Interestingly, we only detected a little amount of gintonin in conventional hot water extracts of Korean red ginseng. However, we can measure gintonin after ethanol extraction of Korean red ginseng marc. Thus, gintonin can be extracted from ginseng with ethanol but not water, and the remaining Korean red ginseng marc can be used to obtain gintonin. These results indicate that the EIA with the anti-gintonin monoclonal antibody can be used to quantify gintonin in various ginseng preparations, including commercial ginseng products.

Oral Administration of Gintonin Attenuates Cholinergic Impairments by Scopolamine, Amyloid-ß Protein, and Mouse Model of Alzheimer's Disease.

Gintonin is a novel ginseng-derived lysophosphatidic acid (LPA) receptor ligand. Oral administration of gintonin ameliorates learning and memory dysfunctions in Alzheimer's disease (AD) animal models. The brain cholinergic system plays a key role in cognitive functions. The brains of AD patients show a reduction in acetylcholine concentration caused by cholinergic system impairments. However, little is known about the role of LPA in the cholinergic system. In this study, we used gintonin to investigate the effect of LPA receptor activation on the cholinergic system in vitro and in vivo using wild-type and AD animal models. Gintonin induced [Ca(2+)]i transient in cultured mouse hippocampal neural progenitor cells (NPCs). Gintonin-mediated [Ca(2+)]i transients were linked to stimulation of acetylcholine release through LPA receptor activation. Oral administration of gintonin-enriched fraction (25, 50, or 100 mg/kg, 3 weeks) significantly attenuated scopolamine-induced memory impairment. Oral administration of gintonin (25 or 50 mg/kg, 2 weeks) also significantly attenuated amyloid-ß protein (Aß)-induced cholinergic dysfunctions, such as decreased acetylcholine concentration, decreased choline acetyltransferase (ChAT) activity and immunoreactivity, and increased acetylcholine esterase (AChE) activity. In a transgenic AD mouse model, long-term oral administration of gintonin (25 or 50 mg/kg, 3 months) also attenuated AD-related cholinergic impairments. In this study, we showed that activation of G protein-coupled LPA receptors by gintonin is coupled to the regulation of cholinergic functions. Furthermore, this study showed that gintonin could be a novel agent for the restoration of cholinergic system damages due to Aß and could be utilized for AD prevention or therapy.

Gintonin stimulates gliotransmitter release in cortical primary astrocytes.

Lysophosphatidic acid (LPA) is a simple and minor phospholipid, but serves as a lipid-derived neurotransmitter via activation of G protein-coupled LPA receptors. Astrocytes abundantly express LPA receptors and contain gliotransmitters that modulate astrocyte-neuron interactions. Gintonin is a novel ginseng-derived G protein-coupled LPA receptor ligand. Gintonin induces [Ca(2+)]i transients in neuronal and non-neuronal cells via activation of LPA receptors, which regulate calcium-dependent ion channels and receptors. A line of evidence shows that neurotransmitter-mediated [Ca(2+)]i elevations in astrocytes are coupled with gliotransmitter release. However, little is known about whether gintonin-mediated [Ca(2+)]i transients are coupled to gliotransmitter release in astrocytes. In the present study, we examined the effects of gintonin on adenosine triphosphate (ATP) and glutamate release in mouse cortical primary astrocytes. Application of gintonin to astrocytes induced [Ca(2+)]i transients in a concentration-dependent and reversible manner. However, ginsenosides, other active ingredients in ginseng, had no effect on [Ca(2+)]i transients. The induction of gintonin-mediated [Ca(2+)]i transients was attenuated/blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5-triphosphate receptor antagonist, and an intracellular Ca(2+) chelator. Gintonin treatment on astrocytes increased ATP and glutamate release in a concentration- and time-dependent manner. BAPTA and Ki16425 attenuated gintonin-mediated ATP and glutamate release in astrocytes. The present study shows that gintonin-mediated [Ca(2+)]i transients are coupled to gliotransmitter release via LPA receptor activation. Finally, gintonin-mediated [Ca(2+)]i transients and gliotransmitter release from astrocytes via LPA receptor activation might explain one mechanism of gintonin-mediated neuromodulation in the central nervous system.

Gintonin, a novel ginseng-derived lysophosphatidic acid receptor ligand, stimulates neurotransmitter release.

Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an intracellular calcium concentration [Ca(2+)]i transient via activation of LPA receptors and regulates calcium-dependent ion channels and receptors. [Ca(2+)]i elevation by neurotransmitters or depolarization is usually coupled to neurotransmitter release in neuronal cells. Little is known about whether gintonin-mediated [Ca(2+)]i transients are also coupled to neurotransmitter release. The PC12 cell line is derived from a pheochromocytoma of the rat adrenal medulla and is widely used as a model for catecholamine release. In the present study, we examined the effects of gintonin on dopamine release in PC12 cells. Application of gintonin to PC12 cells induced [Ca(2+)]i transients in concentration-dependent and reversible manners. However, ginsenoside Rg3, another active ingredient of ginseng, induced a lagged and irreversible [Ca(2+)]i increase. The induction of gintonin-mediated [Ca(2+)]i transients was attenuated or blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5-triphosphate receptor antagonist, and an intracellular Ca(2+) chelator. Repeated treatment with gintonin induced homologous desensitization of [Ca(2+)]i transients. Gintonin treatment in PC12 cells increased the release of dopamine in a concentration-dependent manner. Intraperitoneal administration of gintonin to mice also increased serum dopamine concentrations. The present study shows that gintonin-mediated [Ca(2+)]i transients are coupled to dopamine release via LPA receptor activation. Finally, gintonin-mediated [Ca(2+)]i transients and dopamine release via LPA receptor activation might explain one mechanism of gintonin-mediated inter-neuronal modulation in the nervous system.

Ginseng gintonin activates the human cardiac delayed rectifier K+ channel: involvement of Ca2+/calmodulin binding sites.

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca(2+)]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K(+) (I(Ks)) channel is a cardiac K(+) channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating I(Ks) channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human I(Ks) channel activity by expressing human I(Ks) channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the I(Ks) channel was 0.05 ± 0.01 µg/ml. Gintonin-mediated activation of the I(Ks) channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the I(Ks) channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca(2+)]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on I(Ks) channel. However, gintonin had no effect on hERG K(+) channel activity. These results show that gintonin-mediated enhancement of I(Ks) channel currents is achieved through binding of the [Ca(2+)]i/CaM complex to the C terminus of KCNQ1 subunit.