In vitro and in vivo evaluation of tissue-cultured mountain ginseng on penile erection.

BACKGROUND: Progressed tissue culture techniques have allowed us to easily obtain mass products of tissue-cultured mountain ginseng over 100 yr old (TCMG-100). We investigated the effects of TCMG-100 extract on erectile function using in vitro and in vivo studies. METHODS: To examine the relaxation effects and mechanisms of action of TCMG-100 on rabbit cavernosal strips evaluated in an organ bath. To investigate the long-term treatment effect of TCMG-100, 8-wk administration was performed. After administration of TCMG-100, intracavernosal pressure, cyclic guanosine monophosphate and nitric oxide (NO) levels of cavernosal tissue, serum testosterone level, histological observation of collagen fiber, endothelium, smooth muscle cell, and transforming growth factor-ß1 were investigated. RESULTS: TCMG-100 extract displayed dose-dependent relaxation effects on precontracted rabbit corporal smooth muscle. The TCMG-100-induced relaxation was significantly reduced by removing the endothelium, and treatment with an NO synthase inhibitor or NO scavenger. Eight weeks of TCMG-100 administration increased intracavernosal pressure in a rat model. The levels of cyclic guanosine monophosphate and NO in the corpus callosum and serum testosterone level were also increased by TCMG-100 treatment. Furthermore, histological evaluation of collagen, smooth muscle, and endothelium showed increases in endothelium and smooth muscle, and a decrease in transforming growth factor-ß1 expression. CONCLUSION: These relaxation effects on corporal smooth muscle and increased erectile function suggest that TCMG-100 might be used as an alternative herbal medicine to improve erectile function.

The Natural Substance MS-10 Improves and Prevents Menopausal Symptoms, Including Colpoxerosis, in Clinical Research.

Many natural substances were screened to develop nutraceuticals that reduce menopausal symptoms. A complex of Cirsium japonicum var. maackii and Thymus vulgaris extracts, named MS-10, had significant positive effects. Under a low concentration of estrogen, which represents postmenopausal physiological conditions, MS-10 had beneficial effects on estrogen receptor-expressing MCF-7 cells by reversibly enhancing estrogen activity. In addition, in the ovariectomized rat model, changes in bone-specific alkaline phosphatase activity and osteocalcin, as well as low-density lipoprotein cholesterol and triglyceride levels were significantly decreased by MS-10. These results show that MS-10 protected bone health and reduced metabolic disturbances. Furthermore, in a clinical study, all menopausal symptoms, including hot flushes, parenthesis, insomnia, nervousness, melancholia, vertigo, fatigue, rheumatic pain, palpitations, formication, and headache, as well as colpoxerosis, were significantly improved by taking MS-10 for 90 days. Therefore, the evidence supports that MS-10 is an effective natural substance that can safely improve menopausal symptoms, including colpoxerosis.

Heartwood extract of Rhus verniciflua Stokes and its active constituent fisetin attenuate vasoconstriction through calcium-dependent mechanism in rat aorta.

Rhus verniciflua Stokes (RVS) exert cardiovascular protective activity by promoting blood circulation, but its active ingredients and underlying mechanism have yet to be identified. This study investigated the vascular effects of RVS, focusing on vasoconstriction and smooth muscle Ca(2+) signaling. RVS heartwood extract attenuated contraction of aortic rings induced by the vasoconstrictors serotonin and phenylephrine, and inhibited the Ca(2+) signaling evoked by serotonin in vascular smooth muscle cells. Subsequent activity-guided fractionation identified fisetin as an active constituent exerting a Ca(2+) inhibitory effect. Fisetin could inhibit major Ca(2+) mobilization pathways including extracellular Ca(2+) influx mediated by the L-type voltage-gated Ca(2+) channel, Ca(2+) release from the intracellular store and store-operated Ca(2+) entry. In accordance with Ca(2+) inhibitory effect, fisetin attenuated vasoconstriction by serotonin and phenylephrine. These results suggest that the anticontractile effect, which is presumably mediated by inhibition of Ca(2+) signaling, may contribute to the improvement of blood circulation by RVS.

Antiplatelet effects of Rhus verniciflua stokes heartwood and its active constituents--fisetin, butein, and sulfuretin--in rats.

Rhus verniciflua stokes (RVS) is known to promote blood circulation by preventing blood stasis, although the active ingredients and the underlying mechanism are unclear. Platelets are the primary cells that regulate circulation and contribute to the development of diverse cardiovascular diseases by aggregation and thrombosis. The study assessed the antiplatelet activity of RVS and sought to identify the active constituents. Pretreatment of washed platelets with RVS heartwood extract blunted the aggregatory response of platelets to collagen. In the subfractions, fisetin, butein, and sulfuretin were identified as effective inhibitors of platelet aggregation by collagen, thrombin, and adenosine-5'-diphosphate. Antiplatelet activities of all three compounds were concentration dependent, and fisetin had longer in vitro duration of action compared with butein or sulfuretin. Extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase activation by collagen was prevented by fisetin, whereas butein and sulfuretin failed to inhibit ERK and p38 activation was not affected by any of the compounds. Rats orally administered 100 mg/(kg·day(-1)) fisetin for 7 days were resistant to arterial thrombosis, although total extract of RVS heartwood exhibited little effect at a dose of 1000 mg/(kg·day(-1)). RVS heartwood may have cardiovascular protective activity by inhibiting platelet aggregation. The active constituents are fisetin, butein, and sulfuretin, and fisetin is orally effective against thrombosis.

Dysfunction of vascular smooth muscle and vascular remodeling by simvastatin.

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are widely prescribed for hypercholesterolemia. With the increasing use of statins, numerous reports demonstrated that statins can cause damage to skeletal muscles. However, the toxicities of statins on vascular smooth muscle, which are essential to cardiovascular homeostasis, have not been previously described. Here, we examined the effects of simvastatin on the contractile function and the integrity of vascular smooth muscle in isolated rat thoracic aortic rings, primary cultured vascular smooth muscle cells (VSMCs) in vitro and rats in vivo. In aortic rings, simvastatin suppressed the normal agonist-induced contractile responses in time- and concentration-dependent manners (0.86 g ± 0.11 at 10 µM simvastatin for 24 h compared with 1.89 g ± 0.11 at control). The suppression persisted in the endothelium-denuded aortic rings and was irreversible even after wash-out of simvastatin. Simvastatin suppressed the contraction induced by Bay K8644, an activator of voltage-operated Ca²âº channel (VOCC) in rat aortic rings and abolished agonist-induced intracellular Ca²âº increase in VSMCs. The simvastatin-induced contractile dysfunction was reversed by the supplementation of mevalonate and geranylgeranylpyrophosphate, precursors for protein isoprenylation. Consistently, activation of RhoA, a representative isoprenylated protein, was disrupted by simvastatin in VSMCs and RhoA-mediated phosphorylation of MYPT1 and CPI-17, and tonic tension were also suppressed. Notably, prolonged treatment of simvastatin up to 48 h induced apoptosis of vascular smooth muscle in aortic rings. Most importantly, simvastatin treatment in vivo significantly attenuated the agonist-induced vasoconstriction in rats ex vivo and induced a decrease in luminal area of the vascular wall. Collectively, these results demonstrate that simvastatin can impair the normal vascular contractility by disturbing Ca²âº influx and RhoA activity, ultimately leading to apoptosis and structural remodeling.

Anti-fatigue activity of Hovenia dulcis on a swimming mouse model through the inhibition of stress hormone expression and antioxidation.

Hovenia dulcis (H. dulcis) Thunb., which is distributed in Korea, China, and Japan, has been known to show hepatoprotective and free radical scavenging effects and enhance physical activity. Therefore, the objectives of this present study were to determine the anti-fatigue activity of hot-water extract from H. dulcis peduncle, and to find the reason why H. dulcis extract (HDE)-ingested mice had enhanced physical activity against swimming performance. The mice orally administrated with HDE (HDE-mice) dramatically enhanced their swimming time compared to the control mice. HDE significantly decreased serum levels of stress hormones, such as cortisol and adrenocorticotropic hormone (ACTH) in mice. The levels of thiobarbituric acid reactive substances (TBARS) were dramatically decreased in gastrocnemius muscle from both 100 mg/kg of HDE (LHDE) and 200 mg/kg of HDE (HHDE)-ingested mice compared to the control mice. The liver activities of superoxide dismutase (SOD) were significantly increased in HHDE-mice with increasing tendency in LHDE-mice. In addition, HHDE-mice significantly decreased the levels of blood glucose, total cholesterol (T-Chol), and triglyceride (TG). These results suggest that HDE had a significant anti-fatigue effect via its anti-stress and antioxidant activities, and thereby enhanced physical activity in swimming performance.

Nectandrin B activates endothelial nitric-oxide synthase phosphorylation in endothelial cells: role of the AMP-activated protein kinase/estrogen receptor α/phosphatidylinositol 3-kinase/Akt pathway.

We revealed previously that nectandrin B isolated from Myristica fragrans (nutmeg, Myristicaceae) functions as a potent AMP-activated protein kinase (AMPK) activator and showed its antiobesity effect. In this study, we investigated whether nectandrin B affects phosphorylation of endothelial nitric-oxide synthase (eNOS) in human endothelial cells. Nectandrin B increased the phosphorylation of eNOS and nitric oxide (NO) production in a concentration-dependent manner and maximal effect was found at 10 µg/ml. Nectandrin B activates AMPK, presumably via Ca(2+)/calmodulin kinase II activation and nectandrin B-stimulated eNOS phosphorylation was reversed by AMPK inhibition. Both the enzyme activity of phosphatidylinositol 3-kinase (PI3K) and the estrogen receptor (ER)-dependent reporter gene transcription were enhanced by nectandrin B. ERα inhibition by specific antagonist or small interfering siRNA (siRNA) suppressed nectandrin B-mediated eNOS phosphorylation. Moreover, AMPK inhibition significantly reversed the activation of ER-dependent transcription and PI3K activation in response to nectandrin B. Nectandrin B evoked endothelium-dependent relaxation in rat aortic rings, and this was blocked by inhibition of AMPK, ER, or PI3K. These results suggest that potent AMPK activator nectandrin B enhances NO production via eNOS phosphorylation in endothelial cells and ERα-dependent PI3K activity is required.

Vascular smooth muscle dysfunction and remodeling induced by ginsenoside Rg3, a bioactive component of ginseng.

Ginseng, one of most well-known herbal medicines, is widely and indiscreetly used among the patients with cardiovascular disorders, raising concern over abuse of this medicine and unwanted effects. In this study, we investigated the effects of ginsenoside Rg3 (Rg3), an active ingredient of ginseng, on vascular contractility and structural integrity to explore its potential vascular toxicity. In isolated rat aorta, Rg3 suppressed the normal agonist-induced contractile response. This suppression persisted even after a rigorous washout. In the endothelium-denuded aortic ring, impairment of vascular contractility by Rg3 was retained, suggesting that vascular smooth muscle was affected. In primary vascular smooth muscle cells, Rg3 abolished agonist-induced Ca(2+) increase, indicating that Ca(2+) regulation was disrupted. Rg3 suppressed the contraction induced by Bay K8644, an L-type Ca(2+) channel activator, whereas store-operated Ca(2+) channel or intracellular Ca(2+) store-mediated contraction was not affected, suggesting that the L-type Ca(2+) channel was selectively impaired by Rg3. These in vitro results were further confirmed in vivo where Rg3 treatment significantly attenuated the agonist-induced pressor response. More importantly, 4-week repeated treatment with Rg3 in normal animals induced eutrophic outward remodeling in the thoracic aorta, that is, it brought about an increased luminal area without changes in the wall area. These results suggest that Rg3 can induce the vascular smooth muscle dysfunction by disturbing Ca(2+) influx from the L-type Ca(2+) channel, ultimately leading to impaired vascular contractility and structural remodeling.

Macelignan: a new modulator of P-glycoprotein in multidrug-resistant cancer cells.

The effect of macelignan, a phytoestrogen, on P-gp function was investigated using multidrug resistant cancer cells overexpressing P-gp (NCI/ADR-RES) and the fluorescent P-gp substrates, daunorubicin and rhodamine 123. Macelignan (40 microM) increased the cellular accumulation of daunorubicin by approximately threefold in NCI/ADR-RES cells, whereas it did not alter the cellular accumulation of daunorubicin in MCF-7/sensitive cells. Similarly, the presence of macelignan also enhanced significantly (P < 0.05) the cellular accumulation of rhodamine 123 in a concentration-dependent manner in NCI/ADR-RES cells. Furthermore, cancer cells were more susceptible to the cytotoxicity of vinblastine, a P-gp substrate, in the presence of macelignan. Those results suggest that macelignan has inhibitory effects on P-gp mediated cellular efflux. However, P-gp activity did not affect the cellular accumulation of macelignan itself. Taken all together, macelignan was identified as a novel inhibitor of P-gp activity and may be a promising lead compound for the rational design of more efficacious drugs to reverse multidrug resistance in cancer.

Dual anti-oxidative effects of fraxetin isolated from Fraxinus rhinchophylla.

Atherosclerosis is main cause of arteriosclerosis. The pivotal role of low-density lipoprotein (LDL) oxidation in atherogenesis suggests antioxidants may help prevent cardiovascular disease. Fraxinus rhynchophylla DENCE (Oleaceae) is a traditional medicinal plant from East Asia. During the course of characterizing potential drug candidates from natural products, we isolated two major coumarins, esculetin and fraxetin and found that fraxetin has dual-antioxidative functions. Low concentrations (1-5 microM) of fraxetin potently inhibited LDL oxidation induced by metal and free radicals. Moreover, treatment of vascular smooth muscle cells (VSMCs) with higher concentrations (above 30 microM) of fraxetin significantly increased the protein level of heme oxygenase-1 (HO-1), a key enzyme that inhibits vascular proliferation and atherosclerosis. Subcellular fractionation and reporter gene analysis using an antioxidant response element (ARE) construct revealed that fraxetin increased the level of nuclear factor (NF)-E2-related factor 2 (Nrf2) and reporter activity, and these were associated with the induction of antioxidant enzymes, such as HO-1 and glutathione S-transferase-alpha. In conclusion, fraxetin has direct protective properties against LDL oxidation at lower concentrations, and higher concentrations of fraxetin induce antioxidant enzymes via Nrf2/ARE activation. These effects suggest potential anti-atherosclerosis effects of Fraxinus rhynchophylla D.

The effects of dehydroepiandrosterone (DHEA)/DHEA-sulfate (DHEAS) on the contraction responses of the clitoral cavernous smooth muscle from female rabbits.

INTRODUCTION: Dehydroepiandrosterone (DHEA) is a multifunctional steroid that is increasingly available as a supplement aimed at improving libido and well-being in postmenopausal women in the recent times. Together with its sulfate version, DHEA-sulfate (DHEAS), it is the most abundant steroid in humans. The clitoris is an important component of the female sexual response, with its increased vascular response during sexual arousal that results in erection. AIMS: To elucidate the direct effects of DHEA/DHEAS on the vasomotor reactivity of the rabbit clitoral cavernosum. METHODS: Twenty New Zealand white female rabbits weighing approximately 2.5-3 kg were used in the study. MAIN OUTCOME MEASURES: The contractile response of clitoral cavernous smooth muscle strips in response to phenylephrine (PE; 10(-9)-10(-4) M) were observed in rabbits. Additionally, DHEA/DHEAS effects on phenylephrine-induced contraction and/or acetylcholine-induced relaxation of phenylephrine-induced contraction were measured. RESULTS: DHEA/DHEAS did not elicit any remarkable response in the resting state. However, both DHEA and DHEAS evoked dose-dependent relaxations of PE-induced contraction. The contractile responses to high potassium were significantly decreased in the DHEA/DHEAS-pretreated strips, compared with the DHEA/DHEAS-nontreated strips. Additionally, contractions by Bay K 8644 (10(-7)-10(-6) M) treatment were also significantly inhibited by DHEA/DHEAS. DHEA-induced relaxation responses were stronger than DHEAS-induced relaxation responses. Various K channel blockers, tetraethylammonium (TEA; 1 mM, 10 mM), 4-aminopyridine (10 microM) and glibenclamide (10 microM) did not affect the DHEA/DHEAS-induced relaxation on muscle strips contracted by PE. Relaxation responses by acetylcholine or sodium nitroprusside (SNP) were not changed after DHEA/DHEAS pretreatment. CONCLUSIONS: DHEA/DHEAS was found to induce a relaxation response in rabbit clitoral cavernosal smooth muscle, and this is thought to be mediated by direct inhibition of a voltage-dependent calcium channel.

U-shaped dose response in vasomotor tone: a mixed result of heterogenic response of multiple cells to xenobiotics.

U-shaped response has been frequently encountered in various biological areas including epidemiology, toxicology, and oncology. Despite its frequent observation, the theory of U-shaped response has been crippled by the lack of a robust mechanism underlying and incomplete in vitro and in vivo correlation. In the present study, a novel mechanism is provided for a U-shaped response, based on the findings of agonist-induced vasomotor tone change affected by menadione (MEN) (synthetic vitamin K(3)), a reactive oxygen species generator, and arsenic, an environmental pollutant, which showed typical U-shaped responses in both in vitro aortic contractile response and in vivo blood pressure. U-shaped responses by MEN and arsenic were a combined result from heterogenic susceptibilities and responses of multiple target cells composing blood vessels, that is, endothelium and smooth muscle. Notably, endothelium, a regulator of vasomotor tone, was primarily affected by low-dose stimuli, whereas smooth muscle, an effector of vascular contraction, was affected later by high-dose. The dysfunction of smooth muscle was produced by high-dose MEN-induced hydrogen peroxide, resulting in the attenuation of vascular contractile reactivity, whereas low-dose MEN-induced superoxide led to the quenching of vasodilatory nitric oxide in endothelial cells, resulting in the enhancement of vasoconstriction. This mechanistic theory, the difference in susceptibilities and responses to a common stimulus between regulator and effector components of a system, could give a new insight into the explanation of various U-shaped responses and provide a new evidence for the need of the risk assessment of toxicants with a wider dose range.

Effects of L-norgestrel on the endothelium-dependent relaxation response of rabbit clitoral cavernous smooth muscles.

OBJECTIVE: To determine the effect of a popular oral contraceptive, L-norgestrel (a synthetic progestogen), on relaxing response of clitoral cavernous smooth muscles. DESIGN: Prospective, randomized study. SETTING: Academic facility. ANIMAL(S): Thirty adult female New Zealand White rabbits. INTERVENTION(S): We conducted isometric tension studies with norepinephrine, endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitroprusside) vasodilators, and L-norgestrel. The effects of nonspecific nitric oxide synthase inhibitor (N(w)-nitro-L-arginine methyl ester) and the potassium channel blockers (1 and 10 mM tetraethylammonium as well as 10 microM glibenclamide) on the reactivities of clitoral cavernous strips were investigated. MAIN OUTCOME MEASURE(S): Causation and power of developed tension after treatment. RESULT(S): Acetylcholine, sodium nitroprusside, and L-norgestrel produced concentration-dependent relaxation of the norepinephrine-precontracted strips. Both endothelium removal and treatment with 10 microM N(w)-nitro-L-arginine methyl ester completely inhibited the relaxation response to acetylcholine and L-norgestrel, and supplementation with 10 mM L-arginine partially reversed the inhibition. Incubation with either tetraethylammonium (TEA) or glibenclamide reduced the L-norgestrel-induced relaxation in a dose-independent manner. CONCLUSION(S): The L-norgestrel-induced relaxation of the clitoral cavernous smooth muscle is endothelium and nitric oxide dependent and may be related to more than two types of potassium channels activation.

Sodium pump alpha2 subunits control myogenic tone and blood pressure in mice.

A key question in hypertension is: How is long-term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain-like compound (EOLC) and induces salt-dependent hypertension mediated by Na(+)/Ca(2)(+) exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na(+) pump alpha1 (alpha1(+/-)) or alpha2 (alpha2(+/-)) catalytic subunits. Both low-dose ouabain (1-100 nm; inhibits only alpha2) and high-dose ouabain (> or =1 microm; inhibits alpha1) elevate myocyte Ca(2)(+) and constrict arteries from alpha1(+/-), as well as alpha2(+/-) and wild-type mice. Nevertheless, only mice with reduced alpha2 Na(+) pump activity (alpha2(+/-)), and not alpha1 (alpha1(+/-)), have elevated blood pressure. Also, isolated, pressurized arteries from alpha2(+/-), but not alpha1(+/-), have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB-R7943) normalize myogenic tone in ouabain-treated arteries. Only the NCX blockers normalize the elevated myogenic tone in alpha2(+/-) arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt-dependent and ouabain-induced hypertension. Thus, chronically reduced alpha2 activity (alpha2(+/-) or chronic ouabain) apparently regulates myogenic tone and long-term blood pressure whereas reduced alpha1 activity (alpha1(+/-)) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt-dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing alpha2 Na(+) pump activity and promoting Ca(2)(+) entry via NCX in myocytes.

Antithrombotic activity of LB30057, a newly synthesized direct thrombin inhibitor.

An amidrazonophenylalanine derivative, LB30057, inhibits the catalytic activity of thrombin potently by interaction with the active site of thrombin, and has high water solubility. In the present study, we evaluated the effect of LB30057 on the biological activities of thrombin at various tissues, and determined whether thrombin inhibition by LB30057 could reduce the incidence of occlusive thrombosis in an in vivo animal model. Treatment with LB30057 to human plasma prolonged clotting times in a concentration-dependent manner. LB30057 suppressed significantly thrombin-induced phosphatidylserine (PS) exposure in platelets, suggesting that LB30057 could inhibit blood coagulation accelerated by PS exposure. In human platelets, soluble thrombin- and clot-induced platelet aggregation was inhibited by LB30057 potently. Consistent with this finding, LB30057 showed concentration-dependent inhibitory effects on serotonin secretion and P-selectin expression induced by thrombin in platelets. In the blood vessel isolated from the guinea pig, treatment with LB30057 resulted in a concentration-dependent inhibition of thrombin-induced vascular contraction. In vivo study revealed that LB30057 following oral administration significantly increased the time to occlusion and improved carotid arterial patency using rat carotid artery thrombosis model. All these results suggest that LB30057 is a potent inhibitor of biological activities of thrombin at various target tissues and, therefore, might be developed as an antithrombotic agent for treatment and prevention of thrombotic diseases.